Cellular receptors for viruses with ocular tropism
Transcript of Cellular receptors for viruses with ocular tropism
Cellular receptors for viruses with ocular tropism
Emma Nilsson
Department of Clinical Microbiology Umeå University 2011
Copyright © Emma Nilsson ISBN: 978-91-7459-182-8 ISSN: 0346-6612-1414 Front cover: Sonja Nordström, Print & Media. Picture of Ad37 fk in complex with GD1a, kindely provided by Johannes Bauer, University of Tübingen, Germany. Printed by: Print & Media, Umeå University Umeå, Sweden 2011
To my loved ones
Table of Contents
Abstract 7 Summary in Swedish–Populärvetenskaplig sammanfattning på svenska 9 List of papers 12 Abbreviations 13 Aims of the thesis 15 Introduction 16
History 16 Taxonomy and clinical/pathological aspects 17
Adenovirus 17 Picornavirus 20
Anatomy of the anterior segment of the eye 23 Description of EKC and AHC 25 Viral structure 28
Adenoviruses 28 Major capsid proteins 29 Minor capsid proteins 31 The adenovirus core 33 Non-structural proteins 34
Picornaviruses 35 Structural proteins 35 Non-structural proteins 36
Viral life cycle 38 Receptors 38
Immunoglobulin superfamily (IgSF) 41 Coxsackievirus and adenovirus receptor (CAR) 41 Intercellular Adhesion Molecule-1 (ICAM1) 42 Poliovirus receptor (PVR or CD155) 42
Regulators of complement activation (RCA) family 43 Membrane Cofactor Protein (MCP/CD46) 43 Decay Accelerating Factor (DAF or CD55) 44
Sialic acid 44 Gangliosides 45 Heparan sulfate proteoglycans (HSPGs) 46 Desmoglein-2 46 Integrins 47 Other receptors 47 Soluble components 49
The life cycle after binding to cells; adenoviruses 50 Internalization and trafficking 50 Structure of the genome 51
Gene expression and replication 52 Assembly and release 55
The life cycle after binding to cells; picornaviruses 56 Internalization and trafficking 56 Structure of the genome 58 Translation and replication 59 Assembly and release 59
Antivirals 61 Adenoviruses 61 Picornaviruses 64
Results and Discussion 66 Paper I 66 Paper II 69 Paper III 71 Paper IV 75
Concluding remarks 79 Acknowledgements 81 References 84
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Abstract
Several viruses from different virus families are known to cause ocular
infections, e.g. members of the Adenoviridae, Picornaviridae and the
Herpesviridae families. These infections are spread by contact and in the
case of adenoviruses (Ads) and picornaviruses they are also highly
contagious. The ocular infections caused by Ads and picornaviruses are
called epidemic keratoconjunctivitis (EKC) and acute hemorrhagic
conjunctivitis (AHC), respectively. Historically, EKC is caused mainly by
three types of Ads from species D: Ad8, Ad19 and Ad37. The infection is
characterized by keratitis and conjunctivitis but also involves pain, edema,
lacrimation and blurred vision. AHC is caused mainly by two types of
picornaviruses: coxsackievirus A24v (CVA24v) and enterovirus 70 (EV70),
and is characterized by pain, redness, excessive tearing, swelling and
subconjunctival hemorrhages. In addition, blurred vision, keratitis, malaise,
myalgia, fever, headache and upper respiratory tract symptoms can also be
experienced.
Both infections are problematic in many parts of the world, affecting millions
of people every year. Ads are responsible for appriximately of 20-40 million
cases of EKC every year, and the number of AHC-cases until 2002 have been
estimated to be up to 100 million. EV70 and CVA24v are also known to cause
pandemics and so far, three pandemics have occured. Despite the great need,
the only treatment available today is supportive treatment; no antiviral drugs
are available to combat these common viral infections.
Ad37 has previously been reported to use sialic acid (SA) as its cellular
receptor. Since there is no antiviral treatment available against EKC we
wanted to evaluate the inhibitory effect of SA-based antiviral compounds on
Ad37 binding to and infection of ocular cells. We found that multivalent
compounds consisting of SA linked to a carrier molecule, in this case human
serum albumin, efficiently blocked Ad37 binding and infection at low
concentrations. Further attempts were then made to improve the effect by
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chemically modifying SA monosaccharides. However, no enhanced
inhibitory effect was accomplished and the conclusion was that the best
inhibitors are based on unmodified SA. We next hypothesized that
development of efficient SA-based binding inhibitors may require detailed
knowledge about the structure of the SA-containing receptor. Using a battery
of biological and biochemical experiments, including glycan array, binding
and infection assays, X-ray crystallography and surface plasmon resonance
(SPR) we identified a specific glycan involved in the binding and infection of
Ad37. This glycan turned out to be a branched, di-SA-containing motif
corresponding to the glycan motif of the ganglioside GD1a. However, the
ganglioside itself did not function as a cellular receptor, as shown by a
number of binding and infection assays. Instead, the receptor consisted of
one or more glycoproteins that contain the GD1a glycan motif. This glycan
docked with both its SAs into the trimeric Ad37 knob resulting in a very
strong interaction as compared to most other protein-glycan interactions.
