Investigations of two potential mechanisms which may favour persistence of CDV, the driving force behind the chronic progression of demyelination in canine distemper

Graduate School for Cellular and Biomedical Sciences

University of Bern

PhD Thesis

Submitted by

Dominique Wiener from (Stallikon ZH)

Thesis advisor

Prof. Dr. Andreas Zurbriggen Departement of Clinical Research and Veterinary Public

Health Vetsuisse Faculty of the University of Bern

Accepted by the Faculty of Medicine, the Faculty of Science and the

Vetsuisse Faculty of the University of Bern at the request of the

Graduate School for Cellular and Biomedical Sciences

Bern, Dean of the Faculty of Medicine

Bern, Dean of the Faculty of Science

Bern, Dean of the Vetsuisse Faculty Bern

TABLE OF CONTENTS SUMMARY .......................................................................................................................................... 1 

INTRODUCTION ................................................................................................................................ 3 

1. Infection and disease caused by canine distemper virus ........................................................ 3 

1.1 Natural host range of CDV .................................................................................................... 3 

1.2 Route of infection and virus spread ..................................................................................... 3 

1.3 Clinical signs and tissues infected ....................................................................................... 4 

2. Persistence of CDV in the central nervous system ................................................................... 5 

3. Classification and molecular properties of CDV ........................................................................ 7 

4. Viral proteins ................................................................................................................................... 9 

4.1 Attachment (H) protein .......................................................................................................... 9 

4.2 Fusion (F) protein ................................................................................................................. 10 

4.3 Matrix (M) protein ................................................................................................................. 10 

4.4 Long untranslated region between the M and the F gene (M-F utr) ............................. 11 

4.5 Nucleocapsid (N) protein ..................................................................................................... 12 

4.6 Large (L) protein ................................................................................................................... 12 

4.7 Phosho (P) protein ............................................................................................................... 12 

4.8 C protein ................................................................................................................................ 13 

4.9 V protein ................................................................................................................................. 13 

5. Replication, assembly and release of paramyxoviridae ......................................................... 14 

5.1 Replication ............................................................................................................................. 14 

5.2 Virion assembly and release ................................................................................................ 14 

6. CDV and innate immunity ........................................................................................................... 15 

6.1 Interferons ............................................................................................................................. 16 

6.2 Antiviral functions of IFN ..................................................................................................... 16 

6.3 Interferon induction .............................................................................................................. 17 

6.3.1 TLRs: ............................................................................................................................. 17 

6.3.2 RNA helicases ............................................................................................................. 18 

6.3.3 INF induction ................................................................................................................ 19 

6.4 IFN signaling pathways ....................................................................................................... 20 

7. Objective of the present study: Investigation of two potential mechanisms which may favor persistence of CDV, the driving force behind the chronic progression of demyelination in canine distemper. ......................................................................................................................... 27 

7.1 Investigations about the putative CDV protein M2 .......................................................... 27 

7.2 Molecular mechanisms of innate immune control by wild type CDV V protein .......... 27 

TABLE OF CONTENTS

8. Discussion and perspectives ...................................................................................................... 30 

9. References .................................................................................................................................... 34 

CHAPTER ONE ................................................................................................................................ 40 

CHAPTER TWO ............................................................................................................................... 75 

ACKNOWLEDGEMENTS ............................................................................................................... 91 

CURRICULUM VITAE ..................................................................................................................... 92 

LIST OF PUBLICATIONS ............................................................................................................... 93 

Declaration of Originality ................................................................................................................. 94 

TABLE OF CONTENTS

SUMMARY Canine distemper virus (CDV), a morbillivirus of the paramyxovirus family, closely related

to measles virus (MeV), induces a chronic progressive and relapsing demyelinating

disease in dogs, associated with persistence of the virus in the central nervous system.

This naturally occurring demyelinating disease is considered to be a model for multiple

sclerosis in man. Virus persistence in the central nervous system (CNS) appears to play

an essential role in the chronic progression of the disease. The antiviral immune response

leads to virus clearance in the inflammatory lesions. However, CDV can replicate and

persist outside these inflammatory lesions within the brain. Viral persistence is thus the

driving force behind the chronic progression of the disease. The mechanism of

persistence of CDV in the presence of an effective antiviral immune response is not well

understood. In this study two potential mechanisms of CDV persistence were investigated.

We hypothesized that control of persistence could be influenced by the unusually long

“untranslated” region between the M and the F gene of CDV, which is common to all

morbilliviruses. In particular, previous studies revealed a short potential open reading

frame (ORF) situated at the end of the M gene and is referred to as M2. Intriguingly, even

though the sequence of the MF utr differs dramatically between the different CDV strains,

the region of the putative ORF is conserved in current persistent CDV strains. The

conservation of these putative ORF’s within the MF utr indicates, that there must be some

evolutionary pressure in maintaining these ORF’s and suggests a functional role of this

sequence. However, up to now, there is no evidence that the M2 protein is expressed.

In this study, we investigated whether this short polypeptide was expressed from a

putative ORF located within the 3’ utr of the M mRNA of the highly virulent CDV-A75/17

strain. To this purpose, using reverse genetics, we engineered several recombinant

viruses. Even though M2 could efficiently be expressed in transfection experiments,

similar results could not be obtained in the background of full viral infection. Rather,

several biochemical and immunological assays (such as fluorescence microscopy,

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SUMMARY

immunoblot, flow cytometry and immunoprecipitation) indicated that in viral infection, M2

was not translated. Cell type-specific restriction of M2 expression was unlikely since M2

could not be detected in a range of different cell systems (vero SLAM cells, vero cells,

MDCK SLAM cells, keratinocytes or DBCC’s). All together our results suggest absence of

M2 expression, at least in quantities that could be detected by standard techniques. We

conclude that M2 does not play a role in persistence.

The second mechanism of viral persistence investigated in this study involves immune

evasion of CDV. Morbilliviruses have evolved several strategies to hijack the host cell-

mediated innate immunity. The CDV-V protein has been shown to act as a virulence

factor. Here, we investigated the molecular mechanisms by which the P gene products of

the neurovirulent A75/17-CDV disrupted type I interferons- (IFN-α/β)-mediated antiviral

state. Using recombinant knockout A75/17 viruses, the V protein was identified as the

main antagonist of IFN- α/β -mediated signaling. Importantly, immunofluorescence

analysis illustrated that the latter inhibition correlated with impaired STAT1/STAT2 nuclear

import, though their phosphorylation states were not affected. Co-immunoprecipitation

assays identified the N-terminal region of V (VNT) responsible for STAT1 targeting, which

corroborated with the ability to inhibit the activity of IFN- α/β -mediated antiviral state.

Conversely, while the C-terminal domain of V (VCT) could not function autonomously,

when fused to VNT, it optimally associated with STAT2 and subsequently strongly

suppressed the IFN- α/β -mediated signaling pathway. The latter result was further

supported by a single mutation at position 110 in V resulting in a mutant that lost STAT1

binding, while retaining partial STAT2 association. Taken together, our results identified

VNT and VCT as two essential modules complementing each other to complete IFN- α/β

evasion, which may be involved in CDV-persistence in the CNS. Our experiments also

reveal a novel mechanism of IFN-α/β evasion among the morbilliviruses.

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INTRODUCTION

1. Infection and disease caused by canine distemper virus

CDV causes in dogs a chronic, demyelinating, progressive or relapsing neurological

disease, because it persists in the CNS (Vandevelde and Zurbriggen, 2005). CDV is a

model for multiple sclerosis in man (Appel M.J. and Gillespie J.H., 1972). A

spontaneous MS-like disease with multifocal demyelinating lesions is rare in domestic

animals. Primary demyelination in domestic animals has only been observed for Visna,

a lentivirus infection in sheep and in CDV infection. While demyelination is a rare

complication in Visna, it occurs with high frequency in distemper (Vandevelde and

Zurbriggen, 2005).

1.1 Natural host range of CDV

The host spectrum of CDV is widespread and includes numerous families in the order

of Carnivorae like Canidae, Procyonidae, Mustelidae, Mephtidae, Hyaenidae,

Ailuridae, and Viverridae (Beineke et al., 2009). In addition, in the last years, several

outbreaks occurred in large felids (Appel et al., 1994; Roelke-Parker et al., 1996) and

in collard peccaries (Appel et al., 1991). Furthermore, besides infection with the

closely related phocine distemper virus, seals can become infected by CDV (Kennedy

et al., 2000; Kuiken et al., 2006). Recent outbreaks in St.Gallen, Graubünden,

Appenzell, Liechtenstein and Zürich in foxes and badgers show the presence of CDV

in Switzerland (Basler Zeitung, 17.7.2009; Schweiz Magazin, 12.3.2010).

1.2 Route of infection and virus spread

The incubation period may vary from 1-4 weeks and depends on viral strain, age and

immune status of the host. Disease manifestation ranges from virtually no clinical

signs to severe disease with approximately 50% mortality (Appel, 1970). The virus is

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INTRODUCTION

shed primarily by oro-nasal secretion (but any discharge and secretion can carry the

virus). CDV infects susceptible dogs primarily by inhalation of airborne virus or via

infective aerosol droplets, followed by virus replication in lymphoid-tissue of the

respiratory tract (Beineke et al., 2009). Tissue macrophages and monocytes located in

or along the respiratory epithelium and in tonsils represent the first cell type to pick up

and propagate the virus (Appel, 1970). Then, the virus is disseminated by lymphatics

and blood to distant hematopoietic tissues during the first viraemic phase. After initial

infection of the immune system (lymph nodes, spleen, thymus, bone marrow, mucosa-

associated lymphatic tissues), the second viraemia follows several days later,

frequently associated with high fever, and results in infection of parenchymal and

tissue cells throughout the body (Beineke et al., 2009), including the skin and CNS

where it establishes a persistent infection (Vandevelde and Zurbriggen, 2005; Gröne

et al., 2004).

1.3 Clinical signs and tissues infected

First clinical signs are characterized by lethargy, dehydration, anorexia, weight loss,

development of a biphasic fever, diarrhea, vomiting, mucopurulent and oculo-nasal

discharge, coughing, respiratory distress and possible loss of vision (Beineke et al.

2009; McGavin and Zachary, 2007). Weeks later, nervous signs including ataxia,

paralysis, convulsions, or monoclonus (muscle twitches, tremors and “tics”) occur.

Hard pad disease represents an uncommon cutaneous manifestation of distemper and

is characterized by hyperkeratosis of the footpads and nasal planum. Though the

pathogenesis of this unusual manifestation remains undetermined, it seems that CDV

causes a disturbance of keratinocyte differentiation (Gröne et al., 2003 and 2004;

Beineke et al., 2009).

Canine distemper virus has also the tendency to affect developing tooth buds and

ameloblasts, causing enamel hypoplasia in dogs that recover from infection (McGavin

et al., 2007). A persistence of primary spongiosa in the metaphysic of long bones also

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INTRODUCTION

termed metaphyseal osteosclerosis or growth retardation lattice has been described in

young dogs suffering from systemic canine distemper (Baumgärtner et al., 1995a and

1995b). Of all distemper lesions, demyelinating encephalomyelitis, which develops

late, is the most devastating (McGavin et al., 2007). The virus causes in the CNS

multifocal lesions in the gray and white matter. Generally, demyelinating lesions

prevail. The predilection sites are the white matter of the cerebellum, the

periventricular white matter (especially around the 4th ventricle), the optic pathways

and the spinal cord. CDV enters the brain by infected mononuclear cells penetrating

the blood barrier and more importantly by circulating in the central spinal fluid and

fusing with the ependymal lining of ventricles, hence the supial and periventricular

location of the lesions (Vandevelde and Zurbriggen, 2005). Respiratory manifestation

results in serous to mucopurulent rhinitis, interstitial pneumonia and necrotizing

bronchiolitis, which is often complicated by a suppurative bronchopneumonia due to

secondary bacterial infection. Enteral infection leads to catarrhal enteritis with

depletion of Peyer’s patches. In naturally infected dogs, a pustular dermatitis, also

termed distemper exanthema, of thighs, ventral abdomen and the inner surface of ear

pinnae can be found. Additionally, a generalized depletion of lymphoid organs and an

associated immunosuppression represents an important and common manifestation of

canine distemper (Beineke et al., 2009).

2. Persistence of CDV in the central nervous system The persistence of CDV in the CNS is related to selective spread of the virus from cell

to cell with limited budding and very limited cytolysis, thus delaying immune detection

of the virus. The initial myelin lesion develop during a period of severe

immunosuppression and are not inflammatory, since perivascular cuffs are entirely

lacking and it was shown that demyelination coincides with replication of CDV in glial

cells. Therefore the demyelination in the acute stages of the disease is virus-induced.

In chronic stages of the disease, demyelination of the CNS is compounded by

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INTRODUCTION

immunopathological reactions related to persistence of the virus (Vandevelde and

Zurbriggen, 2005). Infection with the virulent, persistent canine distemper wild-type

strain, called A75/17, is associated with a very limited cytopathic effect, limited

budding, selective cellular spread and with very limited cell-cell fusion in canine

footpad keratinocytes and primary canine brain cells (Zurbriggen et al., 1995). This

phenotype of infection is related, at least in part, to viral genetic factors. Comparison

between an attenuated, cytolytic CDV strain (Onderstepoort) and a virulent, persistent

CDV strain (A75/17) showed profound differences in the way the two viruses spread in

culture. The attenuated CDV spreads randomly to immediately adjacent cells, whereas

persistent CDV spreads selectively to more-distant cells by way of cell processes,

enabling the virus to invade the central nervous system without the need of releasing

much virus into the extracellular space (Zurbriggen et al., 1995). The attenuated

Onderstepoort (OP) CDV strain induces the formation of large multi-nucleated cells

(syncytia), followed by subsequent cytolysis. In addition, OP-CDV has been shown to

bud very efficiently from many different kind of cells, including primary canine brain

cells. These marked differences between the wild and attenuated CDV strains, have

been used to investigate viral molecular determinants related to persistent infection

(Vandevelde and Zurbriggen, 2005). Another study, comparing the two strains,

suggests that both cell-cell spread and limited production of infectious virus are related

to reduced expression of fusogenic complexes on the cell membrane, such as the

fusion (F) and attachement (H) proteins on the cell surface. F and H proteins

colocalized strongly in the cytolytic infection of the attenuated strain, but not in the

persistent strain (Meertens et al., 2003). In lymphoid tissue, however, the wild type

strain causes a severe cytolytic infection and the virus cannot persist. Therefore, the

persistence is, in addition to viral factors, also dependent on the infected tissue. The

lymphotropism of CDV is presumably based on the binding of the H protein of CDV to

the SLAM (signaling lymphocyte activation molecule) receptor, also called CD150,

followed by entry of CDV into the cell. SLAM is consitutively expressed in a variety of

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INTRODUCTION

organs (such as lung, gastrointestinal tract, transitional epithelium) and cells (such as

lymohocytes and macrophages). Upregulation of SLAM expression was observed in

infected dogs, indicating a possible strategy to increase virus amplification in the host

(Wenzlow et al. 2007). Interestingly, SLAM could not be identified either in the footpad

keratinocytes (Wenzlow et al., 2007) or in the brain (Wyss-Fluehmann et al., 2010).

3. Classification and molecular properties of CDV CDV is a non-segmented, negative-stranded, enveloped RNA-virus and belongs to the

morbillivirus genus in the paramyxovirus family. The family of Paramyxoviridae has

been classified into 2 subfamilies, the Paramyxovirinae and the Pneumovirinae (Table

1.). At the present, the subfamily of Paramyxovirinae contain 5 genera (Respirovirus,

Rubulavirus, Henipavirus, Avulavirus and Morbillivirus), while the subfamily

Pneumovirinae contains two (Pneumovirus and Metapneumovirus) (Fontana et al.,

2008). The biologic criteria for this classification are (a) antigenic cross-reactivity

between members of a genus and (b) the presence (Respirovirus, Rubulavirus,

Avulavirus) and absence (Morbillivirus and Henipavirus) of neuraminidase activity

(Lamb et al., 2001). Morbilliviruses in general have been grouped together by their

sequence relatedness and lack of neuraminidase activity. It was found that measles

virus (MeV), CDV, and rinderpest virus (RPV) strains could use SLAMs of their

nonhost species as receptors, albeit at reduced efficiencies. Despite sequence

differences, the structure required for the interaction with morbillivirus H proteins may

be well conserved among SLAMs of many different species. Therefore, the use of

SLAM as a cellular receptor may be included in the characteristic properties of

morbilliviruses (Tatsuo et al., 2001).

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INTRODUCTION

Table 1. Genera and representative species of the family Paramyxoviridae (Fontana et

al., 2008)

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4. Viral proteins The CDV genome consists of six genes, coding for 6 structural proteins, i.e. N

(nucleocapsid), M (matrix), F (fusion), H (attachment), P (phospho) and L (large)

protein, which together form the polymerase complex as well as 2 accessory non-

structural proteins, C and V (Lamb et al., 2001). In addition, in between the M and the

F gene, there is an unusual long untranslated region (MF-utr) with a short putative

open reading frame (ORF) of 52 amino acids (Stettler et al., 1997).

4.1 Attachment (H) protein Attachment of virus particles to receptors on host cell membranes is mediated by the

H protein, an integral membrane glycoprotein on the envelope of the virus. In addition

to the receptor binding function, the attachment protein of the Genera Respirovirus,

Morbillivirus and Avulavirus also agglutinate red blood cells, and the attachment

protein of the Genera Respirovirus, Rubulavirus and Avulavirus possess

neuraminidase activity, whereas Morbilliviruses and Henipavirus lack neuraminidase

activity (Fontana et al., 2008; Lamb et al., 2001). Respiroviruses and rubulaviruses

bind to sialic acid-containing proteins or lipids, while pneumoviruses attach to

glycosaminogylcans containing heparin sulphate and chondroitinsulfate. In contrast,

morbilliviruses and henipaviruses bind specific receptor proteins on the cell surface.

For MeV, two receptors have been identified: CD46 and SLAM. CDV and RPV bind to

canine and bovine homologs of SLAM, respectively. Henipaviruses use Ephrin B2 and

Ephrin B3 as cellular receptors (Fontana et al., 2008). After binding to the SLAM

receptor, the H protein interacts with the F protein. This interaction leads to a

rearrangement of the structure of the F protein, which allows subsequently the viral

genome to enter the host cell (von Messling et al., 2004).

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INTRODUCTION

4.2 Fusion (F) protein Following attachement, the F glycoprotein mediates fusion of the viral envelope with

the plasma membrane of the host cell and release of the nucleocapsid into the

cytoplasm. The F proteins of paramyxoviruses are synthesized as inactive precursors

(F0) that are proteolytically cleaved to biologically active F1 and F2 proteins (Fontana

et al., 2008). The F proteins, are type I integral membrane proteins, which span the

membrane once and contain at their N-terminus a cleavable signal sequence, that

targets the nascent polypeptide chain synthesis to the membrane of the endoplasmatic

reticulum (ER). At their C-termini, a hydrophobic stop-transfer domain anchors the F

protein (as well as the H protein) in the membrane, leaving a short cytoplasmic tail.

Later in infection, the F proteins expressed at the plasma membrane of infected cells

can mediate fusion with neighbouring cells to form syncytia formation (cytopathic

effect, that can lead to tissue necrosis in vivo) (Lamb et al., 2001). Co-expression of

both, the F and the H protein, are necessary and sufficient to induce cellular fusion

(Stern et al., 1995) and both the wild-type F and H proteins as well as the canine

SLAM receptor act in concert to determine the phenotype of infection (Plattet et al.,

2005).

4.3 Matrix (M) protein The M protein is localized beneath the viral lipid bilayer of the envelope and is thought

to be peripherally associated with the membrane and therefore is not an intrinsic

membrane protein. It is considered to be a central organizer of viral morphogenesis,

interacting with the cytoplasmic tails of the integral membrane proteins, the lipid

bilayer and the nucleocapsid (Lamb et al., 2001). In Sendai and Measles virus, the

interaction of the M protein with the two surface glycoproteins and the nucleocapsid

has an influence on cell–cell fusion (Cathomen et al., 1998a and 1998b). Interestingly,

it has been published that, in the absence of the measles M protein, nucleocapsids

were not transported to the cell surface, suggesting that M can drag nucleocapsids

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INTRODUCTION

from inclusion bodies to the plasma membrane (Runkler et al., 2007). Our previously

published study suggests that very limited fusogenicity in virulent CDV infection

(favouring persistence by limiting cell destruction) involves complex interactions

between all viral structural proteins. Fusion efficiency may be determined by the

structure of the viral fusion protein per se but also by its interaction with other

structural proteins of CDV. This was studied by combining genes derived from

persistent and non-persistent CDV strains in transient transfection experiments. It was

found that fusion efficiency was markedly attenuated by the structure of the fusion

protein of the neurovirulent A75/17-CDV and that the interaction of the surface

glycoproteins with the M protein of the persistent strain greatly influenced fusion

activity. Site directed mutagenesis showed that the C-terminus of the M protein is of

particular importance in this respect. Interestingly, although the nucleocapsid protein

alone did not affect F/H-induced cell–cell fusion, maximal inhibition occurred when the

latter was added to combined glycoproteins with matrix protein (Wiener et al., 2007).

4.4 Long untranslated region between the M and the F gene (M-F utr) In between all the genes of CDV untranslated regions of about 100 – 200 nucleotides

can be found, however in between the M and the F gene an unusual long untranslated

region of about 1000 nucleotides, exists in MeV and all other morbilliviruses (Heider et

al., 1997). Its precise function, if any, is unknown but deletion of this region in a ferret

CDV strain led to loss of neurovirulence (Anderson and von Messling, 2008). Previous

studies in CDV revealed a short potential open reading frame (ORF) situated at the 3’

end of the M1 within this untranslated region (referred as M2) just downstream of the

M1 ORF (Stettler et al., 1997). This small putative ORF was also found in other CDV

strains such as the tissue culture adapted strains Rockborn and vero-adapted strain

A75/17v, as well as in Snyder-Hill (derived from a natural case of distemper, passaged

in dog brains). The presence of this additional open reading frame appeared to

correlate with the ability to cause a spontaneous persistent infection in vitro (Stettler et

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al., 1997). It is not yet known whether this ORF is produced by the CDV strains and so

far there is no evidence that the M2 protein is expressed.

4.5 Nucleocapsid (N) protein The N protein has several functions in viral replication, including encapsidation of the

genome RNA into an RNAse-resistant nucleocapsid, association with the P-L

polymerase during transcription and replication and interaction with the M protein

during virus assembly (Lamb et al., 2001). Cherpillod et al. (2000) showed, that it was

sufficient to inoculate the nucleocapsid and the glycoproteins of the virulent strain

A75/17 to protect dogs against distemper by inducing an efficient humoral and cellular

immune response

4.6 Large (L) protein The L protein is the least abundant of the structural proteins. The L gene is the most

promoter-distal in the transcriptional map and thus the last to be transcribed. There

are five short regions in the L gene of high homology near the center that are also

conserved in the RNA-dependent RNA polymerases (RNAP) of other virus families.

Mutational analysis of these highly conserved regions indicates that these regions are

essential for RNAP activity. The P and the L proteins form a complex, and both are

required for polymerase activity (Lamb et al., 2001).

4.7 Phosho (P) protein The P gene represents an extraordinary example of a virus compacting as much

genetic information as possible into a small gene. Apart from the P protein, there are

also the 2 non-structural proteins V and C that can be expressed from the P gene.

Expression of CDV-V depends on the insertion of a non-templated guanine nucleotide

at a precise location, named “editing site”, which generates a messenger RNA that

differs from the one of P by one or two nucleotides. This generates an mRNA with an

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INTRODUCTION

altered ORF downstream of the editing site, and thus, due to this specific mechanism,

the N-terminal domain of P and V are identical, while their C-terminal domains are

unique. The P protein is the only P gene product that is essential for viral RNA

synthesis. It is an essential component of the viral RNAP and the nascent chain

assembly complex. Although the L protein is thought to contain all viral RNAP catalytic

activities, L binds to the N:RNA template via the P protein (Lamb et al., 2001)

4.8 C protein The function of the C protein is not well known, but there is evidence that in measles

virus it plays a role as an infectivity factor (Devaux et al., 2004) and it antagonizes the

proapoptotic and antiviral activities of protein kinase (PKR) (Toth et al., 2009,).

4.9 V protein The V protein is encoded within the P gene. The N-terminus of the V protein is

identical to the P protein, the C-terminal domain of V (VCT) is unique and is known to

contain a conserved cysteine-rich region (Paterson et al., 1995; Thomas et al., 1988)

and recent X-ray studies confirmed that VCT folds into a zinc finger conformation (Li et

al., 2006).The V protein has been identified as the main inhibitor of the IFN-induced

antiviral state, though various molecular mechanisms were unraveled (Fontana et al.,

2008).

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INTRODUCTION

5. Replication, assembly and release of paramyxoviridae

5.1 Replication Intracellular replication of paramyxoviruses begins immediately after release of the

nucleocapsid into the cytoplasm and is catalyzed by the viral RNA-dependent RNA

polymerase (vRNAP). RNA synthesis begins at the 3’ end of the genome, transcribing

the genes into mRNAs in a sequential manner by terminating and reinitiating at each

of the gene junctions. The RNAP occasionally fails to reinitiate the downstream mRNA

at each junction, leading to a loss of further-downstream genes. Hence there is a

gradient of mRNA synthesis that is inversely proportional to the distance of the gene

from the 3’ end of the genome. After primary transcription and translation, when

sufficient amounts of unassembled N protein are present, the viral RNA synthesis

becomes coupled to the concomitant encapsidation of the (+) nascent RNA chain.

Under these conditions, vRNAP ignores all the junctions, to produce an exact

complementary antigenome chain, in a fully assembled nucleocapsid. (Lamb et al.,

2001).

5.2 Virion assembly and release The assembly of viral particles requires cessation of genome replication, preparation

of completed nucleocapsids for packaging and accumulation of genomes and

nucleocapsids at the plasma membrane for budding. Polymerase complexes remain

associated with the packaged nucleocapsid and serve to initate the next circle of

infection. While the nucleocapsid is assembled in the cytoplasm, the glycoproteins are

synthesized in the endoplasmatic reticulum (eR) and undergo maturation during their

transport through the Golgi network to the cell membrane (Fontana et al., 2008). The

folding of the glycoproteins is not a spontaneous event, but it is assisted by numerous

folding enzymes and chaperons. Only correctly folded and assembled proteins are

generally transported out of the eR. As mentioned before, the M protein plays a major

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INTRODUCTION

role in bringing the assembled ribonucleoprotein core to the plasma membrane to form

a budding virion. The glycoprotein cytoplasmic tails make important contacts with the

M protein, which, in turn, associates with the nucleocapsid (Lamb et al., 2001). In

sendai virus (SV) it was shown that expression of SV M protein induced the budding

and release of virus-like particles that contained the M protein only. Expression of the

F protein caused release of virus-like particles as well, but the release was less

efficient. Cells that expressed only the haemagglutinin-neuraminidase (HN) protein

released no HN-containing particles. Coexpression of F and M proteins enhanced the

release of F protein (Takimoto et al., 2001). In measles it was shown, that M stability

and accumulation at the intracellular membranes is a prerequisite for M and

nucleocapsid co-transport to the plasma membrane and for subsequent virus

assembly and budding process. This was found by creating recombinant viruses that

had a mutated M protein and therefore had an increased intracellular turnover but no

defective binding to other proteins. This recombinant virus was barely released from

infected cells, showing, that the defect in assembly was not due to a defective M

binding to other proteins but rather due to a reduced ability to associate with the

cellular membranes, accompanied by a deficient transport of nucleocapsids to the cell

surface (Runkler et al., 2007).

6. CDV and innate immunity The innate immunity refers to defence mechanisms of the host that are present even

before infection and have evolved to specifically recognize microbes and protect the

host against infections. Innate immunity is the first line of defence, because it is

always ready to prevent and eradicate infections. The major components of innate

immunity are epithelial barriers that block entry of environmental microbes, phagocytic

cells (mainly macrophages and neutrophils), natural killer cells and several plasma

proteins (Abbas, 1999).

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INTRODUCTION

6.1 Interferons The interferons (IFN) are a group of secreted cytokines that elicit antiviral effects.

They are grouped in 3 classes called type I, II and III IFNs. Type I interferon comprise

a large group of molecules. Mammals have multiple distinct IFN-α and one to three

IFN-β genes (and other genes, such as IFN-ω, -ε, -δ and –κ). The IFN-α and –β genes

are induced directly in response to viral infection, whereas the other IFNs play less

well-defined roles. Thus IFN type I is rather called IFN-α/β. Type III IFNs have been

described more recently and comprise IFNλ1,-λ2 and -λ3, also referred to as IL-29, IL-

28A and IL-28B, respectively. These cytokines are also induced in direct response to

viral infection and appear to use the same pathway as the IFN-α/β genes to sense

viral infection. Type II IFN has a single member, also called IFN-γ or ‘immune IFN’,

and is secreted by mitogenically activated T cells and natural killer (NK) cells, rather

than in direct response to viral infection (Randall et al., 2008).

6.2 Antiviral functions of IFN Although IFN-α/β, IFN-γ, and IFN-λ share no obvious structural homology, they all

exhibit the ability to generate an ‘antiviral state’ in target cells. The establishment of an

anti-viral state by IFNs involves the induction of a large number of IFN-stimulated

genes (ISGs) encoding cytokines and enzymes that interfere with viral and cellular

processes to block viral replication. For example, double stranded RNA (dsRNA)-

dependent protein kinase (PKR) recognizes dsRNA that is produced during viral

replication and activates itself via autophosphorylation. Activated PKR inhibits protein

synthesis by phosphorylating the subunit of eukaryotic initiation factor 2 (eIF2a) and

also acts on numerous other substrates within the cell to establish an anti-viral state.

Another important way the IFNs limit infection is by inhibiting cell growth and

promoting programmed cell death, or apoptosis, in target cells. In addition to their

direct anti-viral and anti-proliferative properties, the IFNs also play an important role in

regulating host immunity, which can profoundly impact the ability of the host to control

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INTRODUCTION

infection. Both IFN-α/β and IFN-γ promote the upregulation of major histocompatibility

complex (MHC) class I molecules. Because many viruses downregulate the

expression of MHC class I molecules, this is an important function of IFN that

enhances the ability of CD8+ T cells to recognize and kill virally infected cells. IFNs

also directly regulate the activities of cells participating in the innate and adaptive

immune responses. For example, IFN- α/β is critical for the enhancement of NK-cell

cytotoxicity by upregulating levels of perforins and indirectly influencing NK-cell

proliferation, and it has been shown to promote the maturation of dendritic cells. IFN-

α/β also promotes the proliferation of antigen-specific CD8+ T cells, while

simultaneously inhibiting the proliferation of naïve CD8+ T cells (Fontana et al., 2008).

6.3 Interferon induction Most pathways required for the induction of IFN- α/β are linked to interactions between

viral pathogen-associated molecular patterns (PAMPs) and host pattern-recognition

receptors (PRRs). Two major types of proteins are currently recognized as the cellular

PRRs involved in the induction of IFN- α/β: Toll like receptors (TLRs) and RNA

helicases (Fontana et al., 2008).

6.3.1 TLRs: TLRs are membrane molecules that function in cellular activation by a wide range of

microbial pathogens. In general, TLRs 1,2,4 and 6 recognize bacterial products that

are found on the cell surface, and TLRs 3,7,8, and 9 are involved in viral detection and

nucleic acid recognition within endosomes (Snyder, 2007). The primary ligand for

TLR3 is dsRNA, which is a replication intermediate for many viruses. TLR7, TLR8, and

TLR9 are classified in the TLR9 subfamily due to similarities in their amino acid

sequences, but they recognize different ligands and display different expression

patterns. While TLR7 and TLR8 both recognize viral ssRNA, TLR9 recognizes

unmethylated CpG DNA of bacteria and viruses (Table 2.). The TLRs are most

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INTRODUCTION

commonly expressed in cellular endosomal compartments. This particular subcellular

localization is important, because it means that the cell does not need to be infected to

produce IFN- α/β, but rather it can recognize viral RNA from inactivated virus particles

or from dead cells as they are taken into the endosomal compartment (Fontana et al.,

2008). All TLRs contain an extracellular domain characterized by a leucin-rich repeat

motif flanked by a cystein-rich motif (Fig. 1). They also contain a conserved

intracellular signaling domain, Toll/interleukin (IL)-1 receptor (TIR). TLRs and their

pathogen-associated ligands are important recognition molecules for the innate

immune system and trigger a number of antimicrobial and inflammatory responses.

Although the individual TLRs exhibit ligand specificity, they differ in their cellular

expression patterns and the signal pathways they activate. There are constitutively

and inducibly expressed TLRs in different tissues. TLRs regulate cell-recruitment to

sites of infection through up-regulation of the expression of adhesion molecules,

chemokines and chemokine receptors during inflammatory response. TLRs activate

leukocytes and epithelial, endothelial and hematopoietic cells. TLRs are also

hypothesized to be essential for linking the innate immune response to the adaptive

immune response (Snyder, 2007).

6.3.2 RNA helicases In contrast to TLRs, RNA helicases are cytosolic and provide a TLR-independent

mechanism for detecting viral nucleic acids generated in the cytoplasm of an infected

cell by viral replication. The two best studied RNA helicases are retinoic acid inducible

gene I (RIG-I) and melanoma differentiation-associated gene-5 (mda-5). Each of these

proteins contains a domain, which recognizes dsRNA, and two amino-terminal

caspase-recruiting domain (CARD)-like regions, which are responsible for recruiting

downstream signaling molecules. Although both RIG-I and mda-5 recognize dsRNA,

these proteins differ in their recognition of various RNA viruses and of specific RNA

structures. For example, RIG-I but not mda-5 recognizes uncapped, unmodified

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INTRODUCTION

5’triphosphate RNA, which allows detection of MeV and other members of the order

Mononegavirales. In contrast, mda-5 is required to mediate IFN- α/β responses to

polyriboinosinic:polyribocytidylic acid (polyI:C), the synthetic analog of viral dsRNA,

and to encephalomyocarditis (EMC) picornavirus infection in vivo (Fontana et al.,

2008).

6.3.3 INF induction In response to their respective ligands, TLR3, TLR7, TLR8, and TLR9, RIG-I, and

mda-5 each initiate a unique signaling cascade that results in the activation of

transcription factors that promote the induction of IFN- α/β. The ‘classical pathway’ is

most commonly induced by signaling through TLR3, RIG-I, or mda-5, which results in

activation of the main IFN regulatory transcription factors, IFN regulatory factor-3 (IRF-

3) and nuclear factor kB (NF-kB). TLR3 recruits an adapter protein called Toll-IL-1-

receptor domain containing adapter inducing IFN-β (TRIF), which acts as a scaffolding

protein to recruit additional components of two downstream signaling pathways that

result in the activation and translocation to the nucleus of IRF-3 and NF-kB,

respectively.

Upon translocation to the nucleus, these transcription factors bind to the IFN-β

promoter cooperatively with the c-jun/ATF-2 transcription factor to form the

‘enhanceosome,’ which is required for optimal transcription of the IFN-β gene.

IFN-β production that is induced in this way positively feeds back on the cell to

upregulate the IRF-7 transcription factor. IRF-7 is also able to bind to the IFN-β

promoter and enhance the production of IFN-β, or it can activate a ‘second wave’ of

IFN production by promoting the transcription of IFN-α genes (Figure 2A.)

While most cells are able to induce a modest IFN response to viral infection that can

be enhanced through the IRF-7- mediated positive feedback mechanism described

above, plasmacytoid dendritic cells (pDCs) can mount a rapid and extremely robust

IFN response without the need for positive feedback, due to their constitutive

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INTRODUCTION

expression of IRF-7 (Figure 2B). IFN is induced in these cells through TLR7-, TLR8-,

and TLR9-mediated signaling pathways involving the recruitment of the myeloid

differentiation factor 88 (MyD88) adapter protein. Upon activation, MyD88 recruits IL-1

receptor-associated kinase 1 (IRAK-1) and IRAK-4 into a complex that acts as a

scaffold to recruit additional signaling components responsible for activating IRF-7 and

NF-kB. The activation of these transcription factors enables them to translocate to the

nucleus, where they promote transcription of the IFN-β gene and of multiple IFN-α

genes (Fontana et al., 2008). Similarly, RIG-I and mda-5 recruit an adapter protein

called CARD adapter inducing IFN-β (Cardif), which leads to independent activation of

both IRF-3 and NF-kB (Figure 3.) (Randall et al., 2008).

6.4 IFN signaling pathways The IFN-α/β receptor complex consists of two subunits, IFN receptor (IFNAR) 1 and 2,

which are associated with the ‘Janus’ tyrosine kinases, Tyk2 and Jak1, respectively.

Upon binding of IFN- α/β to the receptor, the subunits dimerize and bring these

kinases within sufficiently close proximity to activate each other by

transphosphorylation. Activated Tyk2 phosphorylates tyrosine 466 on IFNAR1, which

serves as a docking site for signal transducer and activator of transcription (STAT)

molecule, STAT2. Tyk2 then phosphorylates STAT2 on tyrosine 690 to recruit STAT1,

which is subsequently phosphorylated at tyrosine 701. The phosphorylated STATs

form a stable heterodimer, which results both in the creation of a novel nuclear

localization signal (NLS) and in the masking of an intrinsic nuclear export signal (NES)

in the carboxyl-terminus of STAT2. It is currently thought that the STAT1/STAT2

heterodimer next translocates to the nucleus, where it associates with the DNA-

binding protein IRF- 9, to form the IFN-stimulated gene factor 3 (ISGF3) complex. The

ISGF3 complex binds to IFN-stimulated response elements (ISREs) within the

promoters of IFN- α/β -inducible ISGs and activates transcription (Figure 4.) (Fontana

et al., 2008).

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INTRODUCTION

TLR LIGAND MICROBIAL SOURCE

TLR2 Lipoproteins

Peptidoglycan

Zymosan

LPS

GPI Anchor

Lipoarabinomannan

Phosphatidylinositol-

Dimannoside

Bacteria

Gram-positive bacteria

Fungi

Leptospira

Trypanosomes

Mycobacteria

Mycobacteria

TLR3 Double-stranded RNA Viruses

TLR4 LPS

HSP60

Gram-negative bacteria

Chlamydia

TLR5 Flagellin Various bacteria

TLR6 CpG DNA Bacteria, protozoans

TLR7 Single-stranded RNA Viruses

TLR8 Single-stranded RNA Viruses

TLR9 CpG DNA Bacteria, viruses

Table 2. Toll-like Receptors (TLRs) and TLR Ligands and their microbial source.

Modified from Kumar V, Abbas AK, Fausto N: Robbins & Cotran pathologic basis of

disease, ed 7, Philadelphia, 2005, Saunders. CpG, Cytosine and guanine-linked

oligonucleotide; GPI, glycosyl phosphatidyl inositol; HSP60, heat shock protein 60;

LPS, lipopolysaccharide

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INTRODUCTION

Figure 1. Signaling by a prototypic TLR, TLR4, in response to bacterial LPS.

An adapter protein links the TLR to a kinase, which activates transcription factors such

as NF-κB and AP-1. TIR, Toll/IL-1 receptor domain. From Robbins & Cotran pathologic

basis of disease, ed 7, Philadelphia, 2005, Saunders.

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INTRODUCTION

Figure 2. Pathways involved in the induction of IFNα/β.

TLR3 recognizes extracellular or endosomal dsRNA and recruits the TRIF adapter

protein. Likewise, RIG-I and mda-5 intracellular RNA helicases recognize cytoplasmic

dsRNA and recruit the Cardif/VISA/MAVS/IPS-1 adapter protein. TRIF and

Cardif/VISA/MAVS/IPS-1 each acts as a scaffold to recruit other signaling molecules

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INTRODUCTION

that are responsible for activating the NF-kB and IRF-3 transcription factors. NF-kB

and IRF-3 bind cooperatively with c-jun/ATF-2 to the IFN-β promoter in order to induce

the ‘first wave’ of IFN-β expression. IFN-β signaling induces the upregulation of the

IRF-7 transcription factor, which both positively feeds back on IFN-β expression and

activates the transcription of a ‘second wave’ of IFN-α expression. (B) The pDC

pathway. TLR7, TLR8, and TLR9 located within the endosomal compartment of pDCs

recognize viral ssRNA or unmethylated CpG DNA upon endocytosis of viral particles.

The MyD88 adapter protein is subsequently recruited and forms a complex with IRAK-

1 and IRAK-4, which acts as a scaffold to recruit other signaling molecules that are

responsible for activating the NF-kB and IRF-7 transcription factors. NF-kB and IRF-7

bind cooperatively with c-jun/ATF-2 to the IFN-β promoter and induce or enhance its

expression. IRF-7 can also directly induce the expression of IFN-α in pDCs without the

need for positive feedback (Fontana et al., 2008).

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INTRODUCTION

Figure 3. Mda-5 and RIG-I-dependent signaling.

Viral RNA, generated in the cytoplasm by uncoating, transcription or replication,

activates the RNA helicases mda-5 and RIG-I. Mda-5 and RIG-I are both activated by

dsRNA, whilst RIG-I can also be activated by RNA molecules with 5’ triphosphates.

Both helicases have N-terminal CARD domains that recruit the adaptor

Cardif/VISA/MAVS/IPS-1. This adaptor, in turn, acts as a scaffold to recruit signaling

components that feed into either the IRF-3 or the NF-kB pathways. Although the

details of these downstream signalling pathways remain incomplete, for Cardif/VISA/

MAVS/IPS-1 activation, they seem very similar to those events described in Fig. 2

downstream of TRIF (Randall et al., 2008).

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INTRODUCTION

Figure 4. Signalling pathway activated by IFN-α/β.

The biological activities of IFN-α/β are initiated by binding to the type I IFN receptor.

This leads to the activation of the receptor associated tyrosine kinases JAK1 and

Tyk2, which phosphorylate STAT1 on tyrosine 701 and STAT2 on tyrosine 690.

Phosphorylated STAT1 and STAT2 interact strongly with each other by recognizing

SH2 domains, and the stable STAT1–STAT2 heterodimer is translocated into the

nucleus, where it interacts with the DNA-binding protein IRF-9. The IRF-9– STAT1–

STAT2 heterotrimer is called ISGF3 and it binds to a sequence motif (the IFN

stimulated response element or ISRE) in target promoters and brings about

transcriptional activation. In addition to the phosphorylation of tyrosine, STAT1 also

requires phosphorylation on serine 727 for function (Randall et al., 2008).

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INTRODUCTION

7. Objective of the present study: Investigation of two potential mechanisms which

may favor persistence of CDV, the driving force behind the chronic progression of

demyelination in canine distemper.

7.1 Investigations about the putative CDV protein M2 As mentioned before, the CDV genome contains an unusual long “untranslated” region

between the M and the F gene, which is common to all morbilliviruses. Studies by

others (Anderson and von Messling, 2008) in a ferret CDV strain have shown that

deletion of this region leads to marked attenuation of the infection in vivo. Previous

studies in our lab revealed a short potential open reading frame (ORF) situated at the

end of the M gene and is referred to as M2. However, its expression has never been

shown. In the present study we performed transient transfection experiments with

several plasmids encoding the M2 ORF, or the M2 ORF fused to an HA-tag or fused to

a red fluorescent protein (RFP) which could show the expression of M2. To validate

this result in the background of full viral infection, several recombinant viruses were

generated with the purpose of detecting expression of M2 by various methods as well

as the production of M2 knock out mutants. Several immunological and biochemical

assays could not reiterate the result of the transfection experiments, therefore

indicating that M2 was not translated. Knocking out M2 did not alter viral growth

kinetics. Altogether, our data provide evidence that M2 is not expressed by CDV, at

least in sufficient amounts to be detected by standard techniques.

7.2 Molecular mechanisms of innate immune control by wild type CDV V protein A pathogen has to evade the innate immune system in order to establish an infection

in the host. In this study we focused on Interferon (IFN) type I. This cytokine is

produced by almost all cells upon viral infection and induces an antiviral state.

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INTRODUCTION

Recently, it has been documented that a virulent CDV strain (5804P) genetically

modified to inactivate V, was attenuated in ferrets, whereas a C-defective CDV was

fully immunosuppressive (von Messling et al., 2006). These findings demonstrate that

wild type CDV suppressed IFN induction but whether additional modulation of IFN-

mediated signaling sustains viral attenuation remains to be determined. In addition,

recent work done with C- and V-deficient MeV and RPV recombinant viruses, indicated

that V and to some extent P contribute to the final control of IFN-α/β-mediated

signaling pathway. However, to analyze the functions of the P gene products, these

recombinant viruses were based on the genetic background of vaccine strains

(Devaux et al., 2007; Nanda and Baron, 2006). Nevertheless, the paramyxovirus V

protein has been identified as the main inhibitor of the IFN-induced antiviral state,

though various molecular mechanisms were unraveled (Horvath, 2004a and 2004b).