Hopefully, this finding will aid development of more potent inhibitors of
Ad37 binding and infection.
The receptor for CVA24v, one of the main causative agents of AHC, has been
unknown until now. We showed that this ocular virus, like Ad37, is also able
to use SA as a receptor on corneal cells but not on conjunctival cells. This
suggested that CVA24v may use two different receptors. As for Ad37, the
receptor used by CVA24v on corneal cells also appears to be one or more
sialic acid-containing glycoproteins. We believe that these findings may be a
starting point for design and development of candidate drugs for topical
treatment of AHC.
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Summary in Swedish–
Populärvetenskaplig sammanfattning på
svenska
Virusinfektioner i ögat är ett stort problem i många delar av världen,
speciellt i tätbefolkade områden. Varje år drabbas miljontals människor av
dessa åkommor vilket i sin tur leder till stora samhällsekonomiska förluster.
Ett antal virus från flera olika virusfamiljer har förmågan att infektera ögat,
t.ex. medlemmar av adenovirus-, picornavirus- och herpesvirusfamiljerna.
Från adenovirusfamiljen är det främst tre typer: 8, 19 och 37, som orsakar
den allvarliga ögoninfektionen epidemisk keratokonjuktivit (EKC). Denna
infektion karaktäriseras av inflammation i horn- och bindhinna men
inkluderar även rinnande, svullna ögon, smärta, irritation och blödningar.
Inflammationen i hornhinnan leder i många fall till synnedsättning.
Infektionen går normalt sett över inom ett par veckor, men i vissa fall kan
synnedsättningen dröja kvar i veckor eller månader. Även permanent
synnedsättning har rapporterats.
Akut hemorrhagisk konjunktivit (AHC) orsakas framförallt av två
medlemmar av picornavirusfamiljen: enterovirus 70 (EV70) och
coxsackievirus A24 variant (CVA24v). Denna infektion karaktäriseras av
inflammation i bindhinnan, rinnande, svullna ögon, smärta, irritation och
blödningar. Man kan även drabbas av hornhinneinflammation, feber,
huvudvärk och övre luftvägs-symptom. Dessa två virus, EV70 och CVA24v,
orsakar epidemiska utbrott av AHC men även pandemier. Sedan upptäckten
av EV70 och CVA24v 1969 så har dessa virus orsakat sammanlagt tre
pandemier och under dessa pandemier har mer än 100 miljoner människor
drabbats. I dagsläget finns ingen medicinsk behandling för ögoninfektioner
som orsakats av adeno- eller picornavirus.
Kolhydraten sialinsyra har tidigare identifierats som den struktur på cellytan
som adenovirus typ 37 (Ad37) binder till. Eftersom det inte finns någon
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antiviral behandling av EKC så var målet med den första studien att
utvärdera den hämmande effekten av sialinsyre-baserade molekyler vid
bindnings och infektions experiment med Ad37. I denna studie så kopplades
sialinsyra till en bärarmolekyl och detta resulterade i en multivalent molekyl
(en molekyl med ett flertal sialinsyror kopplade till bäraren). Resultaten från
denna studie visade tydligt att dessa molekyler är väl fungerande hämmare
av Ad37 orsakade ögoninfektioner in vitro. Som en fortsättning på denna
inledande studie så gjordes försök att genom små förändringar av sialinsyran
förbättra den hämmande effekten. Ett litet bibliotek med tio stycken
varianter av sialinsyra togs fram och utvärderades. Utifrån dessa försök
kunde tydligt utläsas att den hämmande effekten hos de modifierade
sialinsyre-varianterna inte uppnådde samma nivå som den ursprungliga
sialinsyran och därmed drogs slutsatsen att ändringar i den ursprungliga
strukturen inte bidrog till någon ökad effekt. När de sedan kopplades till en
bärarmolekyl så visade det sig att de inte var lika bra hämmare som den
ursprungliga föreningen där sialinsyra kopplats till en bärare.
Tidigare har man visat att även EV70 binder till sialinsyra på utsidan av
ögonceller. Målet med den tredje studien var således att undersöka om så var
fallet även för CVA24v, vars cellulära receptor dittills var helt okänd. Genom
att använda ett antal olika metoder kunde det fastställas att sialinsyra
fungerade som receptor även för detta virus på vissa celler, men det verkar
även finnas ytterligare minst en receptor (på andra celler) som än så länge
inte har identifierats.
Som nämnts ovan så använder Ad37 sialinsyra som sin cellulära receptor och
i den fjärde studien var målet att försöka få fram en mer detaljerad bild av
hur den sialinsyre-innehållande receptorn ser ut. Genom att studera
bindningen av Ad37 till ett stort antal olika sockerstrukturer så
identifierades en specifik sialinsyre-innehållande sockerstruktur som Ad37
band bra till. Denna struktur innehöll två sialinsyror som genererar en
relativt stark bindning till viruset. Den kunskap som erhållits i denna studie
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kan förhoppningsvis bidra till att bättre antiviraler kan tas fram, kanske
baserade på en liknande struktur.