In this study, we investigated the role of the P gene products of the highly virulent

A75/17-CDV strain in counteracting the IFN-α/β-mediated signaling pathway.

Importantly, this strain was isolated from a naturally infected dog and subsequently

kept amplified only in dogs, where it has been reported to maintain its virulence

(Cherpillod et al., 2000). Therefore, this virus has never been adapted to any cell lines.

However, the generation of recombinant virus stocks (rA75/17) with sufficient titers to

work with requires two to three passages in Vero-SLAM cells after virus rescue from

primary full-length cDNA-transfected cells (Rivals et al., 2007). Because these limited

amplification steps might already select viral variants, the entire genome of rA75/17

has been compared by direct sequencing to the one of the parental A75/17 strain and

exhibited no nucleotide difference (Rivals et al., 2007), thereby validating the unique

opportunity to investigate the molecular mechanisms of virus-host cell interactions

based on a demyelinating morbillivirus strain. Hence, recombinant A75/17 viruses and

expression plasmids were generated to investigate the role of the P gene products in

mediating IFN evasion. Infection and transfection experiments were performed in Vero

cells stably expressing the SLAM receptor for CDV. Indeed, Vero-SLAM cells are

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INTRODUCTION

defective in IFN production and thus not only provide an optimal tool to exclusively

study IFN signaling independently of IFN induction, but they also support very efficient

A75/17-CDV replication.

Our results demonstrate that the V protein was the main viral factor responsible for

disrupting the IFN-α/β-mediated signaling pathway. The latter inhibition was neither

due to STAT1 or STAT2 degradation nor to an impairment of their phosphorylation

states upon IFN-α/β treatment. Rather, the CDV-V protein efficiently associated with

both STAT1 and STAT2, which correlated with complete inhibition of both transcription

factors’ nuclear import. Furthermore, transient expression experiments of engineered

V proteins identified both the N-terminal and the C-terminal domains as two

interdependent modules necessary to exhibit optimal IFN evasion.

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INTRODUCTION

8. Discussion and perspectives CDV induces a multifocal demyelinating disease in dogs, similar to multiple sclerosis

in man (Vandevelde and Zurbriggen, 2005). Initial demyelination is associated with

viral replication in the white matter of the CNS. The antiviral immune response, with

invasion of immune cells in the CNS, leads to viral clearance within the inflammatory

lesion. However, despite an apparently effective immune response, CDV can persist

outside of the lesions and continues to replicate and spread to other areas. As a result

of viral persistence, a chronic progressive and relapsing disease develops (Zurbriggen

et al., 1995). Understanding of the mechanisms by which CDV is able to evade the

immune response of the host hence establishing persistence, is essential to design

therapeutic measures and improve prevention of this disease. The present study

focused on two potential mechanisms of persistence of CDV.

We first focused on the untranslated region between the M and F CDV gene. Previous

studies showed that the whole M-F untranslated region of CDV modulates virulence by

controlling F protein expression (Anderson and von Messling, 2008). The latter study,

was done with a ferret-adapted CDV strain (5804PeH) constructed by von Messling et

al. (2003). In MeV, which is closely related to CDV Takeda et al. (2005) found, that the

long utr per se was not essential for MeV replication, but that it regulated MeV

replication and cytopathogenicity by modulating the production of the M and F.

Previous studies in our lab on virulent A75/17CDV revealed a short potential open

reading frame (ORF) situated at the 3’ end of the M1 within this untranslated region

(utr) just downstream of the M1 ORF. Intriguingly, even though the sequence of the

MF utr differs dramatically between the different CDV strains, the region of the putative

ORF is conserved in the current persistent CDV strains. The conservation of these

putative ORF’s within the MF utr indicates, that there must be some evolutionary

pressure in maintaining these ORF’s and suggests a functional role of this sequence

(Stettler et al., 1997). Moreover, it is intriguing that the attenuated cytolytic OP CDV

strain lacks the M2 ORF in contrast to the CDV Rockborn, Snyder Hill and Vero

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INTRODUCTION

adapted A75/17 strains, which in vitro all induce a persistent phenotype. In the present

study we investigated if the M2 protein is expressed and if there is a potential function

of this small protein. Even though M2 could efficiently be expressed in transfection

experiments, the same results could not be obtained in the background of full viral

infection. Several biochemical and immunological assays (such as fluorescence

microscopy, immunoblot, flow cytometry and immunoprecipitation) could not

demonstrate that in the context of a viral infection, M2 is translated. Cell type-specific

restriction of M2 expression was unlikely since M2 could not be detected in a variety of

cellular environments (vero SLAM cells, vero cells, MDCK SLAM cells, keratinocytes

or DBCC’s). In addition, the in vitro behavior of M2 knock out A75/17 Virus did not

differ from that of the parent virus. Thus these negative findings do not support a role

of the M2 ORF in persistence. On the other hand they do not exclude that the MF-utr

region may still play a role maybe not in terms of protein translation but in terms of

stability or controlling up- and/or downstream proteins. It remains intriguing why CDV

conserved this region and even more why it maintained a small putative ORF of 52

amino acids.

Persistence of CDV strongly depends on the ability of the virus to suppress the

immune response of the host. It has recently been described that V knockout CDV

(based on the 5804P virulent strain) was attenuated in infected ferrets, which was

associated, at least in part, with inhibition of IFN-α/β induction in PBMCs (von

Messling et al., 2006). In the present study we showed that the V protein of the highly

virulent A75/17-CDV strain plays a critical role in counteracting innate immunity by

additionally disrupting the IFN-α/β-dependent signaling. Detailed molecular analysis

enabled us to demonstrate that V specifically ablated STATs nuclear import without

affecting their activated phosphorylation states. Furthermore, inhibition of IFN-α/β-

dependent signaling correlated with the capacity of the V protein to efficiently interact

with STAT1 and STAT2. Finally, we identified both the N-terminal and the C-terminal

regions of V as playing a synergistic role in IFN evasion.

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INTRODUCTION

Initial attempts to map the domains of the V protein that interact with STAT1 and

STAT2 revealed that the N-terminal domain of V (VNT) could function as an

autonomous domain interfering with IFN-α/β-induced signaling. Importantly, co-IP

experiments indicated that VNT retained association with STAT1 but failed to co-purify

STAT2. Since the full length V protein (Vwt) could efficiently co-precipitate both STATs

molecules, this suggests that the VNT domain is very likely responsible for STAT1

interaction, whereas VCT is necessary to target STAT2. Anyway, we cannot exclude

that VCT, when fused to VNT, determines a specific conformational state of VNT that

confers the capacity of the N-terminal region to target both STATs molecules.

Nevertheless, we propose that CDV-VNT and -VCT are two interdependent modules

that function synergistically to allow proper folding of the full-length V protein. In turn,

Vwt gains the ability to efficiently interact with STAT1 via its N-terminal region and

STAT2 through its C-terminal domain, which consequently offers optimal conditions to

prevent nuclear import of both STAT molecules and, consequently, to control IFN-α/β-

mediated signaling. Combined, while further work is required to support or reject this

model, this hypothesis is in excellent agreement with our results demonstrating that

CDV-V is not affecting STAT1 and STAT2 phosphorylation.

In addition to V, the CDV-P protein (sharing the identical N-terminal region with V) also

exhibited slight interaction with STAT1, which correlated with partial suppression of

IFN-α/β-mediated signaling. This is consistent with many negative strand RNA virus P

proteins, where a role in regulating innate immunity has been suggested (e.g. MeV or

RPV) (Devaux et al., 2007; Nanda and Baron, 2006;). Nevertheless, our results

obtained in a co-IP assay indicated that P bound STAT1 very inefficiently, suggesting

that a high amount of P is presumably required to act as an effective antagonist of

IFN-mediated signaling.

The peculiar molecular mechanism by which the A75/17-CDV-V protein inhibits the

IFN-α/β-mediated response differs from studies performed with other morbilliviruses.

To our knowledge, all studies performed with MV-V, except the one of Palosaari et al.

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INTRODUCTION

(2003), have reported an inhibition of the phosphorylation of STAT1 (Caignard et al.,

2007; Takeuchi et al., 2003; Yokota et al., 2003) and STAT2 (Caignard et al., 2009;

Devaux et al., 2007; Takeuchi et al., 2003). The reasons for the differences between

CDV and MeV remain unclear. In addition to genuine biological differences between

these two morbilliviruses, the origin and passaging histories of the strains used to

study evasion from IFN action may be a factor. Indeed, we studied a highly virulent

viral strain not adapted to cultured cells, whereas the strain of MeV was attenuated

(Devaux et al., 2007), or persistently infected cells were investigated (Yokota et al.,

2003). Taken together, our results shed light into a unique molecular mechanism

sustained by the neurovirulent A75/17-CDV among the Paramyxovirus family in

controlling IFN-α/β-mediated signaling.

While we do not exclude additional molecular mechanisms, the exclusive strategy

applied by the demyelinating morbillivirus A75/17 strain in controlling the IFN-α/β-

induced response may contribute to the development of viral persistence in the CNS

and ensuing progressive tissue destruction. We are convinced that understanding the

various molecular mechanisms employed by different viruses, even among the same

genus, could inevitably raise the opportunity to develop new virus-specific therapeutic

strategies. Hence, while the precise V peptide domains responsible for STAT1 and

STAT2 association need to be defined, this might represent ideal targets for the

development of antiviral compounds that may be employed for treatment and/or to

improve current vaccines.

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INTRODUCTION

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Cathomen T., Naim H.Y., Cattaneo R. (1998b) Measles virus with altered envelope

protein cytoplasmic tails gain cell fusion competence. J.Virol. 72, 1224-1234

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M., Zurbiggen R., Bruckner L., Roch F., Vandevelde M., Zurbriggen A. (2000) DNA

vaccine encoding nucleocapsid and surface protein of wild type canine distemper virus

protects its natural host against distemper. Vaccine 18, 2927-2936

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Tyrosine 110 in the measles virus phosphoprotein is required to block STAT1

phosphorylation. Virology 360, 72-83

Fontana J.M., Bankamp B., Rota P.A. (2008) Inhibition of interferon induction and

signaling by paramyxoviruses. Immunological Reviews. 225, 46-67

Gröne A., Engelhardt P., Zurbriggen A. (2003). Canine distemper virus infection:

proliferation of canine footpad keratinocytes. Vet.Pathol. 40(5), 574-8

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footpad epidermis. Vet. Dermatol. 15(3), 159-67

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INTRODUCTION

Investigation of a unique short open reading frame within the 3’

untranslated region of the canine distemper virus matrix gene

Dominique Wiener1, Marc Vandevelde2, Andreas Zurbriggen1 and

Philippe Plattet1*

1Department of Clinical Research and Veterinary Public Health and 2Division of

Neurology, Vetsuisse faculty, University of Bern, Switzerland.

*To whom correspondence should be addressed, Bremgartenstrasse 109a, 3001 Bern,

Switzerland. Phone: +4131 631 26 48. Fax: ++4131 631 25 38. E-mail:

philippe.plattet@itn.unibe.ch

Abstract: 250

Text: 5347

Running Title: Investigation of the expression of a novel putative CDV open reading

frame

Keywords: wild-type morbillivirus, matrix gene, 3’ untranslated region, open reading frame, translation

Submitted in Virus Research

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Abstract

Increasing evidence suggest that the long “untranslated” region (UTR) between

the matrix (M) and the fusion (F) genes of Morbilliviruses has a functional role. In

canine distemper virus (CDV), the F 5’ UTR was recently shown to code for a long F

signal peptide (Fsp). Subsequently, it was reported that the M/F UTRs combined with

the long Fsp were synergistically regulating the F gene and protein expression, thereby

modulating virulence. Unique to CDV, a short putative open reading frame (ORF) has

been identified within the wild-type CDV-M 3’ UTR (termed M2). Here, we

investigated whether M2 was expressed from the genome of the virulent and

demyelinating A75/17-CDV strain. An expression plasmid encoding the M2 ORF

tagged both at its N-terminal (HA) and C-terminal domains (RFP), was first

constructed. Then, a recombinant virus with its putative M2 ORF replaced by HA-M2-

RFP was successfully recovered from cDNA (termed recA75/17green-HA-M2-RFP). M2

expression in cells infected with these mutants was studied by immunoprecipitation,

immunofluorescence, immunoblot and flow cytometry analyses. Although fluorescence

was readily detected in HA-M2-RFP-transfected cells, absence of red fluorescence

emission in several recA75/17green-HA-M2-RFP-infected cell types suggested lack of

M2 biosynthesis, which was confirmed by the other techniques. Consistent with these

data, no functional role of the short polypeptide was revealed by infecting various cell

types with HA-M2-RFP over-expressing or M2 knock-out recombinant viruses. Thus,

in sharp contrast to the CDV-F 5’ UTR reported to translate a long Fsp, our data

provided evidence that the CDV-M 3’ UTR does not express any polypeptides.

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1. Introduction

Canine distemper virus (CDV) causes a chronic, demyelinating, progressive or

relapsing neurological disease in dogs, because it persists in the CNS (Vandevelde and

Zurbriggen, 2005). CDV is a non-segmented, negative-stranded, enveloped RNA virus

which belongs to the morbillivirus genus in the paramyxovirus family (Lamb and

Kolakofsky, 2001). Six independent messenger RNAs (mRNAs) are generated by a so-

called “stop-start” sequential transcriptional mechanism driven by the viral RNA-

dependent RNA polymerase (vRdRp) (Lamb and Kolakofsky, 2001). While five out of

the six mRNAs contain 5’ and 3’ untranslated regions (UTR) of short sizes, the

mRNAs encoding the matrix protein (M) and the fusion protein (F) contain unusually

long 3’ UTR and 5’ UTR, respectively (referred to as M 3’ UTR and F 5’ UTR). Since

it is well known that viruses tend to evolve to contain high capacity-encoding genomes

of minimal size to the advantage of efficient viral replication (Domingo and Holland,

1997) the long M/F UTR within the CDV genome can be assumed to offer some

functional advantages to the virus. Indeed, it has been recently documented that the F

5’ UTR of CDV, formerly considered to be truly untranslated, is in fact essential to

translate an unusually long F signal peptide (von Messling and Cattaneo, 2002).

In line with these results, it has recently been shown that ferrets infected with a

recombinant virulent CDV strain (5804P) in which the entire M/F UTR was replaced

by the shorter N/P UTR remained only partially virulent. When the M/F UTR region

was removed including the region coding for the signal peptide of the F protein (Fsp),

the corresponding recombinant virus was fully attenuated (Anderson and von Messling

2008). However, the role of the CDV-M 3’ UTR was not investigated individually and

the results obtained in the latter study could therefore be attributed either to a combined

deletion effect of the Fsp sequence with the M 3’ UTR, the Fsp sequence with the F 5’

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UTR or the Fsp sequence with both UTRs. Moreover, it remains also possible that the

length and specific sequences of both M/F UTRs may not be of primary importance.

Indeed, the N/P cis-acting sequences controlling the vRdRp-dependent upstream gene

transcriptional stop and downstream gene re-initiation signals are different from the

one found in the M/F UTR and may have result in the observed unbalanced F gene

transcriptional control. In the related Morbillivirus, measles virus (MeV), in vitro

studies demonstrated that the long M 3’ UTR and long F 5’ UTR were nonessential for

virus replication, but that they regulate MeV replication and cytopathogenicity by

modulating the production of the M and F proteins. The latter finding suggests a role of

this region in MeV-induced pathogenesis, although in vivo experiments were not

performed (Takeda et al., 2005).

The CDV-M mRNA contains about 1500 nucleotides, from which the M-protein

is translated by a classical ribosomal scanning mechanism. Interestingly, previous

sequence analyses revealed the existence of short putative open reading frame (ORF)

located within the 3’ UTR of the M mRNA of the A75/17-CDV strain and will be

referred in this study as M2 (Stettler et al., 1997). While it seems unlikely that the

ribosomes would efficiently scan 1250 nucleotides before initiating translation at the

first potential M2 initiation codon, unusual translation mechanisms are often utilized by

many viruses to encode proteins, including internal ribosome entry sites, ribosome

shunting, and coupled translation termination/initiation process, all of which are

dependent on sequences and structures present within the mRNA molecule that direct

ribosomal initiation at the desired location (Ahmadian et al., 1999; Hemmings-

Mieszczak and Hohn, 1999; Jang et al., 1988; Latorre et al., 1998; Pelletier and

Sonenberg, 1988; Yueh and Schneider, 1996). Moreover, the putative M2 ORF was

found in the wild-type A75/17-CDV, which exhibits a persistent phenotype in vitro.

Conversely, the M2 ORF was revealed to be absent from the M 3’ UTR of the highly

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attenuated cytolytic Onderstepoort CDV strain (Stettler et al., 1997). We therefore

speculated that M2 could be a hitherto unidentified viral determinant that may

synergize with the Fsp to control CDV-persistence and/ or virulence.

In order to explore this hypothesis, five specific recombinant viruses were

engineered and successfully recovered from cDNA. Subsequently, their corresponding

phenotypes were investigated in different cell types, which were expressing, or not, the

universal Morbillivirus receptor CD150/SLAM. Combined, our results provided strong

evidence that M2 was not expressed from the unusually long M 3’ UTR of the virulent

A75/17-CDV strain, at least not in sufficient amounts to be detected by standard

fluorescence and biochemical techniques. Thus, we here formally demonstrated that the

CDV-M 3’ UTR, as opposed the CDV-F 5’ UTR, does not express any polypeptides,

thereby suggesting different putative mechanism(s) of action.

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2. Materials and Methods

2.1 Cells and viruses

Vero-SLAM cells (kindly provided by V. von Messling, INRS-Institute

Armand-Frappier, University of Quebec, Laval, Quebec, Canada), MDCK-SLAM cells

(Rothlisberger et al., 2010), dog brain cell cultures (Zurbriggen and Vandevelde, 1983),

Vero cells and Bsr-T7 cells (Buchholz et al., 1999) were grown in Dulbecco’s Modified

Eagle Medium (Invitrogen) supplemented with 10% fetal calf serum (FCS), penicillin

and streptomycin. Canine footpad keratinocytes were cultured as described (Engelhardt

et al., 2005). They are maintained in William's Emedium (Bioconcept, Allschwil, CH)

including antibiotic/antimytotic solution (Gibco BRL, Basel, CH), 10% fetal bovine

serum, L-glutamine 2 mM (Bioconcept,Allschwil, CH), 10−10 M cholera toxin (Sigma,

Buchs, CH), and 10 ng/ml of epidermal growth factor EGF (Sigma). The selection of

Vero cells expressing the SLAM receptor was maintained by adding ZeocinTM

(Invitrogen). All cells were kept at 37°C in the presence of 5% CO2. The infection

experiments were performed with recombinant viruses based on the wild-type A75/17-

CDV strain (obtained from M. Appel, Cornell University, Ithaca, NY).

2.2 Antibodies

The anti-M2 antibody was produced against a synthetic peptide (nh2-

PRTCPISSYH-cooh) corresponding to amino acids 18-27 of the putative open reading

frame (M2) located at the untranslated region between the M and the F gene (M/F

UTR) of the A75/17 CDV strain (Primm srl, Custom Antibodies, Milano, Italy). The

anti-HA-tag affinity matrix rat monoclonal antibody (Clone 3F10, Roche) and the anti-

HA-tag mouse monoclonal antibody (Clone 16B12, Covance) were used for

immunoprecipitation (IP) (3F10), western blot and immunofluorescence (16B12). The

monoclonal anti-N antibody (D110) was used to perform western blots.

2.3 Generation of plasmids and recombinant viruses

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In order to generate a suitable control for the different experiments, several

plasmids were generated. First, the pCI-M2 plasmid was generated by amplifying by

PCR (Expand High FidelityPLUS PCR System, Roche) the M2 ORF from the A75/17

full-length cDNA plasmid and cloned into the RsrII-cleaved pCI expression vector. The

HA-tag peptide (YPYDVPDYA) was next added, by PCR technology, in frame to the

N-terminal domain of the pCI-M2 ORF, thus generating pCI-HA-M2. Finally, the

plasmid pCI-HA-M2-RFP was created by adding a linker and a red fluorescence

marker gene in frame with HA-M2 (tD tomato, (Wyss-Fluehmann et al., 2010)) by

PCR technology.

The recombinant viruses are based on a neurovirulent wild-type isolate CDV-

A75/17 (Summers et al., 1984). The recombinant virus recA75/17red was previously

described (Rivals et al., 2007). The latter contains an additional red fluorescence

marker located between the M and F genes. To generate the full-length modified

genomic vector, a shuttle vector was first produced by amplifying a segment containing

the M 3’ UTR and subsequently cloned into the pCI-digested plasmid. Then, PCR

amplicons from vector pCI-HA-M2 and pCI-HA-M2-RFP were cloned into the shuttle

vector. Finally, full-length genomic plasmids were re-formed by cloning the fragments

from the shuttle vector into pA75/17red, thus producing pA75/17green-HA-M2 and

pA75/17green-HA-M2-RFP, respectively. To produce an M2 knock-out plasmid, we

mutated the three potential start codons of M2 into stop codons. The mutations were

obtained applying the QuikChange® Site-Directed Mutagenesis Kit (Stratagene) to the

aforementioned shuttle vector. The pA75/17red-M2ko plasmid was re-generated by

cloning the modified fragment from the shuttle vector into cleaved version of

pA75/17red. All full length cDNA clones respected the rule of six (Calain and Roux,

1993). To rescue the recombinant viruses we proceeded as previously described (Plattet

et al., 2004). Briefly, Bsr-T7 cells were co-transfected with the plasmids containing the

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relevant full-length cDNA and with helper plasmids coding for the N, P and L proteins

of CDV. After two days the transfected Bsr-T7 cells were co-cultured with Vero-SLAM

cells. When a cytopathic effect had developed the cells were lysed by two freeze and

thaw cycles and the viruses (recA75/17red, recA75/17green-HA-M2, recA75/17green-HA-

M2-RFP, recA75/17green-M2Ko and recA75/17HA-M2-RFP) were stored at -80°C. Finally,

the recombinant viruses were amplified in two passages and titrated by limiting

dilution assay. The viral titers were determined by counting red fluorescent single cells

or syncytia as infectious particles per ml.

2.4 Immunofluorescence

Vero cells and keratinocytes were grown on cover slips in 6-well plates and the

following day infected with the recombinant viruses (recA75/17red, recA75/17green-HA-

M2, recA75/17green-HA-M2-RFP, recA75/17green-M2Ko and recA75/17 HA-M2-RFP) at an

MOI of 0.04. At 4 days post infection, cells were fixed with ETOH:acetic acid (95:5)

for 5 minutes at -20°C and washed in PBS. Blocking of unspecific binding sites was

performed in PBS supplemented with 2% FCS for 1 h at RT. Then the first antibody

(anti-F PAb and anti-HA MAb) diluted in PBS (1:500 and 1:1000, respectively) was

added on the cover slips and incubated 1 h at 37°C. Then the slides were washed in

PBS and incubated for 30 min at room temperature with the second antibody (alexa

fluor anti rabbit 555 and alexa fluor anti mouse 488, Invitrogen) diluted in PBS

(1:500). The cells were washed again and the nuclei were stained with TOTO-3

(Invitrogen) following the manufacturer’s advice. The slides were evaluated with a

confocal fluorescence laser microscope (Olympus).

2.5 Western blotting

Immunoblotting was performed as previously described (Plattet et al., 2007).

Vero cells were cultured in 6-well plates and infected (with the various recCDVs) at an

MOI of 0.04 one day later, or left uninfected. At 4-6 days post infection, the cells were

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washed with cold phosphate buffered saline (PBS) and lysed at 4°C in RIPA buffer

(150mM NaCl (500mM NaCl to extract the C protein), 1% NP-40, 0.5% Na-

deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50mM Tris-Cl, pH 7.4) containing

HaltTM Protease Inhibitor Cocktail (SOCOCHIM) and Phosphatase Inhibitor Cocktail

Set II (MERCK) to prevent degradation of the proteins. After centrifugation, Laemmli

Sample Buffer (BIO-RAD) supplemented with DL-dithiothreitol (Fluka) was added to

the samples, they were boiled for 5 min and separated by SDS-polyacrylamide gel

electrophoresis. Afterwards, the proteins were transferred to nitrocellulose membranes

(HybondTM-ECLTM, Amersham Biosciences) which subsequently were blocked in Tris-

buffered saline containing 0.1% Tween 20 (TBS-T) and 5% nonfat dry milk for 1 h.

The membranes were incubated in blocking buffer containing the first antibody at 4°C

over night or 1 h at RT, respectively. Then the membranes were washed three times in

TBS-T and incubated with the horseradishperoxidase-conjugated second antibody for 1

h at RT. After three times washing, ECL (Amersham Biosciences) was used as

substrate and the chemiluminescence signals were detected with a LAS-3000 camera

(FUJIFILM).

2.6 Immunoprecipitation

Vero cells were seeded in 6-well plates and the next day infected at an MOI of

0.04 with recA75/17red, recA75/17green-HA-M2, recA75/17green-HA-M2-RFP,

recA75/17green-M2Ko and recA75/17HA-M2-RFP or left uninfected. In parallel experiments

different expression plasmids were transfected (PCI-HA-M2 and PCI-HA-M2-RFP). At

6 days post infection or 1 day post transfection, the cells were washed with cold PBS

and lysed at 4°C in RIPA buffer (150mM NaCl (500mM NaCl to extract the C protein),

1% NP-40, 0.5% Na-deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50mM Tris-Cl,

pH 7.4) containing HaltTM Protease Inhibitor Cocktail (SOCOCHIM) and Phosphatase

Inhibitor Cocktail Set II (MERCK) to prevent degradation of the proteins. After 10 min

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centrifugation at 13000 rpm at 4°C, aliquots for total protein analyses were separated

and the remaining supernatants were incubated with the anti-HA-tag affinity matrix rat

monoclonal antibody (Clone 3F10, Roche) at 4°C over night. After 1 min

centrifugation at 13000 rpm at 4°C the pellets were washed 4 times with the same

buffer used before and finally dissolved directly in Laemmli sample buffer for western

blotting performed as described above.

2.7 Flow cytometry

Vero cells were infected with the various fluorescent protein-expressing

recCDVs with an MOI of 0.04. After 6 days post infection, cells were washed twice

with PBS and subsequently detached from the wells with PBS-EDTA (50mM) for 45

min at 37°C. The mean fluorescence intensity of 100’000 cells was then measured by

using a FACSCalibur flow cytometer.

2.8 Growth kinetics

To determine growth kinetics, cell-associated progeny viruses from different

recCDVs were taken at the indicated time post-infection. Cell-associated viruses were

recovered by scraping the cells into 1 ml of Opti-MEM (Gibco) followed by two cycles

of freeze-thaw. Cell debris were removed by centrifugation at 2000 rpm for 8 min at 4

°C. For titration, limiting dilutions of the stocks of cell-associated viruses were

performed and subsequently inoculated into Vero-SLAM cells. Two hours later, cells

were washed and DMEM-agar (1%) was applied on each wells. The numbers of

fluorescent syncytia induced by the various recCDVs were counted using a confocal

fluorescence laser microscope three days after initial infection.

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3. Results

3.1 Sequence alignment of the unusually long Morbillivirus M 3’ UTR revealed a

unique ORF in several virulent CDV strains

A sequence alignment of the M 3’ UTR among several CDV strains and other

morbilliviruses was conducted. Figure 1A illustrates the alignment of the M 3’ UTR of

five virulent (5804P, 98-2646, A75/17, Snyder-Hill and CDV3) as well as one highly

attenuated (Onderstepoort) CDV strains. Clearly, the entire M 3’ UTR is very well

conserved among all tested CDV strains. However, while the putative short M2 ORF

(52aa) is present in the five virulent strains, it is absent in the attenuated one (Fig. 1A).

When the A75/17-CDV M 3’ UTR was aligned with three other morbillivirus

sequences (measles virus -MeV-, rinderpest -RPV- and peste-des-petits-ruminants virus

-PPRV-), it turned out that the high degree of conservation observed among the

different CDV strains was totally absent in the three other morbilliviruses (Fig. 1B).

Moreover, we did not find any other significant putative ORFs within the entire M 3’

UTR of MeV, RPV and PPRV. As could be anticipated, however, the only well

conserved sequence among all Morbillivirus M 3’ UTR was the transcriptional stop

motif specifically recognized by their vRdRp (Fig. 1B). Taken together, our sequence

analysis provided evidence that the presence of a putative short ORF within the 3’ UTR

of the M mRNA is restricted to potentially virulent CDV strains.

3.2 Construction of a double tagged M2 protein

In order to facilitate the detection of the short putative M2 ORF, we first

engineered a new M2-expressing plasmid by fusing at its N-terminal domain the well

known HA-tag sequence. In addition, the red fluorescent protein (RFP) was fused to

the C-terminal region of M2. A short linker peptide was also added in between the M2

and RFP proteins in order to limit the possibility that physical constrains impede both

protein functions. Importantly, red fluorescence was readily detected throughout the

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cytosol and the nucleus of HA-M2-RFP-expressing Vero cells (not shown).

Importantly, no difference in cellular localization compared to RFP-transfected cells

was observed (not shown). While we could not directly assess whether the double

tagged M2 construct behaves properly, the above-mentioned results nevertheless

demonstrated that the engineered fusion protein did not dramatically impair the

fluorescence properties of the RFP.

3.3 Generation of recombinant A75/17 viruses

We next engineered five recombinant viruses in order to accurately investigate

whether A75/17-CDV indeed expresses M2 from its M 3’ UTR (Fig. 2). The first virus

consists of the A75/17-CDV genome, which additionally expresses the green

fluorescent protein from a supplementary transcription cassette located between the M

and F genes (recA75/17green). Importantly, in this virus, the M 3’ UTR remained

unaltered. The second virus bears an HA-tag fused to the N-terminal domain of the

putative M2 polypeptide (recA75/17green-HA-M2). In the third virus, in addition to the

HA-tag, the red fluorescent protein (RFP) was fused to the C-terminal part of HA-M2,

thus generating recA75/17green-HA-M2-RFP. The fourth virus was engineered to

potentially over-express the HA-M2-RFP fusion protein. To this purpose, the HA-M2-

RFP gene was removed from its natural position and subsequently cloned in place of

the GFP gene, which is expressed from the additional transcription cassette

(recA75/17HA-M2-RFP). Finally, we modified the A75/17-CDV genome in order to close

the expression of the putative M2 open reading frame (recA75/17green-M2ko). This was

achieved by substituting the three potential initiation codons into stop codons (Fig. 1A)

in order to limit the possibility that modification of the M mRNA 3’ UTR per se causes

indirect influence on viral replication. Then, using reverse genetics technology (Plattet

et al., 2004), all viruses were successfully recovered from their corresponding cDNA

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plasmid. The genetic integrity of the five recombinant viruses was subsequently

confirmed by automated sequencing analysis (not shown).

3.4 Evidence for the absence of M2 expression in several different cellular

environments

To investigate whether M2 was expressed from the A75/17-CDV genome, Vero-

SLAM and MDCK-SLAM cells were initially infected with recA75/17HA-M2-RFP and

recA75/17green-HA-M2-RFP with an MOI of 0.01 and 0.03, respectively (titrated in

Vero-SLAM cells). While the first virus should consistently emit red fluorescence by

over-expressing HA-M2-RFP, the second should indicate whether M2 is expressed or

not. Both viruses exhibited a typical Morbillivirus-mediated cytopathic effect (CPE) in

both cell types within 24 hours. The CPE consisted of massive syncytia formation,

which invariably resulted in cytolysis (Fig. 3). Indeed, syncytium formation is

systematically induced by CDV in cells expressing the universal Morbillivirus receptor

CD150/SLAM. The identical phenotype was observed in the parental recA75/17green-

infeced Vero-SLAM and MDCK-SLAM cells (not shown). Importantly, and in an

anticipated manner, the control virus (recA75/17HA-M2-RFP) readily expressed HA-M2-

RFP since red fluorescence emission was repeatedly captured by confocal laser

fluorescence microscopy (Fig. 3). These results additionally confirmed that the RFP

when fused to the C-terminal domain of HA-M2 also remained fully functional in the

context of a viral infection. However, in striking contrast, no red fluorescence at all

could be monitored in recA75/17green-HA-M2-RFP-infected Vero-SLAM and MDCK-

SLAM cells, whereas bright green fluorescence emission was detected in all syncytia

(Fig. 3). Nonetheless, even though wild-type A75/17-CDV very efficiently replicates in

Vero-SLAM and, to a lesser extent, in MDCK-SLAM cells, they do not represent

natural targets of the virus (the parental Vero and MDCK cells are poorly susceptible to

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wild-type A75/17-CDV replication). Thus, absence of M2 expression in SLAM stably-

transfected cells might be due to an unsuitable cellular environment.

Therefore, two primary SLAM-negative but CDV-susceptible cell types were

infected with both recombinant viruses (canine epithelial keratinocytes and canine glial

cells). Indeed, these cells not only support efficient A75/17-CDV replication but also

represent natural targets of wild-type CDV throughout a course of infection (skin and

brain) (Appel, 1969; Grone et al., 2003; von Messling et al., 2004). Consistent with our

previous results, recA75/17HA-M2-RFP exhibited strong red fluorescence emission in

infected cells, whereas recA75/17green-HA-M2-RFP did not (not shown). In the case of

recA75/17green-HA-M2-RFP-infected cells, however, GFP-expressing cells in both cell

types were easily observed. Immunofluorescence staining (using the anti-F PAb,

(Cherpillod et al., 1999)) confirmed that both viruses efficiently replicated in canine

keratinocytes and glial cells by mediating a typical non-cytolytic cell-to-cell lateral

spread (not shown) (Rivals et al., 2007; Wyss-Fluehmann et al., 2010; Zurbriggen et

al., 1995). Since both viruses bear an HA-tagged M2 protein, indirect

immunofluorescence staining using an anti-HA MAb was additionally performed on

fixed and permeabilized cells. Consistent with our previous results obtained in direct

fluorescence monitoring, only recA75/17HA-M2-RFP-infected cells exhibited positive

staining. Similarly, no stained cells could be detected in recA75/17green-HA-M2-

infected cells (not shown). Taken together, these results strongly suggested that M2

was not translated in these two additional relevant cellular environments.

3.5 Prolonged infection does not induce M2 expression

Since Vero cells do not express high avidity-binding receptors for wild-type

CDV strains, they are poorly susceptible to A75/17-CDV. Therefore, inoculation in

Vero cells with wild-type CDV leads to infection of only few cells, in which the virus

replicates very well without obvious syncytium formation and very limited viral spread.

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Nevertheless, this experiment allowed us to observe infected cells over a long period

(up to 6 days) and to investigate whether M2 expression would perhaps occur over

time, albeit only transiently.

Since the two above mentioned recCDVs either express GFP or RFP from their

additional transcription unit, confocal laser fluorescence microscopy analysis was first

used to monitor viral replication in Vero cells. In an anticipated manner, the percentage

of GFP-expressing Vero cells upon infection with recA75/17green-HA-M2-RFP

remained very low but all infected cells exhibited strong green fluorescence emission,

indicating full replication but very slow viral spread (Fig. 4). Similar results were

obtained with recA75/17HA-M2-RFP but infected cells were emitting red fluorescence.

With both viruses few clusters of a maximum of 5-20 infected cells at 4 dpi could be

observed (Fig. 4). Here again, in almost all green fluorescence-emitting cells,

recA75/17green-HA-M2-RFP did not produce any detectable red fluorescence,

throughout the entire period of incubation (up to 6 days) (Fig. 4). Intriguingly,

however, in extremely rare cells, and only after 4-6 days post infection, very faint red

fluorescence emission could be detected (not shown). To further investigate the

possibility that M2 might be expressed in few selected cells, flow cytometry analysis

was undertaken from infected cells. The results did not support the fluorescence

microscopical findings, since absolutely no red fluorescence signal was reproducibly

recorded (not shown). We thus speculate that the very faint red fluorescence observed

in extremely few infected cells may be due to unspecific emission of recA75/17green-

HA-M2-RFP in Vero cells at late time points post-infection.

3.6 Failure to detect M2 using sensitive biochemical techniques

We next sought to verify the expression of M2 using biochemical methods, such

as protein immunoprecipitation and western blotting. However, all our attempts to

generate functional anti-M2 polyclonal antibodies failed (as controlled with the

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different M2 expression plasmids, see material and methods section). Moreover, the

SLAM molecule used to generate the stable Vero-SLAM and MDCK-SLAM cell lines

bears an HA-tag, thus precluding the use of anti-HA MAbs to accurately assess M2

biosynthesis in these cells. Immunoprecipitation (IP) experiments were thus carried out

from infected Vero cells. In this set of experiments, the parental recombinant viruses

(recA75/17green), the HA-tagged M2-expressing virus (recA75/17green-HA-M2) and the

M2 knockout virus (recA75/17green-M2ko) were additionally included in the analysis.

Hence, cells were infected with the various recCDVs with an MOI of 0.04 (as titrated

in Vero-SLAM cells) and 4 days post-infection cells were lysed using the stringent

RIPA buffer. A rat anti-HA MAb directly coupled to beads was next added to total

protein extracts, thereby allowing for potential HA-tagged proteins

immunoprecipitation. Subsequently, proteins were boiled and loaded on a SDS

polyacrylamide gel, ran and transferred onto a nitrocellulose membrane. Finally, HA-

tagged proteins were revealed using a mouse anti-HA MAb. Results shown in figure 5

(upper panel) indicate that no protein could be immunoprecipitated from Vero cells

infected with recA75/17green and recA75/17green-M2ko, the two untagged viruses.

Conversely, a band which migrated corresponding to proteins with a molecular weight

of about 70 kDa was readily detected in Vero cells infected with the control HA-M2-

RFP over-expressing recombinant virus (recA75/17HA-M2-RFP) (Fig. 5, upper panel).

Importantly, the identical band was not detected using protein extracts derived from

recA75/17green-HA-M2-RFP-infected Vero cells (Fig. 5, upper panel). Immunoblots

were additionally performed from infected Vero cells using the anti-N MAb (D110),

and a band of about 60 kDa was detected in all rCDVs-infected cells, thus confirming

that all recombinant viruses could enter and replicate sufficiently in Vero cells to

express detectable amounts of N protein by western blot (Fig. 5, middle panel). Note

that the intensity of the N-protein revealed by western blotting remained extremely

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faint. This is very likely due to the very limited number of infected cells observed upon

wild-type recCDVs inoculation in Vero cells. Furthermore, direct immunoblotting

using the anti-HA MAb never revealed any bands (also in case of the M2 over-

expressing virus) presumably due to the limit of detection of the western blot assay as

compared to the IP assay (not shown).

3.7 Growth kinetics comparison between recA75/17green and recA75/17green-M2ko in

different cell types

Although the above data suggested that M2 is not expressed in A75/17-CDV-

infected cells, we wished to exclude the possibility that M2 could nevertheless be

translated in a very limited amount (as potentially suspected only in Vero cells), below

the limit of detection of conventional IP and immunoblot assays. It cannot be excluded

that even small amounts of M2 could have an effect on the infection. However,

although we could not formally demonstrate that M2 kept its natural putative function

within the HA-M2-RFP fusion construct, over-expressing M2 did not modify

substantially the phenotype of infection mediated by recA75/17green, and this in all

tested cell types. Figure 6A illustrates a typical example of similar phenotypes of

infection in Vero cells induced by the M2 over-expressing, the M2 knockout and the

wild-type recombinant viruses.

To further validate these results, titration experiments in several cell lines were

performed using the parental recA75/17green virus and the M2-knockout virus

(recA75/17green-M2ko). Strikingly, viral titers of both cell-free (not shown) and cell-

associated viruses remained largely unaltered for both recombinant viruses in the four

cell types tested in this study (Vero-SLAM, MDCK-SLAM, Vero and canine epithelial

keratinocytes) (Fig. 6). In addition, both viruses produced very similar CPE in Vero-

SLAM and MDCK-SLAM cells and induced an indistinguishable non-cytolytic cell-to-

cell spread type of infection in primary canine keratinocytes and glial cells (not

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shown). Taken together, our results provided strong evidence that neither over-

expressing nor deleting M2 from the CDV genome, resulted in detectable phenotypic

differences. This further validated the notion that M2, should it be expressed in very

small amounts, has no obvious effect on viral replication and spread.

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4. Discussion

Viruses have evolved to minimize their genetic information, establishing unique

strategies to translate different viral proteins from the same nucleic acid sequence to

the advantage of rapid and efficient replication (Domingo and Holland, 1997). Previous

studies in CDV revealed a short potential open reading frame (M2) located within an

unusually long UTR at the 3’end of the M mRNA (Stettler et al., 1997). This finding

offered a potential explanation of why CDV has retained the long untranslated region

throughout evolution. Although the M2 ORF location within the M 3’ UTR very likely

excludes classical ribosome-scanning as the main mechanism for its translation, unique

strategies to express proteins have often been observed in different viruses (Ahmadian

et al., 1999; Hemmings-Mieszczak and Hohn, 1999; Jang et al., 1988; Latorre et al.,

1998; Pelletier and Sonenberg, 1988; Yueh and Schneider, 1996). Typically,

paramyxoviruses express at least three proteins from their P gene (P, C and V). While

the P-protein is encoded via a common ribosomal-scanning mechanism, production of

C protein depends on a ribosomal-leaky scanning process that initiates translation at an

alternative, out-of-frame (+1), AUG. Furthermore and unique to paramyxoviruses, an

“editing” mechanism that occurs at a precise location within their P gene, generates the

V protein. In Sendai virus (SeV) infection, it has been reported that the unique Y

proteins (also encoded within the SeV-P gene) were produced through an additional so-

called “ribosomal-shunt” translational mechanism (Latorre et al., 1998). These notions

combined with the fact that the CDV-F 5’ UTR formerly believed to be truly

untranslated has recently been demonstrated to be mostly translated into a long F signal

peptide, prompted us to investigate whether the putative M2 ORF within the CDV-M 3’

UTR is indeed translated. This objective was further supported by sequence analyses,

which revealed that, despite a high degree of “GC” content, the M 3’ UTR were highly

variable among several related morbilliviruses, all of which but CDV lack the M2 ORF.

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However, in sharp contrast, among various CDV strains, the entire M 3’ UTR was

found to be well conserved. Intriguingly, the M2 ORF was present in all sequences

derived from the virulent CDV strains but not in the attenuated strain suggesting that

M2 might be related to viral-induced persistence and/or virulence.

It is now well accepted that CDV, and morbilliviruses in general, mediate a

cytolytic infection in a cellular environments where the universal Morbillivirus

receptor CD150/SLAM is expressed. In contrast, in infection with the wild-type

A75/17-CDV, no obvious cell-cell fusion and subsequent cytolysis is observed in

different cell types lacking SLAM expression. In the latter cellular environments,

A75/17-CDV spreads from cell-to-cell in a non-cytolytic manner with extremely poor

extracellular progeny virus production (Grone et al., 2003; Rivals et al., 2007; Wyss-

Fluehmann et al., 2010; Zurbriggen et al., 1995). In this study, we therefore

intentionally selected several cellular environments representing these two

fundamentally different phenotypes of infection. In order to demonstrate M2

expression in the present study, we based our strategy on the generation of several

recombinant viruses. However, all our attempts to demonstrate M2 expression from the

viral genome of the highly virulent and demyelinating A75/17-CDV were suggestive

for the absence of M2 translation in all cellular environments tested. Several lines of

evidence support the latter conclusion. First, we tagged both the C-terminal (RFP) and

N-terminal (HA) domains of M2 within the viral genome to allow for efficient

immunoprecipitation and assessment of M2 expression in living cells by fluorescence

microscopy. Even though HA-M2-RFP biosynthesis by recA75/17-CDV was

investigated in several cell types, significant red fluorescence emission was never

detected neither by direct fluorescence emission, indirect immunofluorescence staining

nor flow cytometry analysis. In contrast, all these techniques readily detected the

presence of the fluorescent fusion protein in M2 over-expressing recA75/17HA-M2-RFP-

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infected cells. Secondly, due to the presence of the HA-tag, we confirmed by standard

immunoprecipitation and western blot analysis that M2 was indeed not produced in

recA75/17green-HA-M2-RFP-infected cells. Thirdly, because it could not be ruled out

that small amounts of M2 were expressed below the limit of detection of the methods

used, we performed infection experiments with M2 over-expressing, M2 knockout and

wild-type viruses in different cell systems in order to detect any potential differences in

growth kinetics, type of spread and CPE. However, since no differences could be

observed, we concluded that the putative M2 polypeptide, should it be expressed in low

quantities, has no noticeable effect on CDV infection.