Sammanfattningsvis har jag identifierat kolhydraten sialinsyra som receptor
för ytterligare ett ögonvirus (CVA24v) och i samarbete med andra, även
arbetat fram en mer detaljerad bild av den struktur på cellytan som Ad37
binder till. Jag har också analyserat effekten av sialinsyre-baserade
molekyler, vilket i sin tur kan leda till utveckling av bättre antiviraler för
behandlingen av virus-orsakade ögoninfektioner.
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List of papers
I Multivalent sialic acid conjugates inhibit adenovirus
type 37 from binding to and infecting human corneal cells
Johansson SM*, Nilsson EC*, Elofsson M, Ahlskog N, Kihlberg J, Arnberg N.
Antiviral Res. 2007. 73:92-100.
II Design, synthesis, and evaluation of N-acyl modified sialic acids as inhibitors of adenoviruses causing epidemic keratoconjunctivitis.
Johansson S, Nilsson E, Qian W, Guilligay D, Crepin T, Cusack S, Arnberg N, Elofsson M. J Med Chem. 2009. 52:3666-78.
III Sialic acid is a cellular receptor for coxsackievirus A24 variant, an emerging virus with pandemic potential.
Nilsson EC, Jamshidi F, Johansson SM, Oberste MS, Arnberg N. J Virol. 2008. 82:3061-8.
IV The GD1a glycan is a cellular receptor for adenovirus that cause epidemic keratoconjunctivitis
Emma C. Nilsson, Rickard Storm, Johannes Bauer, Susanne M. C. Johansson, Aivar Lookene, Jonas Ångström,
Mattias Hedenström, Lars Frängsmyr, Hugh Willison, Fatima Pedrosa Domelöf, Thilo Stehle and Niklas Arnberg.
Nat Med. 2011. 17:105-9
Paper I-IV have been reprinted with permission from the publishers.
*Contributed equally to this work.
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Abbreviations
aa Amino acid Ad Adenovirus ADP Adenovirus death protein AHC Acute hemorrhagic conjunctivitis AIDS Acquired immunodeficiency syndrome ARD Acute respiratory disease CAR Coxsackievirus and adenovirus receptor CMV Cytomegalovirus CNS Central nervous system CsA Cyclosporine A CVA Coxsackievirus A CVB Coxsackievirus B DAF Decay accelerating factor DBP DNA binding protein DNA Deoxyribonucleic acid DSG-2 Desmoglein-2 ECM Extracellular matrix EKC Epidemic keratoconjunctivitis ER Endoplasmatic reticulum EV Enterovirus fk Fiber knob FMDV Foot and mouth disease virus GAGs Glucosaminoglycans GONs Groups of nine GRP78 Glucose-regulated protein 78 kDa HAVCR Hepatitis A virus cellular receptor HFMD Hand foot and mouth disease HIV Human immunodeficiency virus HRV Human rhinovirus HS Heparan sulfate HSPGs Heparan sulfate proteoglycans HVR Hypervariable region ICAM-1 Intercellular adhesion molecule-1 IFN Interferon IGDD Isoleucine-Glycine-Aspartic acid-Aspartic acid IgSF Immunoglobulin superfamily IRES Internal ribosome entry site ITR Inverted terminal repeat kDa Kilo dalton LDLR Low density lipoprotein receptor Lf Lactoferrin LFA-1 Leukocyte function-associated antigen-1 MCP Membrane cofactor protein MDa Mega dalton MHC-1 Major histocompatibility complex-1
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mRNA Messenger ribonucleic acid Mw Molecular weight NCT N-chlorotaurine NES Nuclear export signal NLS Nuclear localization signal NPC Nulear pore complex NRTI Nucleoside reverse transcriptase inhibitor Pb Penton base PCF Pharyngoconjunctival fever PI3K Phosphatidylinositol-3-OH kinase PKR Protein kinase receptor PSGL-1 P-selectin glycoprotein ligand-1 PV Poliovirus PVP-I Povidone-iodine PVR Poliovirus receptor RCA Regulators of complement activation RdRP RNA-dependent RNA polymerase RGAD Arginine-Glycine-Alanine-Aspartic acid RGD Arginine-Glycine-Aspartic acid RISC RNA-induced silencing complex RNA Ribonucleic acid RSV Respiratory syncytium-forming virus SA Sialic acid SCARB2 Scavenger receptor B2 SCR Short consensus repeat siRNA Small interfering RNA STP Serine-threonine-proline SV40 Simian virus 40 TP Terminal protein UTR Untranslated region VA-RNA Virus-associated RNA VLA-2 Very late activation antigen-2 VLDLR Very low density lipoprotein receptor VPg Virus protein genome linked
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Aims of the thesis
Overall aim
The overall aim of this thesis was to identify and characterize the
interactions between ocular viruses and their cellular receptors.
Specific aims
Aim 1: Evaluate the inhibitory effect of multivalent sialic acid-containing
compounds on Ad37 binding and infection of ocular cells and to characterize
the mechanism behind this inhibitory effect.