It has been clearly documented that the whole CDV-M/F untranslated region

combined with the Fsp modulated virulence in ferrets by controlling F transcription and

protein expression (Anderson and von Messling, 2008). However, Anderson and

colleagues did not determine whether the M 3’ UTR and/or the F 5’ UTR acted

synergistically with the Fsp to attenuate CDV. While the latter question merits further

investigation, the results obtained in our study appear to exclude that the putative ORF

within the M 3’ UTR plays a role in CDV infection. Interestingly, although in the

closely related measles virus (MeV) no long signal peptide is translated from the F 5’

UTR, the extremely long M/F UTR per se was not essential for MeV replication but

could indirectly regulate MeV replication and cytopathogenicity by modulating the

levels of M and F protein synthesis (Sidhu et al., 1995; Takeda et al., 2005). In

particular, the M 3’ UTR seemed to be responsible for stabilizing the mRNA in turn

enhancing the production of M, which was associated with enhanced replication.

Further studies are therefore required to demonstrate whether i) yet unidentified

specific regulating sequences, ii) transcriptional cis-acting sequences, or iii) the length

of the CDV-M 3’ UTR per se are of primary importance for synergizing with the CDV-

Fsp to modulate the infection. In any case, these mechanisms may well be important in

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modifying the CDV-M protein expression level, which in addition to F, would in turn

regulate CDV-mediated replication and disease induction.

In conclusion, while we cannot exclude that M2 is selectively translated in cells

that were not tested in this study, our data provided evidence that M2, potentially

translated from a unique and short open reading frame located within the M 3’ UTR of

the virulent A75/17-CDV strain, is not a viral determinant synergising with the Fsp to

control virulence. The intriguing question remains why CDV evolved to retain more

than 400 untranslated nucleotides within the M mRNA, in its otherwise very compact

and tightly tuned genome .

Acknowledgments

We are grateful to Veronika von Messling for having provided the Vero-SLAM

cells.

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Figure legends

Figure 1. A) Sequence alignment of the M 3’ UTR among several CDV strains.

GenBank numbers for each virus sequence are: AY386316.1 (CDV, 5804P strain),

AY542312.2 (CDV, 98-2646 strain), AF164967.1 (CDV, A75/17 strain), GU138403.1

(CDV, Snyder Hill strain), EU726268.1, (CDV, CDV3 strain), AF305419.1 (CDV,

Onderstepoort strain). B) Sequence alignment of the M 3’ UTR among several

morbilliviruses. GenBank numbers for each virus sequence are: AF164967.1 (CDV,

A75/17 strain), AB016162.1 (measles virus, ICB strain), NC_006296.2 (Rinderpest

virus, Kabete O strain), EU267273.1 (Peste-des-petits-ruminants virus, ICV89 strain).

Red boxes highlight the putative M2 ORF. The different potential initiation codons are

also represented in red. Furthermore, the black box in figure 1B illustrates the

transcriptional stop cis-acting sequence. Stars below the sequences show identical

residues in all sequences.

Figure 2. Schematic representation of the five generated recombinant A75/17-CDVs.

All viruses bear an additional expressing unit coding for the indicated fluorescence

protein located between the M and F genes. The different constructs (shown above each

genome) that were cloned in place of the natural putative M2 ORF are also represented.

Below every genome is shown the putatively entire generated M mRNA. The length of

each genome is also indicated below its corresponding viral genome’s drawing. RFP:

red fluorescent protein, GFP: green fluorescent protein, M2: putative M2 ORF located

within the M 3’ UTR, HA: influenza hemagglutinin-specific tag sequence. Drawings

are not in scale.

Figure 3. Monitoring of the fluorescence induced by the indicated recombinant viruses.

Vero-SLAM and MDCK-SLAM cells were infected with an MOI of 0.01 and 0.03,

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respectively. Two days post-infection infected cells were screened for green and/or red

fluorescence emission by confocal laser fluorescence microscopy. Moreover, phase

contrast images of representative fields of view illustrating the typical cytopathic effect

induced in both cell types by the recCDVs, are shown.

Figure 4. Monitoring of the fluorescence induced by the indicated recombinant viruses.

Vero cells were infected with an MOI of 0.04. Two days post-infection infected cells

were screened for green and/or red fluorescence emission by confocal laser

fluorescence microscopy. Moreover, phase contrast images of representative fields of

view illustrating no obvious cytopathic effect induced by the recCDVs, are shown.

Figure 5. Investigation of M2 translation by immunoprecipitation assay. Vero cells

were infected at an MOI of 0.04 with the various recCDVs or left uninfected, and

subsequently lysed 4 dpi for immunoprecipitation assay. To assess M2 expression, HA-

M2-RFP was immunoprecipitated (IP) with an anti-HA monoclonal antibodies (rat)

directly coupled to G sepharose beads. Then, western blot analysis, using an anti-HA

monoclonal antibody (mouse) to detect the HA-M2-RFP fusion protein, was performed

(upper panel). Total cell lysates (TL) were subjected to immunoblot analysis to

investigate the endogenous the viral N-protein expression (middle panel). As a loading

control, we revealed the actin protein using an anti-actin MAb (bottom panel).

Figure 6. No phenotypic differences are observed in the M2 over-expressing and the

knockout virus-infected cells as compared to the parental wild-type rA75/17green virus.

A) recCDVs lateral spread in Vero cells. Vero cells were infected with recA75/17green-

M2ko, recA75/17HA-M2-RFP and rA75/17green with an MOI of 0.04. Fluorescence emission

from the marker protein expressed from the additional transcription unit was captured

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by confocal laser fluorescence microscopy 1, 3 and 6 days post-infection. B) Growth

kinetics of the recCDVs in various cell types. Vero-SLAM, MDCK-SLAM,

keratinocytes and Vero cells were infected with recA75/17green and recA75/17green-

M2ko. Viral titers of cell associated viruses, taken at the indicated time post-infection,

were determined by limiting dilution assay.

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CDV-5804P ATCATTGGTTCATGAACTAAAAATCAAATGCCTTGGTGGCATTGTCCAGGATCTCTTAAT 60

CDV-98 2646 ATCATTGGTTCATGAACTAAAACTCAAATGACTTGGTGGCATTGTCCAGGATCCCTTAAT 60

CDV-A75/17 ATCATTAGTTCATGAACTAAAACTCAAATGACTTGGTGGCATTGTCCAGGATCCCTTAAT 60

CDV-SH ATCATTAGTTCATGAACTAAAACTCAAACGCCTTAGTAGCATTGCCCAAGATCCCTTGAT 60

CDV-CDV3 ATCATTAGTTCATGAACTAAAACTCAAACGCCTTAGTAGCATTGCCCAAGATCCCTTGAT 60

CDV-OP ATCATCAGTTCATGAACTTAAAAGCAAACGCCTTAGTAGCACTGCCCAAGATCCCTTGAT 60

***** *********** *** **** * *** ** *** ** *** **** *** **

CDV-5804P CCCCTCAGACAAGAATTGAGGCTACAAGTATCAACTGTCTCGATGTCGCTCCTGCACTTT 120

CDV-98 2646 CCCCTCAGACAAGAATTGAGGCTACAAGTATCAACTGTCTCGATGTTGCTCCTGCATTTT 120

CDV-A75/17 CCCCTCGAACAAGGATTGAGGCTACAAGTATCAACTGTCTCGATGTTGCTCCTGCATTTT 120

CDV-SH CCCCGCAAGCGAGGATTGAGGGTATAAATATCGACTGTCTAGATGTTGCTCCTGCATTTT 120

CDV-CDV3 CCCCGCAAGCGAGGATTGAGGGTATAAATATCGACTGTCTAGATGTTGCTCCTGCATTTT 120

CDV-OP CCCCGCAAGCGAGGATTGAGGGTATAAGCACCGACCATCCAGACGTTGCTCCTGCATTTT 120

**** * * ** ******* ** ** * * ** ** ** ** ********* ***

CDV-5804P AAGCGTGTTCTATAGGTTTCTAAACTGCTCTTTCGTGCCTACTACTCTGGTGGCTCTGCA 180

CDV-98 2646 AAGCGTGTTCTATAGGTTTCTAAACTGCTCTTTCGTGCCTACTACTCTGGTGGCTCTGCA 180

CDV-A75/17 AAGCGTGTTCTATAGTTTTCTAAGCTGCTCGTTCGTGCCTGCTATTCTGGTGACTCTGCA 180

CDV-SH GAGCGTGGCCTATAGGTTTCTAAACTGCTCATCCGTGCCCACAATTCCAGTGACGCCTCA 180

CDV-CDV3 GAGCGTGGCCTATAGGTTTCTAGACTGCCCATCCGTGCCCACAATTCCAGTGACGCCTCA 180

CDV-OP GAGTGTGTCCCATAAGCCTCCAAACCGCTCACTCGTGCCCACAACTCCAGTGACGCCTCG 180

** *** * *** ** * * ** * ****** * * ** *** * * *

CDV-5804P ATATGAAGACAGCTGAATCAAACCAATTCATGCCTAAGAGTAGGTTGATCATTATCGGAC 240

CDV-98 2646 ATATGAAGACAGCTGAATCAAACCAATTTATGCCTAAGAGTAGGTTGATCATTATCGGAC 240

CDV-A75/17 ATATGAAGACAGCTGAATCAAACCAATTCATGCCTAAGAGTAGGTTGATCATTATCGGAC 240

CDV-SH ATATGTGAAAATAGCTGAATCAAAACAGTTCTTGCTTAAGATTAGGTTGATCATTATCGGAC 240

CDV-CDV3 ATATGATGAAAATAGCTGAATCAAAACAGTTCTTGCTTAAGATTGGGTTGATCATTATCGGAC 240

CDV-OP ATACGAAAGCATCCGAACCAAAACAGCTCTTGCCCAAGATTAGGTTGATCATTATCGGAC 240

*** *** * * *** **** ** * *** **** * ******************

CDV-5804P CAAGAAATTTATGGATGCTTGGGGTTTTGAACTTCGCCTCTAGGAATCTCACTTTAACAA 300

CDV-98 2646 CAAGAAATTTATGATGGATGCTTGGGGTTTTGAACTTCGCCTCTAGGAATCTCACTTTAACAA 300

CDV-A75/17 CAAGTAATGTATGGATGCTTGGGGTTTTGAACTTCGCCTCTAGGAATCTCACTTTAACAA 300

CDV-SH CAAGAAATGAATGGATGCCTGGGGTTTTGAGCTTCGCTTCTAGGATTCTCACTTTAACAA 300

CDV-CDV3 CAAGAAATGAATGGATGCCTGGGGTTTTGAGCTTCGCTTCTAGGAATCTCACTTTAACAG 300

CDV-OP CAAGAAATGAATGGATGCCTGGGGTTTTTAGCTTCGCTTCTAGGTATCTCACTTTAACAA 300

**** *** ******** ********* * ****** ****** *************

CDV-5804P TTATACTTCCACGCACTTGCCCGAGCTCAAACTATCACTAGTAGTCCTGTTTCACGAAAT 360

CDV-98 2646 TTATACCTCCACGCACTTGCCCGATCTCAAGCTATCACTAGTAGTCTTGTTTCACGAAAT 360

CDV-A75/17 TTATACCTCCACGCACTTGCCCGATCTCAAGCTATCACTAGTAGTCTTGTTTCACGAAAT 360

CDV-SH TTATACTCCCACGCACTTGCCTGATCTCAAGCCATCACTAGTAGTCTTGTTTCACGGAGT 360

CDV-CDV3 TTATACTCCCACGCACTTGCCTGATCTCAAGCCATCACTAGTAGTCTTGTTTCACGGAGT 360

CDV-OP TTATACTCCCACGCACTTGCCTGATCTCAAGCTATCACTAGTAGTCCTGTTTCACGGAAC 360

****** ************* ** ***** * ************* ********* *

CDV-5804P TGTGACTGTCTATCTTTCTATCACCAATCGTTAATAATTAATCAAAA 407

CDV-98 2646 TATGACTGTCTATCTTTCTATCACCAATCGTTAATAATTAATCAAAA 407

CDV-A75/17 TATGACTGTCTATCTTTCTATTACCAATCGTTGATAACTAATCAAAA 407

CDV-SH TATGACTGTCCATCTTTCTATCACAGCTCATTAATAATTAATCAAAA 407

CDV-CDV3 GATGACTGTCCATCTTTCTATCACAGCTCATTAATAATTAATCAAAA 407

CDV-OP TATGACTGTCCATCTTTCTATCACAGCTCATTAATAATTAATCAAAA 407

******** ********** ** ** ** **** *********

Figure 1

A

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CDV-A75/17 -----------------------ATCATTAGTTCATGA--ACTAAAACTCAAATGACTTG 35

MeV-ICB -----------ACCGCAGTGCCCAGCAATACCCGAAAACGACCCCCCTCATAATGACAGC 49

RPV-kabeteO ---------------ACTCACCCAGCATCACTCCA------CCCCCGCCGCACCGTCCCG 39

PPRV-ICV89 ACCGGTCGCCGGACAATAGACCCACCTTCAAGAAGTGGCTCCCCCTTCCCCCGTCCAAAA 60

* * * *

CDV-A75/17 GTGGCATTGTCCAGGATCCCTTAATCCCCTCGAACAAGG-------ATTGAGGCTACAAG 88

MeV-ICB CAGAAGGC-CCGGACAAA--AAAGCCCCCTCCAGAAGACTCCACGGA-CCAAGCGAGAGG 105

RPV-kabeteO CAGAGACC-CACAGCAGGGCAGAACGCCCGACACCAGCCTCAACCCAGCCGAACAACAAG 98

PPRV-ICV89 GACACACC-CCAAGACCCCCCAAAACAGCTCCAACATGGCCATCCTCCCAACGGGACGGG 119

* * * * * *

CDV-A75/17 TA--TCAACTGTCTCGATGTTGCTCCTGCATTTTAAGCGTGTTCTATAGTTTT--CTAAG 144

MeV-ICB CCAGCCAGCAGCCGACAGCAAGTGTGGACACCAGGCGGCCCAAGCACAGAACAGCCCCGA 165

RPV-kabeteO GC--CTAGACCCCCACAGCGCAGCCCCCCACCCCGAACTCAACACACGGAGCA--CCCAG 154

PPRV-ICV89 GCCGACCTCCCCCACGAACCCGGTCCGGCAGGAGGGGCCCCCCCCGCAACCCA--CCGGG 177

* * ** *

CDV-A75/17 C--TGCTCGTTCGTGCCTGCTATTCTGGTGACTCTGCAATATGATGAAGACAGCTGAAT---- 198

MeV-ICB CACAAGGC-----CACCACC-AGCCATCCCAATCTGCGTCCTCCTCGTG---GGAC---C 213

RPV-kabeteO CTCTGGGCA---GCACCCGC-ACCCCACCTGCCCTGCACCCCCACCTGGTCCGGAC---C 207

PPRV-ICV89 CGCCGACCGCGTGGGCCAGC-AGGACGCCCCCAAGAGGGCCCGACCGGGACCGGGCACCC 236

* * ** * * *

CDV-A75/17 -CAAACCAATTCATGATGCCTAAGAGTAGGTTGATCATTATCGGACCAA---GTAATGTATGATGG 254

MeV-ICB CCCGAGGACCAACCCCGAAGGTCGCT-CCGAACACAGACCACCAACCGCATCCCCACAGC 272

RPV-kabeteO TCCCCACGGCTCCGCCCACCGCCGCAGCCGACCAAGGCCCGACCAACACGCGGCCGCTGC 267

PPRV-ICV89 TCCCCCCAAAAAAACCCCCCGACACATCCGAGGCCAGGC-GCCAG--GCACTCCCAATCC 293

* * * ** * *

CDV-A75/17 ATGCTTGGGGTTTTGAACTTCGCCTCTAGGAATCTCACTTTAACAATTATACCTCCACG- 313

MeV-ICB TCCCGGGAAAGGA-ACCCCCAGCAACTGGAAGGCCCCTCCCCCCCTCC--CCCAACGCAA 329

RPV-kabeteO ACATCCAGAGCGC-ACACCCGACAAC-----AACCCCGACAGTCCCCT--ACCGAAAGGA 319

PPRV-ICV89 CGCCACCGGGGGG-ACAAGCAGCCAAGACAGGGCCCCCCCACCCAAACGGACCGCCAGGG 352

* * * * **

CDV-A75/17 CACTTGCCCGATCTCAAGCTA------TCACTAGTAGTCTTGTTTCACGAAATTATGACT 367

MeV-ICB GAA-CCCCACA-ACCGAACCGCACAAGCGACCGAGGTGACCCAACCGCAGGCATCCGACT 387

RPV-kabeteO CAA-CCCCAGACACCCAACCA---GGGCCAACAGAGGAAGGAAACCACAGGAACCAGACA 375

PPRV-ICV89 GAGGCCCCACCGACCCAGCACAGACCCGGCCCAAACAAAGGAGACTCCAAAGACGAAACC 412

* ** * * * * **

CDV-A75/17 GTCT---ATCTT--TCTATTACCAATCGTTGATAACTAATCAAAA 407

MeV-ICB CCTT--AGACAG---ATCCTCTCCCCCCGGCAT-ACTAAACAAAA 426

RPV-kabeteO CCCCCGAGACGAGGCAACCTACCCACCATGAAT-ACCAAACAAAA 419

PPRV-ICV89 GCCC---AGCGC---ACCCTACTCATC-------ATCAAACAAAA 444

* * * * ** *****

Figure 1

B

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putative M2 polypeptide

N P M F H LGFP

N P M F H LGFP

HA-M2

recA75/17 green

recA75/17 -HA-M2green

N P M F H LGFP

HA-M2-RFP

RFPrecA75/17 -HA-M2-RFPgreen

N P M F H L

HA-M2-RFP

RFPrecA75/17 HA-M2-RFP

N P M F H LGFPrecA75/17 -M2green ko

Figure 2

1 16470

1 16500

1 17970

1 16470

1 17400

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Vero-SLAM cells

recA75/17 -HA-M2-RFPgreenrecA75/17 HA-M2-RFP

Figure 3G

FP e

mis

sio

np

has

e co

ntr

ast

MDCK-SLAM cells

recA75/17 HA-M2-RFP recA75/17 -HA-M2-RFPgreen

RFP

emis

sio

n

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Vero cells

recA75/17 -HA-M2-RFPgreenrecA75/17 HA-M2-RFP

Figure 4

GFP

em

issi

on

ph

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con

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tRF

P em

issi

on

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recA

75/17

-H

A-M2-R

FP

green

recA

75/17

HA-M2-R

FP

Figure 5

uninfe

cted

IP

recA

75/17

green

recA

75/17

-M

2

green

ko

HA-M2-RFP

recA

75/17

-H

A-M2

green

TL

TLN

actin

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Vero-SLAM MDCK-SLAM

Vero Keratinocytes

1

2

3

4

5

6

log

i

nfe

ctio

us

un

its/

ml

10

1

2

3

4

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log

i

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ctio

us

un

its/

ml

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24h 36h 48h 24h 36h 48h

24h 48h 72h 96h 120h 72h 120h 168h

Figure 6

B

recA

75/17

green

recA

75/17

-M

2

green

recA

75/17

HA-M2-R

FP

1 dpi

3 dpi

6 dpi

A ko

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JOURNAL OF VIROLOGY, July 2010, p. 000 Vol. 84, No. 130022-538X/10/$12.00 doi:10.1128/JVI.01878-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Two Domains of the V Protein of Virulent Canine Distemper VirusSelectively Inhibit STAT1 and STAT2 Nuclear Import�

Anne Rothlisberger,1† Dominique Wiener,1† Matthias Schweizer,2 Ernst Peterhans,2Andreas Zurbriggen,1 and Philippe Plattet1*

Department of Clinical Research and Veterinary Public Health, Vetsuisse Faculty, University of Bern, Switzerland,1

and Institute of Veterinary Virology, Vetsuisse Faculty, University of Bern, Switzerland2

Received 4 September 2009/Accepted 18 April 2010

Canine distemper virus (CDV) causes in dogs a severe systemic infection, with a high frequency of demy-elinating encephalitis. Among the six genes transcribed by CDV, the P gene encodes the polymerase cofactorprotein (P) as well as two additional nonstructural proteins, C and V; of these V was shown to act as a virulencefactor. We investigated the molecular mechanisms by which the P gene products of the neurovirulent CDVA75/17 strain disrupt type I interferon (IFN-�/�)-induced signaling that results in the establishment of theantiviral state. Using recombinant knockout A75/17 viruses, the V protein was identified as the main antagonistof IFN-�/�-mediated signaling. Importantly, immunofluorescence analysis illustrated that the inhibition ofIFN-�/�-mediated signaling correlated with impaired STAT1/STAT2 nuclear import, whereas the phosphory-lation state of these proteins was not affected. Coimmunoprecipitation assays identified the N-terminal regionof V (VNT) responsible for STAT1 targeting, which correlated with its ability to inhibit the activity of theIFN-�/�-mediated antiviral state. Conversely, while the C-terminal domain of V (VCT) could not functionautonomously, when fused to VNT it optimally interacted with STAT2 and subsequently efficiently suppressed theIFN-�/�-mediated signaling pathway. The latter result was further supported by a single mutation at position 110within the VNT domain of CDV V protein, resulting in a mutant that lost STAT1 binding while retaining a partialSTAT2 association. Taken together, our results identified the CDV VNT and VCT as two essential modules thatcomplement each other to interfere with the antiviral state induced by IFN-�/�-mediated signaling. Hence, ourexperiments reveal a novel mechanism of IFN-�/� evasion among the morbilliviruses.

Virulent canine distemper virus (CDV) causes a severe sys-temic infection in dogs that is characterized by high fever,diarrhea, and pneumonia. Large-scale immunosuppression is ahallmark of infection, and some virus strains additionally in-vade the central nervous system to cause chronic demyelinat-ing encephalitis. The molecular mechanisms differentiating vir-ulent from attenuated strains are poorly understood. However,the fact that dogs can be protected from infection with virulentCDV by vaccination with attenuated strains suggests that reli-able induction of adaptive immunity is possible, provided thatthe critical early stage of infection is successfully mastered bythe host. During the early stage of infection, host defensedepends on the innate immune system, which is also respon-sible for generating signals that activate the adaptive immuneresponse (27). The interferons of type I (IFN-I, e.g., IFN-�/�)are a critical element of the innate immune defense againstviruses (13, 36, 41). Virtually all nucleated cells are capable ofsensing viral infection by receptors such as Rig-I, MDA-5, orToll-like receptor-3 (16). Activation of these receptors initiatesa signal cascade that results in transcription, translation, andrelease from the cells of IFN-�/�. This part of the IFN defenseis referred to as the induction stage. IFN action is initiated bythe binding of IFN to type I IFN receptors that activates the

receptor-associated tyrosine kinases JAK1 and Tyk2, which, inturn, phosphorylate the signal transducers and activators oftranscription (STATs) (21, 41). Subsequently, the activatedSTAT1 and STAT2 together with IFN regulatory factor 9(IRF9) form a complex, the IFN-stimulated gene factor 3(ISGF3), which, once translocated to the nucleus, binds theIFN-stimulated response element (ISRE) sequence (39, 45).This initiates the expression of well over 100 proteins which areresponsible for the antiviral effect of IFN (36). In recent years,gene products targeting specific steps of IFN induction oraction have been found in virtually all viruses studied, indicat-ing the crucial role of IFN evasion in any successful interactionof viruses with their hosts.

CDV, a Morbillivirus of the Paramyxoviridae, contains a non-segmented, single-stranded, negative-sense RNA genome. Thegenome consists of six genes expressing the structural nucleo-capsid (N), matrix (M), fusion (F), hemagglutinin (H), andlarge (L) proteins and the phospho-protein (P) (20). The P andthe L proteins together form the RNA polymerase. In additionto the P protein, the nonstructural C and V proteins are alsoexpressed from the P gene (20). Recently, it has been docu-mented that a virulent CDV strain (5804P) genetically modifiedto inactivate V was attenuated in ferrets, whereas a C-defectiveCDV was fully immunosuppressive (47). These findings demon-strate that wild-type (wt) CDV suppressed IFN induction, butthe issue of whether additional modulation of IFN-mediatedsignaling sustains viral attenuation remains to be determined.In addition, recent work done with C- and V-deficient recom-binant measles virus (MV) and rinderpest virus (RPV) indi-cated that V and, to some extent, P contribute to the final

* Corresponding author. Mailing address: Department Clinical Re-search and Veterinary Public Health, Bremgartenstrasse 109a, 3001Bern, Switzerland. Phone: 4131 631 26 48. Fax: 4131 631 25 09. E-mail:philippe.plattet@itn.unibe.ch.

† A.R. and D.W. contributed equally to this study.� Published ahead of print on ●●●●●●●●.

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control of the IFN-�/�-mediated signaling pathway. However,to analyze the functions of the P gene products, these recom-binant viruses were based on the genetic background of vac-cine strains (8, 25). Nevertheless, the paramyxovirus V proteinhas been identified as the main inhibitor of the IFN-inducedantiviral state though various molecular mechanisms wereunraveled (12, 14, 15). Expression of the CDV V protein(CDV-V) depends on the insertion of a nontemplated guaninenucleotide at a precise location, called an “editing site,” whichgenerates an mRNA which differs from that of P by one or twonucleotides. This produces an mRNA with an altered open read-ing frame (ORF) downstream of the editing site, and, thus, due tothis specific mechanism, the N-terminal domain of P and V areidentical, whereas their C-terminal domains are unique. The C-terminal domain of V (VCT) is known to contain a conservedcysteine-rich region (31, 43), and recent X-ray studies confirmedthat VCT folds into a zinc finger conformation (22).

In this study we investigated the role of the P gene productsof the highly virulent CDV A75/17 strain in counteracting theIFN-�/�-mediated signaling pathway. Importantly, this strainwas isolated from a naturally infected dog and subsequentlykept amplified only in dogs, where it has been reported tomaintain its virulence (6). Therefore, this virus has never beenadapted to any cell lines. However, the generation of recom-binant virus stocks (rA75/17) with sufficient titers to work withrequires two to three passages in Vero cells expressing signal-ing lymphocyte activation molecule (Vero-SLAM) after virusrescue from primary full-length cDNA-transfected cells (37).Because these limited amplification steps might already selectviral variants, the entire genome of rA75/17 was compared bydirect sequencing to that of the parental A75/17 strain andshowed no nucleotide differences (37), thereby validating theunique opportunity to investigate the molecular mechanisms ofvirus-host cell interactions based on a demyelinating morbilli-virus strain. Hence, recombinant A75/17 viruses and expres-sion plasmids were generated to investigate the role of the Pgene products in mediating IFN evasion. Infection and trans-fection experiments were performed in Vero cells stably ex-pressing the SLAM receptor for CDV. IFN production fromVero-SLAM cells is defective, and thus they not only providean optimal tool to exclusively study IFN signaling indepen-dently of IFN induction but also support very efficient CDVA75/17 replication.

Our results demonstrate that the V protein was the mainviral factor responsible for disrupting the IFN-�/�-mediatedsignaling pathway. The latter inhibition was neither due toSTAT1 or STAT2 degradation nor to an impairment of theirphosphorylation states upon IFN-�/� treatment. Rather, theCDV-V protein efficiently associated with both STAT1 andSTAT2, which correlated with a complete inhibition of thenuclear import of both transcription factors. Furthermore,transient expression experiments of engineered V proteinsidentified both the N-terminal and the C-terminal domains astwo interdependent modules necessary to exhibit optimal IFNevasion.

MATERIALS AND METHODS

Cells and viruses. Vero-SLAM cells (kindly provided by V. von Messling,INRS-Institute Armand-Frappier, University of Quebec, Laval, Quebec, Can-ada), MDCK-SLAM cells, and Bsr-T7 cells(stably expressing T7 RNA polymer-

ase) (3) were grown in Dulbecco’s modified Eagle medium (Invitrogen) supple-mented with 10% fetal calf serum (FCS), penicillin, and streptomycin. Theselection of Vero cells expressing the SLAM receptor was maintained by addingzeocin (Invitrogen). All cells were kept at 37°C in the presence of 5% CO2. Thetransfection and all the infection experiments were performed in Vero-SLAMcells, which are easily infected by CDV. The infection experiments were per-formed with recombinant viruses based on the wild-type CDV A75/17 strain.

Antibodies. In Western blotting the viral proteins were detected by a rabbitanti-P, anti-V, and anti-C antibody and by monoclonal mouse anti-N antibodyD110 (2). The first two antibodies were formerly produced in our laboratory.Briefly, the P-specific and the V-specific domains (both C-terminal) were pro-duced in bacteria, purified, and injected in rabbits to produce P and V antisera.The anti-C antibody was produced against a mix of two synthetic peptides(nh2-RSAASETKPATQARRMEPQACRK-cooh and nh2-RQSSPLKMTSNQDLE-cooh) corresponding to amino acids (aa) 24 to 46 and 85 to 99 of the Cprotein (Primm srl; Custom Antibodies, Milano, Italy). The cellular STAT pro-teins were detected by anti-STAT1 (Cell Signaling), anti-STAT2 (A-7; SantaCruz Biotechnology), anti-phospho-STAT1 (Tyr 701; 58D6; Cell Signaling), andanti-phospho-STAT2 (Tyr 690; Cell Signaling) antibodies. For immunofluores-cence staining the anti-N antibody D110, STAT1� p91 (C-24; Santa Cruz Bio-technology), STAT2 (C-20; Santa Cruz Biotechnology), and anti-phospho-STAT1 (Tyr 701; 58D6; Cell Signaling) were used in combination with AlexaFluor 555 and 488 (Invitrogen) anti-mouse and anti-rabbit antibodies, respec-tively. The coimmunoprecipitations (co-IPs) were performed with either anti-STAT1 p84/p91 (C-136; Santa Cruz Biotechnology) antibody or anti-STAT2(A-7; Santa Cruz Biotechnology). In selected co-IP experiments, the anti-hem-agglutinin (HA) tag monoclonal antibody (MAb) coupled with Sepharose beads(3F10; Roche) was used. Anti-�-actin (Sigma) served as a loading control. Theanti-HA tag monoclonal antibody 16B12 (Covance) was used for immunoblot-ting and immunofluorescence analyses.

Generation of recombinant viruses and plasmids. The recombinant virusesare based on a neurovirulent wild-type isolate, CDV A75/17. The mutations toknock out either the C (Cko) or the V (Vko) protein or both proteins (CVko) wereperformed on a shuttle vector containing the sequences of the N, P, and L genesand produced from a full-length cDNA clone (pFL-A75/17, [18]). The mutationswere obtained by applying a QuikChange Site-Directed Mutagenesis Kit (Strat-agene). After digestion with NotI and PmeI, the M, F, and H genes of CDVA75/17 and a red fluorescence marker gene (tD-tomato; kindly offered by D.Garcin, University of Geneva, Switzerland) (23) placed between the first twogenes were cloned into the shuttle vectors, which contained the mutated P genes,using T4 DNA ligase (New England BioLabs). To rescue the recombinant viruseswe proceeded as previously described (33). Briefly, Bsr-T7 cells were cotrans-fected with plasmids containing the relevant full-length cDNA and helper plas-mids coding for the N, P, and L proteins of CDV. After 3 days the transfectedBsr-T7 cells were cocultured with Vero-SLAM cells. When a cytopathic effecthad developed, the cells were lysed by two freeze-thaw cycles, and the viruses(referred to as rA75/17red, rA75/17red Cko, rA75/17red Vko, and rA75/17red CVko)were stored at �80°C. Finally, the recombinant viruses were amplified in twopassages and titrated by a plaque assay in Vero-SLAM cells, starting at a mul-tiplicity of infection (MOI) of 0.02. The virus titers were determined by countingred fluorescent single cells or syncytia as infectious particles per ml.

To generate the expression plasmids encoding the P, V, and C proteins (calledpCI-P, pCI-V, and pCI-C, respectively), the three relevant sequences of therA75/17red cDNA P gene were amplified by using an Expand High Fidelity PlusPCR System (Roche). The PCR products were digested with RsrII and cloned ina pCI mammalian expression vector (Promega) by applying the T4 DNA ligase(New England BioLabs). To silence the C gene, the same mutations as for theknockout viruses were performed on the plasmids pCI-P and pCI-V, and theadditionally required G was introduced at the editing site in plasmid pCI-V bysite-directed mutagenesis (Stratagene). To generate pCI-RFP (where RFP is redfluorescent protein), the marker gene was amplified from the cDNA clone byPCR and cloned into the pCI expression vector previously digested with theidentical restriction sites as described above. Then, a small linker (SGGSGGTG)and the HA-tagged peptide (YPYDVPDYA) were added by PCR technology inframe to the C-terminal domain of the RFP ORF (pCI-RFP-Linker-HA). Next,pCI-RFP-HA-Vwt (where Vwt is the wt, or full-length, V protein), pCI-RFP-HA-VNT, and pCI-RFP-HA-VCT plasmids were generated by PCR amplifica-tion of the different V domains from the pCI-V vector and subsequently clonedinto the pCI-RFP-Linker-HA-cleaved plasmid. Finally, the single substitutionY110D in V was generated by site-directed mutagenesis (Stratagene), thus pro-viding the pCI-Vwt Y110D or pCI-RFP-HA-Vwt Y110D expression plasmids.HA-tagged versions of the following plasmids were produced by PCR technol-ogy: pCI-HA-Vwt, pCI-HA-P, pCI-HA-C, pCI-HA-VNT, pCI-HA-VCT, and

2 ROTHLISBERGER ET AL. J. VIROL.

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pCI-HA-Vwt Y110D. The green fluorescent protein (GFP) gene was amplifiedby PCR technology from a plasmid pgA75/17-V cDNA clone (33) and sub-sequently digested and ligated into the pCI-cleaved plasmid, thus generatingpCI-GFP.

Luciferase assay and transient transfections. Vero-SLAM cells were grown in24-well plates and transfected the following day with pISRE-Luc (encoding anIFN-inducible firefly luciferase), pTK-RL (coding for a constitutively expressedRenilla luciferase as a transfection control; both plasmids were kindly providedby D. Garcin, University of Geneva, Switzerland), and pCI-P, -V, -C (or thederivative RFP constructs), the empty pCI, or the control plasmid pCI-GFPusing Lipofectamine 2000 (Invitrogen) and Opti-MEM (Invitrogen). The nextday the cells were treated (or left untreated) with 1,000 IU/ml universal IFN-�/�(IFN type I; PBL) for 6 h. Then the cells were lysed, and the luciferase activitywas measured by applying a dual-luciferase reporter assay system (Promega)according to the manufacturer’s recommendation. The luminescence signals ofthe firefly and the Renilla luciferase were measured with a TD-20/20 Luminom-eter (Promega), and their ratio was called relative luciferase activity, with theratio of the empty vector pCI set to 1. For MDA5 signaling assays, cells weretransfected with a FLAG-tagged MDA5 construct, p�-IFN-fl-lucter (both vec-tors kindly provided by D. Garcin, University of Geneva), and pTK-RL as wellas with an RFP-expressing plasmid or one of the different V protein-expressing

plasmids. After 24 h of transfection, the cells were stimulated with 1.5 �g ofpoly(I:C)/ml (Sigma) by transfection with Fugene HD (Roche). The cells wereharvested after 15 h and assayed for firefly and Renilla luciferase activity asdescribed above, with the ratio of the pCI-RFP-expressing vector set to 1.

Immunofluorescence. Vero-SLAM or Vero cells were grown on cover slides insix-well plates, and the following day cells were either infected with the recom-binant viruses (rA75/17red, rA75/17red Cko, rA75/17red Vko, and rA75/17red

CVko) at an MOI of 0.02 or transfected with the desired expression plasmids. At24 h postinfection or transfection, cells were treated (or left untreated) with1,000 IU/ml IFN-�/� for 30 min at 37°C and then fixed with phosphate-bufferedsaline (PBS) containing 4% paraformaldehyde (PFA). After permeabilizationwith PFA and 0.1% Triton X-100, the cells were washed in PBS. Blocking ofnonspecific binding sites and incubation with the first antibody were performedin PBS supplemented with 2% FCS for 1 h at room temperature and for 1.5 h at37°C, respectively. Then, the slides were washed in PBS and incubated for 1 h atroom temperature with the second antibody diluted in PBS. The cells werewashed again, and the nuclei were stained with TOTO3 (Invitrogen) according tothe manufacturer’s protocol. Pictures were taken with a confocal microscope(Olympus).

Western blotting. Immunoblotting was performed as previously described(32). Briefly, Vero-SLAM cells were cultured in six-well plates and infected (with

FIG. 1. Generation of expression plasmids and recombinant viruses to assess the A75/17 CDV-P gene products responsible for inhibitingIFN-�/�-mediated signaling. (A) Schematic representation of the full-length A75/17 CDV cDNA clone bearing an additional transcription unitencoding RFP. (B) Schematic presentation of the mutations performed on the P gene of rA75/17red to enable V and P expression without C. Thestart codon for the C was mutated, and an additional stop codon was introduced (performed to obtain rA75/17red Cko, rA75/17red CVko, pCI-P,and pCI-V). To inactivate V expression, three nucleotides were changed at the V editing site (applied for rA75/17red Vko and rA75/17red CVko).All mutations were performed without influencing the P open reading frame. (C) The V protein substantially inhibits the IFN-induced activationof the ISRE promoter in a dual luciferase assay. Vero-SLAM cells were transfected with the expression plasmids for P, V, C, an irrelevant GFPcontrol plasmid, and the empty pCI as well as the two plasmids coding for the IFN-inducible and the constitutively expressed luciferases. At 24 hpost transfection they were treated (or left untreated) with IFN-�/� for 6 h and subsequently lysed for luciferase analysis. (D) P, V, and Cexpression control by SDS-PAGE immunoblotting of Vero-SLAM cells at 2 days posttransfection with the expression plasmids pCI-P, pCI-V,pCI-C, and an empty plasmid (pCI). Each plasmid expresses only one protein (detected by polyclonal antibodies). (E) The recombinant virusesexpress the expected P gene products. Vero-SLAM cells were either infected with rA75/17red, rA75/17red Cko, rA75/17red Vko, and rA75/17red CVko

at an MOI of 0.02 or left uninfected and lysed 2 days postinfection for subsequent Western blot analysis. The viral proteins were detected byimmunoblotting with three polyclonal antibodies. (F) The four recombinant viruses show similar growth curves in Vero-SLAM cells. The diagramindicates the titers of cell-associated viruses at 8, 24, 32, and 48 h postinfection in infectious particles per ml.

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the various recombinant CDVs [rCDVs]) at an MOI of 0.02 1 day later ortransfected with the different expression vectors. At 2 days postinfection orposttransfection, the cells were washed with cold PBS and lysed at 4°C inradioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl [500 mM NaCl toextract the C protein], 1% NP-40, 0.5% Na-deoxycholate, 0.1% sodium dodecylsulfate]SDS], 50 mM Tris-Cl, pH 7.4) containing Halt Protease Inhibitor Cock-tail (Socochim) and Phosphatase Inhibitor Cocktail Set II (Merck) to protectphosphorylated STAT proteins. After centrifugation, Laemmli sample buffer(Bio-Rad) supplemented with DL-dithiothreitol (Fluka) was added to the sam-ples, and they were boiled for 5 min and separated by SDS-polyacrylamide gelelectrophoresis. Afterwards, the proteins were transferred to nitrocellulosemembranes (Hybond-ECL; Amersham Biosciences), which subsequently wereblocked in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) and 5%nonfat dry milk (or bovine serum albumin for detection of the C protein) for 1 h.The membranes were incubated in blocking buffer containing the first antibodyat 4°C overnight. Then the membranes were washed three times in TBS-T andincubated with the horseradish peroxidase-conjugated second antibody for 1 h atroom temperature. After the membranes were washed three times, ECL sub-strate (Amersham Biosciences) was used, and chemiluminescence signals weredetected with an LAS-3000 camera (Fujifilm). Relative protein amounts werecalculated with AIDA Image Analyzer software (Raytest Schweiz AG, Wetzikon,Switzerland).

Coimmunoprecipitation. Vero-SLAM cells were seeded in six-well plates, andthe next day cells were infected at an MOI of 0.02 with rA75/17red or rA75/17red

Vko or left uninfected. In parallel experiments different expression plasmids weretransfected. At 2 days postinfection or 1 day posttransfection, the cells weretreated (or left untreated) with 1,000 IU/ml IFN-�/� for 30 min at 37°C and thenwashed with cold PBS and incubated on ice with 50 mM Tris, 150 mM NaCl, 2mM EDTA, and 1% NP-40 (pH � 7.5) complemented with Halt Protease

Inhibitor Cocktail (Socochim) for 45 min until complete lysis. After a 10-mincentrifugation at 5,000 � g at 4°C, aliquots for total protein analyses wereseparated, and the remaining supernatants were incubated for 3 h with the firstantibody (anti-STAT1, anti-STAT2, anti-V, or anti-HA) or without antibody,followed by the addition of protein G-Sepharose beads at 4°C overnight. After a1-min centrifugation at 5,000 � g at 4°C, the pellets were washed four times withthe same buffer used earlier and finally dissolved directly in Laemmli samplebuffer for Western blotting performed as described above.

Generation of MDCK cells constitutively expressing the CDV receptor CD150/SLAM. MDCK cells were transduced with pRRL lentivirus vectors at an MOI of5. Subsequently, a highly SLAM-expressing clone was selected by limiting dilu-tion and was used for further experiments.

The lentivirus vector pRRL as been described elsewhere (7) (kindly providedby Patrick Salomon, University of Geneva). Stock of lentivirus vectors wasgenerated in 293T/17 cells as previously described (7).

Production of cIFN-�. Standard reverse transcription-PCR (RT-PCR) tech-niques were employed to amplify and clone the canine IFN-� (cIFN-�) cDNAinto the pCI vector (primers are available upon request). To express cIFN-�,293T cells were transfected with pCI-cIFN-� for 3 days. Then, the supernatantwas harvested, filtered through a 0.45-�m-pore-size filter, and concentratedusing 10-kDa size exclusion Centricon columns (Millipore).

RESULTS

The A75/17 CDV-V protein inhibits the IFN-�/�-mediatedsignaling pathway. In order to investigate any potential role ofthe CDV-P gene products in modulating the IFN-�/�-medi-

FIG. 2. Neither degradation nor inhibition of STAT1 and STAT2 phosphorylation occurs after infection with the various rCDVs. (A) Cyto-pathic effect induced by the different rCDVs in Vero-SLAM cells. Vero-SLAM cells were infected at an MOI of 0.02, and representative fieldsof view were then photographed at 2 days postinfection. (B) To assess whether the various rCDVs affected the endogenous expression of STAT1and STAT2, Vero-SLAM cells were infected with rA75/17red, rA75/17red Cko, rA75/17red Vko, and rA75/17red CVko at an MOI of 0.02. Two dayspostinfection, cells were treated (or left untreated) with IFN-�/� for 30 min and subsequently lysed. Cell lysates were then subjected to immunoblotanalysis using specific anti-STAT1 or anti-STAT2 antibodies. To assess the phosphorylated state of both STAT molecules in the presence orabsence of IFN, Vero-SLAM cells were infected with rA75/17red, rA75/17red Cko, rA75/17red Vko, and rA75/17red CVko at an MOI of 0.02. At 2days postinfection, cells were treated (or left untreated) with IFN-�/� for 30 min and subsequently lysed. Cell lysates were then subjected toWestern blot analysis using specific anti-phospho-STAT1 or anti-phospho-STAT2 monoclonal antibodies.

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FIG. 3. rCDVs expressing the V protein inhibit STAT1 and STAT2 nuclear translocation. (A) Immunofluorescence images of Vero-SLAMcells stained for STAT1. Vero-SLAM cells were infected with the various rCDVs or left uninfected. At 1 day postinfection, cells were treated for

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ated signaling pathway, we first generated expression vectorsencoding the P, V, and C proteins. In addition, specific nucle-otides in the P and V genes were mutated to close the openreading frame (ORF) of the C protein without affecting theORFs of P and V (Fig. 1B, top). Western blot analysis con-firmed that all proteins were efficiently expressed and migratedaccording to their expected molecular weights in SDS-PAGE(Fig. 1D). Next, a dual luciferase assay was performed to assessthe role of each single protein in controlling IFN-�/�-inducedsignaling. As expected, after treatment with IFN-�/�, the rel-ative luciferase activity clearly increased in cells transientlytransfected with both an empty control plasmid (pCI) and witha plasmid expressing the irrelevant control green fluorescentprotein (GFP) (Fig. 1C). This observation confirmed the bio-activity of the recombinant human IFN in Vero-SLAM cells. Incontrast, the activation of the ISRE promoter was strikingly

inhibited in cells transfected with the V expression plasmid.Interestingly, while we did not observe any effect at all in cellsexpressing the C protein, we noticed a partial inhibition in cellstransiently expressing the P protein (Fig. 1C). Taken together,these results demonstrated that the CDV-V protein potentlyinhibited the activation of the ISRE promoter, whereas the Pprotein could only slightly control the IFN-�/�-mediated sig-naling pathway.