Aim 2: Increase the inhibitory effect previously observed with the
multivalent compound by structural modification of sialic acid. If the
binding between sialic acid and Ad37 could be increased then this would lead
to a more potent antiviral compound.
Aim 3: Identify the so far unknown cellular receptor(s) for CVA24v.
Aim 4: Further characterize the cellular receptor for Ad37 with focus on the
structure of the sialic acid-containing motif.
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Introduction
History
The first isolation of adenoviruses (Ads) was achieved in 1953 by Rowe et al.
when searching for agents of acute respiratory infections in adenoid tissue
[1]. Almost at the same time Hilleman and Werner identified an agent in
respiratory samples from military recruits suffering from respiratory disease
[2]. The first clinical description matching the clinical features of epidemic
keratoconjunctivitis (EKC) was recognized already in 1889 in Austria and it
was named keratitis punctuate superficialis [3-7]. The following years there
were case reports of EKC from United Kingdom, Japan and the US [8-10]. In
1901 the first report regarding epidemic disease in Bombay, India was
published [11]. At this time the epidemics were concentrated to the Indian
subcontinent but in the 1930:s there was an epidemic outbreak in the US
[12]. In 1941, there was a large outbreak in the marine shipyards of Pearl
Harbor involving more than 10,000 individuals [13]. This coincided with a
number of EKC outbreaks in shipyards along the west coast in the US, this is
why the disease was named “shipyard eye” [14, 15]. Even though a filterable
agent was identified at that time it took until 1955 to isolate the first EKC-
causing Ad, which was Ad type 8 [16]. Many contributions to our
understanding of viral and cellular gene expression and regulation, DNA
replication, cell cycle control and cellular growth regulations have been made
by studying Ad-infected cells. Probably, the biggest contribution to modern
biology from the Ad research field was the discovery of messenger RNA
(mRNA) splicing by Sharp and Roberts in 1993. For this discovery they were
awarded the Nobel Prize in physiology/medicine.
The picornavirus family is one of the “oldest” known virus families. The first
known animal virus discovered was the foot-and-mouth disease virus
(FMDV) in 1898 and only 10 years later the well-known poliovirus was
discovered [17, 18]. Picornaviruses have also, like Ads, played an important
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role in the development of modern virology. Some of the advances where
picornaviruses have contributed are: the discovery of the first RNA-
dependent RNA polymerase, the demonstration of proteolytic processing of
a polyprotein, and the propagation of virus in cell culture (viral replication)
[19-21]. The more recent advances made by picornavirus researchers are e.g.
infectious DNA clones and three-dimensional structures by X-ray
crystallography [22-24]. In June 1969 a new type of picornavirus-caused
conjunctivitis was reported and it was first named epidemic hemorrhagic
conjunctivitis or “Apollo 11 disease” because it coincided with the moon
landing of the Apollo 11 spaceship. Later it was re-named to acute
hemorrhagic conjunctivitis (AHC) [25, 26].
Taxonomy and clinical/pathological aspects
Adenovirus
Taxonomy
The family of Adenoviridae contains five different genera according to the
International Committee of Taxonomy of Viruses (ICTV): Mastadenovirus
isolated from mammals, aviadenovirus isolated from birds, atadenovirus
isolated from reptiles, birds, mammals and a marsupial, siadenovirus
isolated from reptiles and birds, and the newest genus ichtadenovirus
isolated from fish. The genus of mastadenovirus contains all the 51 human
serotypes known today and they are further divided into species A-F. A
number of new Ad types have been suggested: Ad53 and 54 that cause EKC
will probably be classified as species D types and also Ad52 (species G), 55
and 56 have been suggested [27-31]. However, these have not been
characterized with classical serology. The classification of new types is
however under debate (manuscripts in press in J. Virol: Aoki et al. and Seto
et al.)
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Historically, the classification of Ads is based on the ability to agglutinate red
blood cells, oncogenicity in rodents, DNA homology and tropism [32]. The
classification of different serotypes is based on the resistance to
neutralization by antisera of already known types [33]. The types are then
separated into different species based on lack of cross-neutralization and a
phylogenetic distance of more than 10%. If the phylogenetic distance is less
than 5%, some of the other grouping criteria can be used to place the types in
the same species even if they are isolated from different hosts [34]. To be
identified as a new serotype the cross-reaction with previously known types
must yield a titer greater than 16 [35].
Clinical and pathological aspects
Ads are common pathogens that infect a wide range of hosts in a highly
species specific manner [36]. In many cases, Ad infections are asymptomatic
but when disease occurs it is usually in the form of respiratory, ocular or
gastrointestinal disease (see table 2) [37]. Infections of the urinary tract,
lymphoid tissue, liver and other organs have also been reported. Only about
half of the known Ad types are linked to illness. Respiratory disease in
children is commonly caused by Ads from species C: Ad1, 2, 5 and 6,
sometimes also Ad 3 and 7 from species B. In the adult population it is the
other way around, Ad1, 2, 5 and 6 rarely cause disease but Ad 3, 4 and 7 do.
Acute respiratory disease (ARD) has been a problem amongst the military
recruits in the US. This population is probably more susceptible due to the
crowded sleeping situation and the constant exhaustion that they experience.