CDV infection neither induces STAT degradation nor inhib-its STAT phosphorylation. We then investigated at which stepthe CDV-V protein was able to affect the IFN-�/�-mediatedsignaling pathway. However, conclusions drawn from single-protein overexpression experiments may differ from resultsobtained with the same protein in the context of a viral infec-tion (25). In order to overcome this problem, we generatedrecombinant C and/or V knockout viruses based on the highly

30 min with IFN-�/� and subsequently fixed, permeabilized, and stained for STAT1 localization using an anti-STAT1 antibody. Then, an AlexaFluor 488-conjugated secondary antibody was employed (green), and images were captured with a scanning confocal laser microscope (Olympus).The nuclei were counterstained with TOTO3 (blue). Infected cells were localized by the expression of RFP. (B) Immunofluorescence images ofVero-SLAM cells stained for STAT2. Experimental settings were identical to those of panel A. Filled arrowheads indicate nuclei without STAT1(or STAT2) accumulation, and open arrowheads indicate nuclei with STAT1 (or STAT2) accumulation. (C) Nuclear translocation of STAT1 andSTAT2 in Vko and CVko recombinant A75/17red viruses is dependent on IFN treatment. Immunofluorescence images show Vero-SLAM cellsstained for STAT1 and STAT2. Experimental settings were identical to those of panel A with the exception that IFN was not added to the cells.In all panels, specific fields of view of the cell monolayer were selected to illustrate infected and noninfected cells in the identical areas.

FIG. 4. The A75/17 CDV-V protein inhibits STAT1 nuclear import. (A) Subcellular localization of the NES-GFP-SV5-V fusion construct in thepresence and absence of leptomycin B (LMB). Vero-SLAM cells were transfected with the fusion protein NES-GFP-SV5-V and treated (or leftuntreated) with LMB for 3 h. Subsequently, cells were fixed and permeabilized, and nuclei were counterstained with TOTO3. Green and bluefluorescence emissions were captured with a scanning confocal laser microscope. (B) Vero-SLAM cells were infected with rA75/17red or rA75/17red Vko

for 1 day. Then, cells were treated (or left untreated) with LMB for 3 h and with IFN-�/� for the last 30 min. Subsequently, cells were fixed andpermeabilized, and nuclei were counterstained with TOTO3. Moreover, STAT1 localization was investigated using an anti-STAT1 antibody followed byan Alexa Fluor 488-conjugated secondary antibody. Green, blue, and red fluorescence emissions were captured with a scanning confocal laser microscope.Filled arrowheads indicate nuclei without STAT1 accumulation, and open arrowheads indicate nuclei with STAT1 accumulation.

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virulent CDV strain A75/17 (Fig. 1A). Mutations ablating theC ORF were identical to those applied in the P and V expres-sion vectors. Furthermore, specific nucleotide substitutionswere performed at the P gene editing site, which preserved theP reading frame while silencing the production of the VmRNA (Fig. 1B, bottom). All viruses were successfully rescuedfrom cDNA, and subsequently the expression of the P geneproducts in cells infected with rA75/17red, rA75/17red Cko,rA75/17red Vko, and rA75/17red CVko was confirmed by immu-noblot analysis. As shown in Fig. 1E all recombinant virusesexpressed the expected pattern of P, V, and C proteins.Growth kinetics indicated that the absence of the C and Vproteins scarcely affected the efficiency of viral replication inVero-SLAM cells since all the recombinant viruses showedvery similar growth curves (Fig. 1F).

Different molecular mechanisms were documented for sev-eral viruses among the family Paramyxoviridae in order toevade the IFN-�/�-induced antiviral state. For instance, the Vproteins of rubulaviruses are responsible for a direct degrada-tion of the pool of STAT molecules in the cytoplasm, whereasthe V proteins of MV (depending on the viral strain) and RPVinhibit the phosphorylation of STAT1 and STAT2 upon IFNtreatment (5, 8, 25, 30, 42). In line with these results, we nextexamined by immunoblotting whether the CDV-V-mediatedinhibition of the IFN-�/�-mediated signaling pathway was dueto either STAT degradation or inhibition of STAT phosphor-ylation. Figure 2A documents that after 2 days of infection inVero-SLAM cells, all rCDVs mediated a typical cytopathiceffect, with the formation of syncytia involving about 80% ofthe cells. Even though only about 20% of the cells remainednoninfected, the expression and stability of both STAT1 andSTAT2 were not modulated in infected cells compared tomock-treated cells (Fig. 2B). Furthermore, none of the recom-binant viruses was able to block the phosphorylation of STAT1and STAT2. Indeed, immunoblotting revealed no major dif-ferences in the amount of phospho-STAT molecules detectedin cells infected with any of the recombinant viruses comparedto mock-infected, IFN-�/�-treated, cells (Fig. 2B). Taken to-gether, these results indicate that the aforementioned role of Vin inhibiting an IFN-�/�-dependent response is due neither tothe degradation of STAT1 and STAT2 nor to the inhibition oftheir phosphorylation.

CDVs expressing the V protein inhibit the nuclear accumu-lation of STATs. Upon IFN-�/� treatment, accumulation ofSTAT1 and STAT2 in the cytoplasm rather than in the nucleuswas reported to occur in cells infected with measles and rinder-pest morbilliviruses (MV and RPV, respectively) (8, 25, 28).Thus, taking advantage of our newly generated recombinantviruses, we next assessed by immunofluorescence analysiswhether the nuclear accumulation of STAT1 and STAT2 wasmodulated by these viruses. In mock-infected Vero-SLAMcells, STAT1 staining exhibited the anticipated shift betweencytoplasmic to mainly nuclear localization upon treatment withIFN-�/� (Fig. 3A, mock). The identical phenotype was ob-served in cells infected with rA75/17red Vko and rA75/17red

CVko. Strikingly, however, cells infected with the rA75/17red

and rA75/17red Cko viruses (the two viruses expressing the Vprotein) exhibited a clear accumulation of STAT1 in the cyto-plasm (Fig. 3A). Identical results were observed for STAT2under corresponding conditions (Fig. 3B). Next, to verify

whether STAT1 nuclear accumulation was dependent on IFNaddition, STAT1 cellular localization was assessed in infectedbut non-IFN-treated cells. In the absence of IFN treatment,rA75/17red Vko and rA75/17red CVko (data not shown) couldnot mediate STAT1 nuclear translocation (Fig. 3C). Rather,STAT1 was readily detected in the cytoplasm of rA75/17red-infected but non-IFN-treated cells, thereby validating the no-tion that STAT1 is not degraded in rA75/17red-infected cells.The identical phenotypes were observed when the cellular lo-calization of STAT2 was assessed (Fig. 3C). These observa-tions are in agreement with the results found in our transienttransfection assay and confirmed a key regulating role of theCDV-V protein in inhibiting IFN-�/�-dependent activation ofinnate immunity.

STAT1 cytoplasmic accumulation is caused by a nuclear im-port inhibition mechanism. Upon IFN-�/� treatment, STATmolecules are first phosphorylated, then translocate into thenucleus, and finally relocate to the cytoplasm through theirnuclear export signal (NES) (1, 24). Hence, CDV-V-depen-dent STAT cytoplasmic accumulation may result either fromnuclear import inhibition or from an accelerated nuclear ex-port mechanism. To discriminate between these two possibil-ities, the Crm1-dependent nuclear export inhibitor leptomycinB (LMB) was used. Immunofluorescence analysis was thenperformed to assess the cellular localization of STAT1 in cellsinfected with rA75/17red or rA75/17red Vko. As a control, weused the simian virus 5 (SV5) V protein ([SV5-V] known to betargeted to the nucleus [34]) fused at its N-terminal region withan NES-tagged green fluorescent protein (NES-GFP-SV5-V).As expected, upon LMB treatment, the fluorescent fusion pro-

FIG. 5. The rA75/17 CDV-V protein associates with endogenousSTAT1 and STAT2. Vero-SLAM cells were infected at an MOI of 0.02with rA75/17red or rA75/17red Vko or left uninfected and subsequentlylysed 2 days postinfection for coimmunoprecipitation assay. STAT1 orSTAT2 was first immunoprecipitated (IP) with an anti-STAT1 or anti-STAT2 monoclonal antibody, which was followed by addition of proteinG-Sepharose beads. Then, Western blot analysis using an anti-V poly-clonal antibody to detect any potential association or anti-STAT1 and-STAT2 antibodies to control for direct IP, was performed. Total celllysates taken prior to IP (TL) were subjected to immunoblot analysis(WB) to investigate the endogenous STAT1 and STAT2 expressions orthe viral V protein expression. In addition, immunoprecipitations wereperformed either with an anti-HA monoclonal antibody or in the absenceof any monoclonal antibody (no antibody) to control the co-IP assay.

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tein remained accumulated mainly in the nucleus while it wasclearly relocated in the cytoplasm in the absence of the drug(Fig. 4A). Importantly, in IFN- and LMB-treated cells, STAT1was found to be strongly accumulated in the nucleus in recom-binant V knockout virus-infected cells. Conversely, in cellsinfected with the wild-type rA75/17red virus, STAT1 could notbe detected in nuclei (Fig. 4B). Together, these results indicatethat cytoplasmic accumulation of STAT1 in cells infected withCDVs expressing the V protein is caused by nuclear importinhibition rather than by an accelerated export mechanism.

The A75/17 CDV-V protein efficiently binds to endogenousSTAT1 and STAT2. Coimmunoprecipitation (co-IP) from in-fected cells was next performed to assess whether the A75/17CDV-V protein may influence STAT nuclear accumulation bybinding to endogenous STAT1 and/or STAT2. Thus, STAT1from lysates of infected and noninfected cells was immunopre-cipitated with an anti-STAT1 monoclonal antibody (MAb),followed by immunoblotting using an anti-CDV-V poly-

clonal antibody. The identical strategy was employed usingan anti-STAT2 MAb to determine whether V can associatewith endogenous STAT2. An anti-HA MAb immunoprecipi-tation or immunoprecipitation in the absence of antibodywas performed in parallel to validate the co-IP assay. In-deed, in rA75/17red-infected cells, V could be efficientlycopurified after STAT1 and STAT2 immunoprecipitation(Fig. 5). Moreover, these interactions were formed indepen-dently of the activation of the signaling pathway since V wascoprecipitated in both the presence and the absence of IFN-�/� treatment (data not shown). As expected, V was not co-precipitated in rA75/17red Vko-infected cells or when the anti-HAMAb or no MAb was used for immunoprecipitation (Fig. 5).Western blotting performed with cell lysates taken prior toimmunoprecipitation revealed the expected pattern of V ex-pression. Indeed, V was produced by rA75/17red but not byrA75/17red Vko (Fig. 5). Finally, Western blotting using anti-STAT1 and anti-STAT2 antibodies demonstrated that under

FIG. 6. Identification of V domains responsible for inhibiting the IFN-�/�-mediated signaling pathway. (A) Schematic diagram of the A75/17CDV-V protein and engineered variants. (B) Sequence alignment of several canine distemper virus P genes. (C) Effect of the V protein andvariants in inhibiting the IFN-induced activation of the ISRE promoter in a dual luciferase assay. Vero-SLAM cells were transfected with thevarious expression plasmids at two different concentrations (500 ng and 100 ng) or with an empty plasmid (pCI) as well as with the two plasmidscoding for the IFN-inducible and the constitutively expressed luciferases. At 24 h posttransfection, cells were treated (or left untreated) withIFN-�/� for 6 h and subsequently lysed for luciferase analysis. (D) Schematic diagram of the A75/17 CDV-V protein and variants fused to RFP.(E) Experimental settings were identical to those in panel C but with the RFP-fused proteins.

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all conditions both endogenous transcription factors wereexpressed in very similar amounts (Fig. 5). These resultsstrongly suggest that the A75/17 CDV-V protein controlsIFN-�/�-induced signaling by efficiently forming a complexwith STAT1 and STAT2.

Both the N-terminal and C-terminal domains of V are nec-essary to completely inhibit IFN-�/�-induced activity. To ini-tiate the mapping of the CDV-V-dependent STAT-interactingdomains, various HA-tagged expression vectors were engi-neered (Fig. 6A). A first construct, composed of the sharedN-terminal domain between P and V, was produced (VNT; 240aa) (Fig. 6A). The second encompassed the cysteine-rich C-terminal domain of V (VCT; 78 aa) (Fig. 6A). Next, a singlesubstitution (Y110D) was introduced into the N-terminal re-gion of the full-length HA-tagged V protein (308 aa) (Fig. 6A).This tyrosine is, indeed, highly conserved among morbillivi-ruses and has been shown for MV-V to be responsible forspecific STAT1 binding (4, 5, 8, 28, 35). Interestingly, sequencecomparison between several wild-type CDV strains and thelarge plaque-forming variant of the Onderstepoort (OP) vac-cine strain of CDV revealed a substitution at that preciselocation (Y110D) (Fig. 6B). Finally, we also fused the HA tagsequence to the N-terminal part of the P gene construct toallow immunoprecipitation under similar conditions (Fig. 6A).

In order to verify the activity of the HA-tagged full-lengthconstructs and to determine the role of each individual V subdo-main in inhibiting IFN-�/�-mediated signaling, ISRE luciferasereporter gene assays were performed from IFN-treated Vero-SLAM cells transfected with the various expression plasmids.Two different plasmid concentrations were used for transfectionin order to control for the amount of protein expressed. As ex-pected, HA-Vwt strongly suppressed IFN-�/�-mediated signalingin a concentration-dependent manner, whereas HA-P caused apartial inhibition at its higher concentration only (Fig. 6C).Similarly, the HA-VNT and the HA-Vwt Y110D mutantsshowed partial inhibition as well when expressed at highconcentrations. Conversely, the HA-VCT construct alone wasnot sufficient to control IFN-�/�-mediated signaling.

To confirm the expression of the various mutants, immuno-blot analysis from total cell extract of transfected Vero-SLAMcells was undertaken using an anti-HA MAb to detect thevarious HA-tagged proteins. Figure 7A (bottom panel) docu-ments that all proteins, except HA-VCT, were correctly ex-pressed and migrated according to their expected molecularweights in the SDS polyacrylamide gel. It is possible that theextremely small size of HA-VCT affected proper expressionand/or stability. Thus, to overcome these putative defects, Vwt,Vwt Y110D, VNT, and VCT were fused to RFP. In addition,these fusion proteins were designed to contain both a smalllinker peptide and the HA tag sequence to retain both proteinfunctionalities and to facilitate detection and immunoprecipi-tation, respectively (Fig. 6D). Indeed, using this strategy, allengineered proteins were properly expressed, as demonstratedby immunoblot analysis (Fig. 7B, bottom panel). ISRE lucif-erase reporter gene assays were performed in order to assessthe ability of these fusion proteins to control IFN-�/�-medi-ated signaling. Figure 6E illustrates that all proteins modulatedthe IFN-induced activity to the same extent as the identicalnonfused V mutant proteins (Fig. 6C). These results indicatethat the CDV-VCT module alone lacks the capacity to con-

trol IFN-�/�-induced activity, whereas VNT was able tofunction as an autonomous module, albeit to a limited ex-tent compared to Vwt.

To verify the proper folding of the C-terminal region of V inHA-VCT- and RFP-HA-VCT-expressing cells, their ability todisrupt signaling by the RNA helicase protein MDA5 wasinvestigated. Indeed, it has been previously reported that themeasles virus VCT domain was sufficient to suppress theMDA5-mediated signaling pathway (35). The results shown inFig. 7C indicate that HA-VCT presumably does not fold into abiologically active conformation since this domain was not ableto control the MDA5-dependent signaling pathway to theIFN-� promoter reporter gene. In contrast, RFP-HA-VCT wasfully able to ablate MDA5-mediated signaling, as were Vwt and

FIG. 7. Only Vwt efficiently interacts with both STAT molecules.Results of coimmunoprecipitation of endogenous STATs with V pro-tein fragments (A) or with the corresponding RFP-fused proteins(B) are shown. Vero-SLAM cells were transfected to express the var-ious HA-tagged V proteins, and cell extracts were prepared for immu-noprecipitation (IP) with an anti-HA MAb, followed by overnightincubation with protein G-Sepharose beads. The eluates were evalu-ated by STAT immunoblotting. The middle and bottom panels illus-trate total lysate results of samples taken prior to immunoprecipitation(total; representing 1/10 of total cell extracts), whereas the upperpanels indicate the results obtained after HA immunoprecipitation(coIP; representing 9/10 of total cell extracts). (C) Effect of the differ-ent V constructs on the suppression of the MDA5-mediated signalingpathway. Vero-SLAM cells were transfected with p�-IFN-fl-lucter andpTK-RL reporter genes along with the different V-expressing plasmidsand the FLAG-tagged MDA5 construct. At 24 h posttransfection, cellswere left untreated or were additionally transfected with 1.5 �g ofpoly(I:C)/ml (15 h) prior to luciferase assays.

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Vwt Y110D, but not VNT, with or without fusion to RFP (Fig.7C). We thus concluded that VCT alone was very likely misfoldedand thus not functional but that it could fold into an active con-formation if fused to an irrelevant, stabilizing protein. Impor-tantly, this result furthermore confirms the selective incapacity ofCDV-VCT to control IFN-�/�-induced activity on its own.

Only Vwt efficiently associates with STAT1 and STAT2. Acoimmunoprecipitation assay was used to investigate the abilityof the different V domain mutants to bind the endogenousSTAT1 and STAT2 proteins (in the absence of IFN treat-ment). The anti-HA 3F10 monoclonal antibody was used toimmunoprecipitate the different V constructs, followed by im-munoblot analysis using anti-STAT1 or anti-STAT2 antibodiesfor detection. Figure 7A and B document that, independentlyof whether Vwt was fused to the RFP, both STAT1 and STAT2could efficiently be copurified (Fig. 7A and B, upper panels).Interestingly, although VNT retained some interaction withSTAT1, it displayed significantly reduced binding to STAT2,whereas VCT, even when fused to RFP, showed no bindingactivity at all to either STAT molecule. The latter results are inagreement with the data obtained in the ISRE luciferase re-porter gene assay. Moreover, while the single point mutantVwt Y110D almost completely lost the capacity to associatewith STAT1, we detected a slight interaction with STAT2 (Fig.7A and B, upper panels). Finally, the ability of the P and Cproteins in binding STAT1 and STAT2 was also investigated(HA-tagged versions were constructed). The C protein was

unable to bind both STAT1 and STAT2, whereas the P proteinweakly interacted with STAT1 only (Fig. 7A). Immunoblotanalysis of STAT1 and STAT2 prior to immunoprecipitationrevealed that both transcription factors were expressed to verysimilar amounts under all conditions. These results suggestthat VCT, when fused to VNT, confers the capacity of Vwt toenhance STAT1 binding and to associate with STAT2.

Selective inhibition of nuclear import of STAT molecules byVNT and Vwt Y110D. We next assessed whether the differen-tial binding capacities of the V mutants correlated with thenuclear import inhibition mechanism described above. To thispurpose, Vero-SLAM cells were transfected with the variousexpression plasmids and treated with IFN-�/�, and STAT1/STAT2 cellular localization was subsequently investigated byimmunofluorescence analysis (Fig. 8 and 9, for STAT1 andSTAT2, respectively). Clearly, while HA-Vwt efficiently inhib-ited STAT1 and STAT2 nuclear import, HA-P partially re-tained STAT1 but not STAT2 in the cytoplasm (Fig. 8A and9A). Interestingly, the extent of STAT1 nuclear import inhibi-tion seemed to correlate with the level of P expression intransfected cells (Fig. 8A). Conversely, HA-C did not at allsuppress IFN-�/�-induced STAT1 and STAT2 nuclear accu-mulation (Fig. 8A and 9A), confirming the data obtained in theco-IP assay (Fig. 7).

In order to assess the ability of VNT, VCT, and Vwt Y110Dto modulate STAT1 and STAT2 nuclear translocation, theidentical experiments were performed using the RFP fusion

FIG. 8. Vwt and VNT inhibit STAT1 nuclear translocation. Vero-SLAM or Vero cells were transfected with the various expression vectors.One day posttransfection, cells were treated with IFN-�/� for 30 min. Subsequently, cells were fixed and permeabilized, and STAT1 localizationwas investigated using an anti-STAT1, which was followed by addition of an Alexa Fluor 488-conjugated secondary antibody (green) (A). The viralHA-tagged proteins were stained using an anti-HA monoclonal antibody, followed by addition of an Alexa Fluor 555-conjugated (red) secondaryantibody. STAT1 localization was investigated with an anti-STAT1 antibody, followed by addition of an Alexa Fluor 488-conjugated (green)secondary antibody, and RFP fusion proteins were directly visualized for red fluorescence emission (B). Nuclei were counterstained with TOTO3(blue). Green, blue, and red fluorescence emissions were captured with a scanning confocal laser microscope. Filled arrowheads indicate nucleiwithout STAT1 accumulation, and open arrowheads indicate nuclei with STAT1 accumulation.

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proteins. Here again, only RFP-HA-Vwt efficiently inhibitedboth STAT1 and STAT2 nuclear import (Fig. 8B and 9B).Intriguingly, whereas RFP-HA-VNT was able to efficiently re-tain STAT1 in the cytoplasm, STAT2 mainly localized in thenucleus in the presence of the same construct though theseexperiments were performed in the presence of IFN. Remark-ably, the exact reverse correlation was observed in RFP-HA-Vwt Y110D-transfected Vero-SLAM cells. In this case, STAT1efficiently translocated to the nucleus in the presence of the Vmutant, whereas STAT2 exhibited a clear cytoplasmic accumu-lation. In contrast, and in agreement with the co-IP assay,RFP-HA-VCT did not function autonomously since bothSTAT molecules were located in the nucleus in IFN-�/�-treated cells (Fig. 8B and 9B). These results demonstrate thatVNT and VCT need to be fused to efficiently control nuclearimport of both STAT molecules.