Historically, ARD is mainly caused by Ad 4 and 7, sometimes also Ad3 and
more recently Ad14. The Ad-caused respiratory infections can be
accompanied by ocular infections as well and the disease is then named
pharyngoconjunctival fever (PCF). The most common PCF-causing Ads are
Ad3 and 7. A more severe ocular infection called epidemic
keratoconjunctivitis (EKC) is caused by Ad8, 19 and 37 from species D and
also the new types Ad53 and 54. Since Ads are known to replicate in the
intestine and regularly are isolated from stool they were believed to cause
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diarrhea, but studies often showed the same amount of virus in the controls
suggesting asymptomatic replication in the intestine [38]. Only the two Ads
from species F, Ad40 and 41, are known to cause gastroenteritis to any larger
extent, especially among young children [39].
Even though Ad infections are usually mild in the immunocompetent host
they are severe and sometimes lethal in immunocompromized patients. The
problem is increasing with the raising numbers of transplant recipients and
AIDS patients. The first descriptions of fatal outcome of disseminated Ad
infection were reported already in the 1960s [40]. Ad infections in the
immunocompromized hosts can present very high fatality rates [41]. In
many of the immunodeficient patients, Ads from species A, B and C are the
most common. Ad31 from species A has been shown to be a main contributor
to fatal disease [41].
Table 2. Classification and tropism of human adenoviruses.
Species Type Tropism
A 12, 18, 31 Intestine
B:1
B:2
3, 7, 16, 21
11, 14, 34, 35, 50
Respiratory tract, eye
Respiratory tract, urinary tract, eye
C 1, 2, 5, 6 Respiratory tract
D 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, 42-49, 51
Eye, intestine
E 4 Respiratory tract, eye
F 40, 41 Intestine
G 52 Intestine
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Picornavirus
Taxonomy
The picornavirus family is divided into 12 different genera according to the
International Committee of Taxonomy of Viruses (ICTV; table 3):
These genera are then further divided into different species, and the species
are divided into types. The genus of enterovirus contain ten different species,
seven of them contain human types (see table 3). The first classification of
types was based on type of disease in humans and in newborn mice e.g.
coxsackievirus A (CVA) caused acute flaccid paralysis in newborn mice and
coxsackievirus B (CVB) caused spastic paralysis in newborn mice. Problems
with this classification method lead to development of other criteria like:
immunological response in humans, receptor usage and protection from
disease. This type of classification also lead to problems since there was
cross-reactivity between types and there was also antigenic variation within
types [42]. Serological methods are still used for identification and
classification, but thanks to the development of molecular methods it is
easier to compare sequences. Criteria to fulfill to be classified within a
species in the enterovirus genus are based on molecular methods [43]:
Share greater than 70% aa identity in P1 (domain encoding the
capsid proteins)
Share greater than 70% identity in the non-structural proteins 2C
(ATPase)+3CD (proteinase)
Share a limited range of host receptors and a limited natural host
range
Have a genome base composition (G+C) which varies by no more
than 2.5%
Share a significant degree of compatibility in proteolytic
processing, replication, encapsidation and genetic recombination.
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Table 3. Classification and tropism of human enteroviruses.
Species Types Tropism
HEV-A Coxsackievirus A 2-8, 10, 12, 14, 16
Enterovirus 71, 76, 89, 90-92
CNS, skin and/or airways
Skin and/or GI tract
HEV-B Coxsackievirus B 1-6
Coxsackievirus A 9, 23
Enterovirus 69, 73-75, 77-88, 93, 97, 98, 100, 101, 106, 107
Echovirus 1-9, 11-21, 24-27, 29-33
Airways, heart, pancreas and/or CNS
CNS, skin and/or airways
Airways, skin and/or GI tract
Airways, GI tract and/or CNS
HEV-C Poliovirus 1-3
Coxsackievirus A 1, 11, 13, 17, 19-22, 24, 24v
Enterovirus 95, 96, 99, 102, 104, 105, 109
CNS
CNS, skin, airways and/or eye
Airways, skin and/or GI tract
HEV-D Enterovirus 68*, 70, 94 Eye, airways and/or CNS
HRV-A Human rhinovirus 1, 2, 7-13, 15, 16, 18-25, 28-34, 36, 38-41, 43-47, 49-51, 53-68, 71, 73-78, 80-82, 85, 88-90, 94- 96, 98, 100
Airways
HRV-B Human rhinovirus 3-6, 14, 17, 26, 27, 35, 37, 42, 48, 52, 69, 70, 72, 79, 83, 84, 86, 91-93, 97, 99
Airways
HRV-C Serotypes not yet assigned Airways
*Previously identified as human rhinovirus 87 (HRV87).
Clinical and pathological aspects
Enterovirus (EV) infections are many times asymptomatic but a number of
different symptoms are seen in the cases where they cause disease [44]. The
diseases caused by enteroviruses includes severe symptoms such as
poliomyelitis, meningitis, acute flaccid paralysis and fatal paralytic
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poliomyelitis, and less severe symptoms such as ”common cold”, diarrhea,
myalgia, conjunctivitis and skin rashes (see Table 3).
Poliovirus (PV) infections are in many cases asymptomatic but when
symptoms occur it is usually in the form of a disease called abortive
poliomyelitis. This is a mild febrile illness that sometimes includes
gastrointestinal symptoms. About one of 200 symptomatic infections results
in the severe paralytic disease known as poliomyelitis [44]. Other
enteroviruses are also able to cause poliomyelitis e.g. EV70 and EV71 [45,
46]. Since the eradication period for polio started, EV71 has emerged as the
major neurotropic EV. EV70, the major causative agent of acute hemorrhagic
conjunctivitis (AHC) has also been shown to cause paralytic disease.
Aseptic meningitis is frequently caused by EVs as seen in the US were up to
95% of the cases are caused by EVs [47]. Typical symptoms of aseptic
meningitis are fever, headache and meningeal signs that usually resolve
within a week. The symptoms can be more severe and also lead to death,
especially in neonates [48]. The EVs that are overrepresented as causative
agents of aseptic meningitis are PV, EV70, 71, echovirus 7, 9, 11, 30 and
CVB5. However, other types can also cause this disease [49].
Some of the EVs, in particular CVBs, have been associated with acute cardiac
disease. A variety of different studies have detected EVs in heart tissue by
immune assays, blotting and RT-PCR [50-52]. A link has also been suggested
between EVs and dilated cardiomyopathy [50].
Respiratory disease is commonly caused by EVs, in particular by human
rhinoviruses (HRV). These viruses usually infect the upper respiratory tract
leading to “common cold”, but they also contribute to chronic respiratory
diseases of the lower respiratory tract [53].
Acute hemorrhagic conjunctivitis (AHC) is a severe epidemic and sometimes
pandemic ocular infection mainly caused by EV70 and coxsackievirus A24
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variant (CVA24v) [54]. These viruses can also cause respiratory illness,
gastrointestinal and neurological dysfunctions [44].
Herpangina and hand-foot-and-mouth disease (HFMD) are common EV
infections among children [44]. Herpangina is characterized by lesions of the
palate, tonsils and pharynx that usually disappear within a few days. The
major causative agents of herpangina are: echoviruses, members of the CVAs
and CVBs and also EV71 [55]. HFMD is characterized by blisters of the
hands, feet and mouth and is caused mainly by CVA16 and EV71 [56].
Anatomy of the anterior segment of the eye
The sclera, “the white part of the eye”, is a fibrous tissue that protects the
eye. The tissue consists of collagen bundles and elastic fibers. In the front
part of the eye, the sclera proceeds into the cornea. The cornea is the clear
outermost tissue of the eye. The cornea contains no blood vessels and
receives its nourishment and protection from tears and the fluid in the
anterior chamber behind it [57]. The corneal tissue is divided into five
different layers (see figure 1):
Figure 1. (a) Anatomy of the anterior segment of the human eye. (b) Cross-
section of the human cornea showing the five different layers. Reprinted
from [58] with permission from the publisher.
(a) (b)
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Epithelium: This is the outermost layer of the cornea; it is mounted on a
basement membrane and is only five to six cell layers thick. The main
functions are to protect the cornea from foreign material and to provide a
smooth surface that absorbs nutrients and oxygen. The epithelium consists
of: basal cells, wing cells and superficial cells [58]. The outermost cells have
microvilli to increase the cell surface area contributing to a close contact with
the tear film [59]. The cornea is highly sensitive to pain due to all the nerve
endings that are present in the epithelium. In fact this is the tissue with the
highest number of nerve fibers in the body. The corneal epithelium has the
ability to regenerate due to the stem cells present in the corneal-sclera
junction. This area is called the limbus.
Bowman´s Layer: This transparent layer anchors the epithelial cells and is
composed of collagen, and if injured it can form scars when healing, which
can affect the vision.
Stroma: The stroma corresponds to approximately 90% of the thickness of
the cornea, and the main components are water, collagen and keratinocytes
or fibroblasts, but it also contains glucosaminoglycans and proteoglycans
[58]. It contributes to the strength and elasticity of the corneal tissue. Due to
the fact that there are no blood vessels in the cornea, the stroma together
with the endothelial cells serve as the circulatory system of the cornea.
Descemet´s Membrane: This is a thin, but strong tissue located just beneath
the stroma. It is composed by collagen, which is produced by the endothelial
cells beneath and serves as a barrier to protect the eye from pathogens and
injuries. These cells are responsible for nutrient transportation and of
maintaining the optimal hydration to prevent corneal edema [59].
Endothelium: The endothelium is the thin, innermost layer of the cornea.
These cells regulate transport of fluid and other components in and out of
the cornea. If it is injured the stroma swells up, becomes cloudy and in the
end also opaque leading to visual damage. These cells do not regenerate.
25
The conjunctiva covers the inside of the eyelids and extends to the edge of
the cornea which is called the limbus. It is composed of two layers, the
stratified epithelia and the stroma. The thickness of the conjunctiva ranges
between 2-10 cell layers depending on the part of the conjunctiva studied.