Evidence for cytoplasmic accumulation of phospho-STAT1in CDV-V-expressing cells. We next confirmed the notion thatthe phosphorylation state of STAT1 is not affected by theCDV-V proteins. To this aim, immunofluorescence analysesusing an anti-phospho-STAT1 MAb were performed in cellstransfected with RFP-HA-Vwt, -VNT, -VCT, and -Vwt Y110Dand treated with type I IFN. As expected, nuclear translocationof phospho-STAT1 was observed in IFN-treated and RFP-transfected cells. Importantly, in RFP-HA-Vwt- and RFP-HA-VNT-transfected cells, phospho-STAT1 accumulated in thecytoplasm to a greater extent than in nontransfected cells of

the same area (Fig. 10A). In contrast, phospho-STAT1 wasclearly detected in the nucleus of RFP-HA-VCT- and RFP-HA-Vwt Y110D-transfected cells (Fig. 10A).

The identical experiments were repeated in rCDV-infectedcells. Hence, in rA75/17red- and rA75/17red Cko-infected cells,phospho-STAT1 could be detected in the cytoplasm, whereasnuclear staining was observed in rA75/17red Vko- and rA75/17red CVko-infected cells (Fig. 10B). We noticed less stainingof phospho-STAT1 in the cytoplasm of infected cells than intransfected cells, probably as a result of the formation of largesyncytia by the different recombinant viruses. Taken together,the above data clearly validate the notion that the CDV-Vprotein inhibits STAT1 nuclear import without affecting itsphosphorylation state both in transfected and infected cells.

Viruses lacking V expression are more sensitive to IFN-�/�treatment. Taken together, the above results suggest that V isessential in counteracting IFN-�/�-dependent signaling al-though the P protein was able to exert partial control. To verifywhether the different phenotypes described above correlatewith differences in growth kinetics, Vero-SLAM cells wereinfected with the various recombinant CDVs (Fig. 11). Next,cells were treated (or left untreated) with IFN-�/� at 3, 12, 24,and 36 h postinfection. Finally, at 48 h postinfection, virustiters of cell-associated viruses were determined by limitingdilution assay. Interestingly, all viruses had reduced viral titerscompared to those obtained in IFN-untreated cells (Fig. 9).This is probably because we used an MOI of 0.02 and treated

FIG. 9. Vwt and Vwt Y110D inhibit STAT2 nuclear translocation. Vero-SLAM or Vero cells were transfected with the various expressionvectors. One day posttransfection, cells were treated with IFN-�/� for 30 min. Subsequently, cells were fixed and permeabilized, and STAT2localization was investigated using an anti-STAT2, which was followed by addition of an Alexa Fluor 488-conjugated secondary antibody (green)(A). The viral HA-tagged proteins were stained using an anti-HA monoclonal antibody, followed by addition of an Alexa Fluor 555-conjugated(red) secondary antibody. (B) STAT2 localization was investigated with an anti-STAT2 antibody, followed by addition of an Alexa Fluor488-conjugated (green) secondary antibody, and RFP fusion proteins were directly visualized for red fluorescence emission. Nuclei werecounterstained with TOTO3 (blue). Green, blue, and red fluorescence emissions were captured with a scanning confocal laser microscope. Filledarrowheads indicate nuclei without STAT2 accumulation, and open arrowheads indicate nuclei with STAT2 accumulation.

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the cells with IFN as early as 3 h postinfection. Thus, most ofthe uninfected cells had probably established an antiviral statebefore being infected, in turn affecting proper viral growtheven in the case of V-expressing viruses. Nevertheless, viruseslacking V expression had approximately 10 times less progenyvirus production, confirming that V is crucial for counteractingthe IFN-�/�-mediated antiviral state. It is important to note

that the concentration of IFN used in these experiments (1,000units/ml) was 10 to 50 times higher than the minimal concen-tration required to completely inhibit a VSV-induced cyto-pathic effect in Vero-SLAM cells (data not shown). Neverthe-less, albeit not to the same extent, all viruses were able to grow,which suggested that a viral component(s) in addition to V mayprovide partial control of innate immunity.

FIG. 10. Phospho-STAT1 is accumulated in the cytoplasm of both CDV-V-transfected cells and rA75/17red-infected cells. (A) Vero-SLAM cellswere transfected with the various expression vectors. One day posttransfection, cells were treated with IFN-�/� for 30 min. Subsequently, cells werefixed and permeabilized, and phospho-STAT1 localization was investigated using an anti-phospho-STAT1 MAb, which was followed by additionof an Alexa Fluor 488-conjugated secondary antibody (green). RFP fusion proteins were directly visualized for red fluorescence emission.(B) Vero-SLAM cells were infected with the corresponding recombinant viruses at an MOI of 0.02. Phospho-STAT1 localization was investigatedwith an anti-phospho-STAT1 MAb, followed by addition of an Alexa Fluor 488-conjugated (green) secondary antibody, and the RFP expressedby the various viruses was directly visualized for red fluorescence emission. In both panels, nuclei were counterstained with TOTO3 (blue). Green,blue, and red fluorescence emissions were captured with a scanning confocal laser microscope. Filled arrowheads indicate nuclei withoutphospho-STAT1 accumulation, and open arrowheads indicate nuclei with phospho-STAT1 accumulation. Specific fields of view of the cellmonolayer were selected to illustrate infected and noninfected cells in the identical areas.

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The CDV-V protein inhibits STAT1 nuclear import in ca-nine cells. To confirm the findings that CDV-V controls IFN-induced STAT1 nuclear import not only in Vero but also incanine cells, we sought to determine the effect of CDV-V inMDCK cells. Indeed, these cells are functional in both IFN in-duction and IFN action (data not shown). First, a cell line stablyexpressing the universal morbillivirus receptor CD150/SLAM wasproduced in order to allow the virulent virus to replicate. Second,since none of the tested recombinant IFN (rIFN) molecules wasfunctional in these cells (human, bovine, feline, and universalrIFN), we cloned the canine IFN-� obtained from primary caninekeratinocytes. Finally, the recombinant canine IFN-� was pro-duced in 293T cells. Figure 12 illustrates that upon addition ofcIFN-�, STAT1 was, indeed, readily translocated into the nucleusof the engineered MDCK-SLAM cells.

As expected, in cells infected with rA75/17red (Fig. 12A) andrA75/17red Cko viruses (data not shown), STAT1 nuclear im-port was inhibited, whereas in rA75/17red Vko-infected cells(Fig. 12A and B) and rA75/17red CVko-infected cells (data notshown), STAT1 was located in the nucleus. The identical phe-notypes were observed for STAT2 (data not shown). Thus, themain conclusion obtained from Vero-SLAM cells correlatedwith results in canine cells, which are characterized by an intactIFN response system. In addition, we performed titration exper-iments at 2 days postinfection in the presence and absence ofcanine IFN-� in rA75/17red- and rA75/17red Vko-infected cells.The Vko virus grew less efficiently than rA75/17red (Fig. 12C) evenin the absence of IFN, presumably because the Vko virus is defi-cient in suppressing both the IFN action and induction signalingpathways. Nevertheless, IFN treatment reduced the replication ofthe Vko virus about 2 orders of magnitude, whereas the V-ex-pressing virus was reduced only about 10-fold.

DISCUSSION

It has recently been reported that V knockout CDV (basedon the 5804P virulent strain) was attenuated in infected ferrets,which was associated, at least in part, with inhibition of IFN-�/� induction in peripheral blood mononuclear cells (PBMCs)(47). We now show that the V protein of the highly virulent

CDV A75/17 strain also counteracts IFN action by additionallydisrupting the IFN-�/�-dependent signaling. Importantly, thisdoes not seem to be valid in only Vero-SLAM cells as prelim-inary experiments in canine MDCK-SLAM cells providedstrong evidence that the effects observed in Vero cells are alsoactive in canine cells. Detailed molecular analysis enabled us todemonstrate that CDV-V specifically ablated the nuclear im-port of STAT1 and STAT2 without affecting their activatedphosphorylation states. Furthermore, inhibition of IFN-�/�-dependent signaling correlated with the capacity of the V pro-tein to efficiently interact with both STAT molecules. Finally,we identified both the N-terminal and the C-terminal regionsof V as playing a synergistic role in IFN evasion.

Initial attempts to map the domains of the V protein thatinteract with STAT1 and STAT2 revealed that the N-terminalregion of V was able to function as an autonomous domaininterfering with IFN-�/�-induced signaling. Importantly, co-IPexperiments indicated that VNT retained association withSTAT1 but failed to copurify STAT2. Since the full-length Vprotein (Vwt) efficiently coprecipitated both STAT molecules,this suggests that VNT is very likely responsible for STAT1interaction, whereas VCT is necessary to target STAT2. Inagreement with this notion, the single-amino acid mutant VwtY110D retained slight STAT2 binding but almost completelylost STAT1 interaction. Nevertheless, we cannot exclude thepossibility that VCT, when fused to VNT, determines a specificconformational state of VNT that confers the capacity of theN-terminal region to target both STAT molecules. Recentwork done with MV is consistent with the former hypothesissince the N-terminal domain of MV-V was assigned to STAT1binding, whereas STAT2 has been discovered to be the maintarget of the MV-VCT module (4, 5, 35). The main differencethat we observed in this study between both morbillivirus Vprotein functionalities is that the CDV-VCT domain expressedalone could not disrupt both the IFN-�/�-mediated and theMDA5-mediated signaling pathways, thus suggesting improperfolding and/or protein degradation. Remarkably, when stabi-lized by an irrelevant protein, the VCT domain selectivelysuppressed MDA5-mediated signaling but not signaling in-duced by IFN-�/�. A sequence alignment of the MV- andCDV-VCT domains shows only about 50% amino acid iden-tity, which may explain the different functions (Fig. 13). It maybe possible that VCT, when fused to RFP but not in the wtprotein, adopts a conformational state that remains functionalin inhibiting the MDA5-mediated signaling pathway but losesits intrinsic ability to bind STAT1 and/or STAT2 to suppressthe IFN-�/�-mediated signaling. Alternatively, VCT may foldsimilarly when fused to the RFP than in the wt protein. In thiscase, the CDV-VCT domain may be responsible for (i) dis-rupting the MDA5-mediated signaling pathway and (ii) con-ferring proper folding to the VNT domain, which consequentlywill efficiently engage STAT1 and STAT2 to control IFN-�/�-mediated signaling. Since Vwt elicited enhanced binding avid-ity to STAT1 compared to VNT alone, this indeed suggeststhat VNT’s conformational state is modulated by the presenceof VCT. Taking these observations together, we propose thatCDV-VNT and -VCT are two interdependent modules thatfunction synergistically to allow proper folding of the full-length V protein. In turn, Vwt gains the ability to efficientlyinteract with STAT1 and STAT2, which offers optimal con-

FIG. 11. Viruses lacking V expression showed enhanced sensitivityto IFN-�/� treatment. Vero-SLAM cells were infected with the differ-ent rCDVs at an MOI of 0.02. Then, IFN-�/� was added (or not) at 3,12, 24, and 36 h postinfection. Virus titers of cell-associated viruseswere determined by limiting dilution assay at 48 h postinfection.

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ditions to prevent nuclear import of the two STAT mole-cules and, consequently, to control IFN-�/�-mediated sig-naling. These results contrast with those obtained with thehenipavirus family, where the C-terminal region of V was to-tally dispensable for STAT binding and IFN evasion (38, 40).The requirement of both the N- and C-terminal domains of V

to evade innate immunity is consistent with the studies of therubulavirus family, though in that case both domains of V werenecessary to mediate STAT1 (9, 19, 22, 30, 49), STAT2 (17, 18,26, 29, 30, 50), or STAT3 (44) proteasomal degradation ratherthan STAT nuclear import inhibition without affecting theirphosphorylation states.

FIG. 12. STAT1 nuclear import is suppressed in MDCK-SLAM cells after treatment with canine interferon-� (cIFN-�). (A) Immunofluores-cence images of MDCK-SLAM cells stained for STAT1. MDCK-SLAM cells were infected with the rA75/17red and rA75/17red Vko viruses or leftuninfected. One day postinfection, cells were treated (or left untreated) for 30 min with IFN-�/� and subsequently fixed, permeabilized, and stainedfor STAT1 localization using an anti-STAT1 antibody. Then, an Alexa Fluor 488-conjugated secondary antibody was employed (green), and imageswere captured with a scanning confocal laser microscope (Olympus). The nuclei were counterstained with TOTO3 (blue). Infected cells werelocalized by the expression of RFP. (B) Close-up view of the nuclear STAT1 translocation in rA75/17red Vko-infected cells. Staining is identical tothat described in panel A. (C) Titration experiments in the presence or absence of canine IFN-� in rA75/17red- and rA75/17red Vko-infected cells.MDCK-SLAM cells were infected with both rCDVs at an MOI of 0.02. IFN-�/� was added (or not) at 9 h postinfection, and virus titers ofcell-associated viruses were determined by limiting dilution assay 48 h postinfection in Vero-SLAM cells.

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Results obtained in ISRE luciferase reporter gene assaysclearly demonstrated the capacity of both VNT and VwtY110D to interfere with IFN activity although to a limitedextent compared to Vwt. Remarkably, immunofluorescenceanalysis of STAT1 and STAT2 intracellular localizations in thepresence of IFN and in the presence of these two V mutantsrevealed an unanticipated reverse localization of both tran-scription factors. Indeed, while VNT specifically disruptedSTAT1 (but not STAT2) nuclear import, Vwt Y110D impairedSTAT2 (but not STAT1) nuclear localization. These resultsseem to be in contradiction with the current model of theIFN-�/�-induced signaling pathway, which states that phos-phorylated STAT1 and STAT2 dimerize upon IFN treatmentprior to being translocated to the nucleus. We therefore hy-pothesize that the association of VNT with STAT1 and theassociation of Vwt Y110D with STAT2 compete for the dimer-ization of phosphorylated STAT1 and STAT2. Subsequently,in the presence of VNT and IFN, phosphorylated STAT1 ac-cumulates in the cytoplasm, whereas phosphorylated STAT2would translocate and accumulate in the nucleus. Conversely,in the presence of Vwt Y110D, which exhibits STAT1 bindingimpairment but efficient STAT2 association, the reverse pheno-type for STAT cellular localization is observed. This hypothesis isin excellent agreement with our results demonstrating thatCDV-V does not affect STAT1 and STAT2 phosphorylation, butfurther experiments are required to consolidate this model.

In addition to V, the CDV-P protein (sharing the identicalN-terminal region with V) was found to retain weak interac-tion with STAT1, which correlated with partial suppression ofIFN-�/�-mediated signaling. The biological relevance of thislimited interaction with STAT1 was supported by the fact thatalthough the growth of all rCDVs was reduced, they were notcompletely inhibited in the presence of IFN. This effect of theCDV-P is consistent with observations made with P proteins ofother negative-strand RNA viruses, e.g., MV, RPV, Nipahvirus, and rabies virus (8, 10, 25, 46). Nevertheless, our resultsobtained in the co-IP assay indicated that P bound STAT1rather inefficiently, suggesting that a high concentration of Pmay be required for effective inhibition of IFN signaling. Sup-porting this notion, we noticed that at late time points afterinitial infection (48 h postinfection) all knockout viruses wereultimately able to disrupt nuclear import of STATs (data notshown). It is noteworthy that a differential effect of MV-P inIFN evasion was recently documented. Indeed, results suggestedthat the origin of the virus strain determined the extent of the Pfunctionality (11). Thus, further investigations are required todemonstrate whether the extent by which the CDV-P proteincounteracts the IFN-�/� response is also regulated by the originof the strain and/or by the host cell environment.

The mechanism by which the A75/17 CDV-V protein inhib-

its the IFN-�/�-mediated response differs from that of othermorbilliviruses. To our knowledge, all studies performed withMV-V, with the exception of that by Palosaari et al. (28), havereported an inhibition of the phosphorylation of STAT1 (5, 42,48) and STAT2 (4, 8, 42). The reasons for the differencesbetween CDV and MV remain unclear. In addition to genuinebiological differences between these two morbilliviruses, theorigin and passaging histories of the strains used to studyevasion from IFN action may be a factor. Indeed, we studied ahighly virulent viral strain not adapted to cultured cells,whereas the strain of MV was attenuated (8) or persistentlyinfected cells were investigated (48). There are also similari-ties, however, between CDV and other morbilliviruses. Con-sistent with the findings in CDV, MV-V was shown to be morepotent in inhibiting IFN-�/� signaling than the C protein, andthis observation was equally true for all strains independent oftheir virulence (11). Similarly, using recombinant knockoutviruses based on a vaccine strain, RPV-V was shown to blockIFN-mediated phosphorylation of STAT1 and STAT2 withoutcausing the degradation of these proteins (25).

Taken together, our results shed light on a unique molecularmechanism by which a highly virulent CDV strain interfereswith IFN action by disrupting signaling for the synthesis ofantiviral proteins. While the mechanism of CDV virulence islikely to be complex and may involve different host cells andinteractions between cells, disruption of the IFN defense maydeprive the host of an early mechanism known to limit viralreplication and spread at a critical stage of infection. Under-standing the precise mechanisms of IFN evasion may also leadto the rational design of vaccines that combine optimally bal-anced stimulation of the innate immune system, which isknown to be essential for activation of an effective adaptiveimmune response (27).

ACKNOWLEDGMENTS

We thank D. Garcin for offering the red fluorescent marker protein,the luciferase plasmids pISRE-Luc, p�-IFN-fl-lucter, and pTK-RL,and the NES-GFP-SV5-V and FLAG-tagged MDA5-expressing plas-mids, and we thank V. von Messling for providing the Vero-SLAMcells. We are grateful to Ruth Parham for linguistic improvement ofthe manuscript.

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FIG. 13. Sequence alignment of amino acids 233 to 300 (VCT domain) in the carboxyl-terminal segment of V proteins of two wild-type CDVstrains and two MV strains. GenBank numbers for each virus sequence are as follows: AB016162.1 (measles, ICB strain), AB254456.1 (measles,subacute sclerosing panencephalitis strain Kobe-1), AY386316.1 (canine distemper, A75/17 strain), BAA01203.1 (canine distemper, 5804P strain).Gray boxes represent identical residues in all four sequences.

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ACKNOWLEDGEMENTS I would like to express a genuine gratitude to my supervisor, Dr. Philippe Plattet, for

his support, patience, flexibility and friendliness during the entire PhD work.

I am sincerely grateful to my supervisor, Prof. Dr. Andreas Zurbriggen, for the

opportunity to accomplish my PhD thesis in his research group, for his kind support

and his excellent scientific advices

I would like to thank my co-referee, Prof. Dr. Ernst Peterhans for his great scientific

propositions and for evaluating my PhD work.

I would like to thank my mentor, Prof. Dr. Peter Bütikofer, for the involvement in my

scientific development.

I would like to thank Prof. Dr. Marc Vandevelde for his good scientific suggestions and

for his instructional, congenial neurological teaching.

Many thanks to Ljerka Zipperle for giving me technical help, for a day-to-day support

and an enjoyable, friendly atmosphere.

Also my thanks go to all the people of my department, for being always friendly and

making fun.

Special thank to Anne Röthlisberger for the nice and enjoyable co-work and for being

my friend.

My deepest thank to my mother and father, for always having time for me and their

understanding and caring comportment. Without you, I would never have

succeeded.

Thanks to all my friends and family for your support and an enjoyable and amusing

distraction every once a while.

ACKNOWLEDGEMENTS

CURRICULUM VITAE First Name Dominique

Surname Wiener

Date of birth 02.08.1977

Native place Stallikon (ZH)

Education

3.1.2007 – currently PhD student

Department for Clinical Research and Veterinary

Public Health

Vetsuisse Faculty

University of Bern, Switzerland

Thesis title: “Investigation of two potential

mechanisms which may favor persistence of CDV,

the driving force behind the chronic progression of

demyelination in canine distemper”

1.7.2005 – currently Residency/PhD program in animal pathology

Vetsuisse Faculty

University of Bern, Switzerland

1.2.2004 – 31.6.2005 Doctoral thesis

Department for Clinical Research and Veterinary

Public Health

Vetsuisse Faculty

University of Bern, Switzerland

Thesis title: “Effect of structural proteins derived

from cytolytic and persistent canine distemper virus

strains on cell-cell fusion in co-transfection studies”

15.2.1998 – 5.12.2003 Academic study of veterinary medicine

University of Bern

92

CURRICULUM VITAE

LIST OF PUBLICATIONS

Röthlisberger A., Wiener D., Schweizer M., Peterhans E., Zurbriggen A., Plattet P.

(2010) Two domains of the V protein of virulent canine distemper virus selectively

inhibits STAT1 and STAT2 nuclear import, J. Virol. Apr 28. [Epub ahead of print]

Brachelente C., Wiener D., Malik Y., Huessy D. (2007) A case of necrotizing fasciitis

with septic shock in a cat caused by Acinetobacter baumannii. Vet Dermatol.

18(6):432-8.

Pfister P., Geissbuehler U., Wiener D., Hirsbrunner G., Kaufmann C. (2007)

Pollakisuria in a dwarf goat due to pathologic enlargement of the uterus. Vet Q.

29(3):112-6.

Wiener D., Plattet P., Cherpillod P., Zipperle L., Doherr M.G., Vandevelde M.,

Zurbriggen A. (2007) Synergistic inhibition in cell-cell fusion mediated by the matrix

and nucleocapsid protein of canine distemper virus. Virus Res. 129(2):145-154.

Plattet P., Cherpillod P., Wiener D., Zipperle L., Vandevelde M., Wittek R., Zurbriggen

A. (2007) Signal peptide and helical bundle domains of virulent canine distemper virus

fusion protein restrict fusogenicity. J Virol. 81(20):11413-11425.

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LIST OF PUBLICATIONS

Declaration of Originality Last name, first name: Wiener Dominique Matriculation number: 97-119-499 I hereby declare that this thesis represents my original work and that I have used no

other sources except as noted by citations.

All data, tables, figures and text citations which have been reproduced from any other

source, including the internet, have been explicitly acknowledged as such.

I am aware that in case of non-compliance, the Senate is entitled to divest me of the

doctorate degree awarded to me on the basis of the present thesis, in accordance with

the “Statut der Universität Bern (Universitätsstatut; UniSt)”, Art. 20, of

17 December 1997.

Place, date Signature

……………………………………… …………………………………………………

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