The conjunctival epithelia contain goblet cells that have as their main
function to produce mucins that lubricate the eye. In contrast to the cornea,
the conjunctiva is highly vascularized. The conjunctiva is divided into three
different areas: Palpebral conjunctiva, bulbar conjunctiva and the fornix.
The palpebral conjunctiva covers the inside of the eyelids, the bulbar
conjunctiva covers the sclera (until the limbus region) and the fornix is
where the palpebral and bulbar conjunctiva meets.
The tear fluid layer continuously covers the surface of the eye and
protects the cornea and conjunctiva from the external environment, and
maintain the transparency of the cornea [60]. Other functions of the tear
film are e.g. keeping the eye moist, creating a smooth surface, supplying
oxygen and nutrients and protecting the eye against pathogens. The tear film
consists of three layers: the lipid, the aqueous and the mucous layer [61]. The
outermost lipid layer is present to prevent the evaporation of the aqueous
layer beneath. The aqueous layer is responsible for taking the main nutrients
and oxygen to the cornea and at the same time get rid of waste products.
Since the epithelial plasma membrane of the corneal cells is hydrophobic,
the hydrophilic mucin layer is needed to prevent the aqueous layer from
rolling off. The mucous layer is produced by the goblet cells of the
conjunctiva.
Description of EKC and AHC
Epidemic keratocconjunctivitis (EKC)
Epidemic keratoconjunctivitis is a severe ocular infection caused by viral
infection of the cornea and conjunctiva. Ads are the main causative agents of
the infection, especially Ad8, 19 and 37 but also Ad4 can cause a milder form
26
of the infection [62, 63]. More recently, two new types have been associated
with EKC, Ad53 and 54 [27, 28]. However, EKC can also be caused by viruses
from other families e.g. herpesviruses and picornaviruses. EKC is
characterized by an acute and severe onset with swelling, lacrimation,
discharge or photophobia [64, 65]. Follicular hypertrophy, edema,
hyperemia and petechial hemorrhage are caused by viral replication in the
palpebral and bulbar conjunctiva [65-70]. Replication in the cornea results
in focal epithelial keratitis, which is seen as punctuate opacities and leads to
a “foreign body” sensation [69, 71]. In severe cases chemosis, iritis and
formation of pseudomembranes are also observed [65, 67, 68]. The infection
normally lasts for 18-21 days, but the corneal opacities can last for months
up to years and even be permanent [72-76]. The infection is usually bilateral,
meaning that it starts in one eye and spreads to the other. Only about 25% of
the EKC cases display a unilateral infection [77]. The infection is spread by
contact and is therefore highly contagious causing outbreaks in densely
populated areas of the world. In many cases the outbreaks starts in eye
clinics, ophtalmologists´offices, schools or military bases [62]. Ad-induced
EKC is endemic in Japan where half a million to one million individuals are
infected every year, leading to substantial socioeconomical losses [63, 78].
Acute hemorrhagic conjunctivitis (AHC)
Acute hemorrhagic conjunctivitis is a highly contagious infection mainly
caused by two members of the picornavirus family; CVA24v and EV70 [79].
The disease has a rapid onset with pain, redness, excessive tearing, swelling
of the eyelid or photophobia within six to twelve hours after infection [80].
The infection usually spreads to the other eye within 24 hours. Another
typical symptom of the infection is subconjunctival hemorrhages, usually
beneath the bulbar conjunctiva covering the sclera (white part of the eye). In
addition, blurred vision, malaise, myalgia, fever, headache, keratitis or upper
respiratory tract symptoms may also appear [81]. There are some differences
in the given symptoms depending on the virus type e.g. fever, headache and
upper respiratory symptoms are more common in CVA24v infections and for
27
EV70 infections there are more pronounced subconjunctival hemorrhages
[82-85]. There have also been reports about neurological symptoms like
mild cranial nerve palsy or acute flaccid paralysis in EV70 infections [86].
The infection normally resolves within 7-10 days without any long-lasting or
permanent problems [81, 85]. So far these viruses have caused three
pandemics, 1969 to 1971 [87], 1980 to 1981 [88] and the most recent 2002 to
2004, which probably started in South Korea 2002 [89, 90] and thereafter
spread to Malaysia [91], India [92, 93], Nepal [94-96], Tunisia [97], Congo
and Marocco [98], Nicaragua, Honduras, Guatemala, El Salvador, Caribbean
countries [99], French Guiana, the West Indies [100], Puerto Rico [101] and
Brazil [102, 103]. Since the causative agents of AHC was identified it has
been estimated that up to 100 million cases have occurred until 2002 [104].
28
Viral structure
Adenoviruses
Ads are nonenveloped double-stranded DNA viruses with an icosahedral
capsid structure. The particle has a diameter of approximately 90 nm, a mass
of 150 MDa and approximately 87 % is constituted of proteins. Eleven
structural proteins have been identified by SDS-PAGE analysis and named
II-XII after their decreasing molecular weight [105]. The capsid has 20
surfaces and 12 vertices with a fiber protein protruding from each vertice
(see figure 2) [106].
Figure 2. A schematic picture of the Ad structure showing the major
proteins (capsid proteins), the minor proteins and the core proteins. The
picture has been reprinted from [107] with permission from the publisher.
29
Major capsid proteins
Hexon protein (pII)
The capsid contains 720 hexon monomers forming 240 trimers; this makes
the hexon protein the main contributor to the Ad capsid (63 % of the total
protein mass). The structure of the >900 amino acid hexon protein is
complex: Each monomer consists of a base with two antiparallel β-barrels
stabilized by an internal loop and three protruding loops on the external side
[108]. The loops contain hypervariable regions (HVRs), which gives the type-
specific epitopes (see figure 3a). This complex structure with a number of
intermolecular contacts renders the trimeric hexon proteins extremely stable
[105]. The hexons surrounding the pentons are called the peripentonal
hexons and the hexons covering the centre of the 20 facets of the capsid are
called “groups of nine” (GONs) [109, 110].
Penton base (pIII)
The penton base is a 340 kDa protein that consists of five monomers of
polypeptide III and is located at the twelve vertices of the icosahedral capsid
(see figure 2). The structure of the penton base monomer is similar to the
hexon, consisting of a β-barrel domain and two loops in the distal end. The
pentameric protein contains a central cavity that recognizes, and bind the N-
terminal domain of the fiber protein [111]. Ad internalization into host cells
is mediated by penton base binding to cellular integrins [112, 113]. The
binding to cellular integrins leads to a clustering of the integrins, which in
turn might lead to cell-signal induced endocytosis [114]. The integrin binding
is mediated through the Arg-Gly-Asp (RGD) motif that is present in a loop
on the penton base in all Ads except Ad40 and Ad41 (see figure 3b) [112, 115-
117]. These two viruses carry other motifs: Ad40 has an RGAD motif and
Ad41 has an IGDD motif. Ad41 has been suggested to use another entry
pathway than the typical clathrin mediated endocytosis that is used by other
Ads [118].
30
Fiber protein (pIV)
The 182 kDa fiber protein is a trimer situated at the twelve vertices of the
capsid in the center of the penton base. It consists of an N-terminal tail, a
shaft and a C-terminal globular domain known as the knob, which is the
receptor binding part of the virus (see figure 3 c & d). The N-terminal tail of
the protein contains the highly conserved FNPVYPY sequence that anchors
the protein to the center of the cavity in the penton base [111, 119]. The fiber
shaft is built up by various numbers of repeating motifs of approximately 15
residues, rendering the difference in fiber length of different serotypes e.g.
Ad3 has six repeats compared to Ad2 and 5 that have 22 repeats [120, 121].
Like the hexon protein and the penton base protein, the terminal knob
domain contains β-barrels building up the core of the knob and loops that
are exposed [122]. The three globular domains forming the knob of the virus
give rise to a central cavity on the top of the knob, which was first thought to
be the CAR-binding domain [123]. But, structural studies of the Ad12 knob
in complex with an extracellular domain of CAR indicated that the binding
site for CAR was surface loops at lateral subunit interfaces rather than the
central cavity [124]. The same has been seen for the CD46-binding Ad11
where the binding also takes place on the outside of the knob, but, in
contrast to CAR-binding, CD46-binding leads to a conformational change of
the receptor [125]. The sialic acid-binding Ads from species D (Ad19 and
Ad37) on the other hand interact with their receptor using the central cavity,
which can bind three sialic acid saccharides [126]. Three of the serotypes,
Ad40, Ad41 and Ad52 have a unique characteristic. They express two
different fiber proteins on their capsid surface, one long, CAR-binding and
one short with an as yet unknown receptor [127-129].
31
Figure 3. Structure of the capsomeres. (a) Ad5 hexon trimer model shown
in side view and from the top with the hypervariable loops in colors. (b)
Cryo-EM image of the Ad5 penton base shown as a side view with one of the
RGD loops highlighted. (c) A ribbon representation of the fiber knob of
Ad35 showing the three monomers in green, blue and pink. (d) Space-filling
model of the Ad2 fiber based on the atomic structure, with some receptor-
binding sites indicated. Reprinted from [107] with permission from the
publisher.
Minor capsid proteins
The minor capsid proteins are far less studied than the major capsid
proteins. They function as capsid cement managing the different
requirements for accurate assembly of the virion, its fluctuating stability and
its efficient disassembly [109].
32
Protein IIIa (pIIIa)
pIIIa is a 63.5 kDa protein with a somewhat unknown function but it is
believed to be important for correct encapsidation of the DNA since pIIIa
mutants form empty capsids [130, 131]. The protein is phosphorylated to
some extent, but the role of the phosphorylation is not known [132-134]. The
location of this protein is on the interior side of the viral capsid where it
binds the penton base to the peripentonal hexons and it also interacts with
pVIII [135].
Protein VI (pVI)
pVI is a 22 kDa protein located on the internal side of the capsid adjacent to
the hexons suggesting it to be in contact with two peripentonal hexons [136,
137]. But it might also be situated in the central cavities of the hexons as
suggested by cryo-EM studies [138, 139]. During the infection, pVI is
involved in the endosomal escape by inducing a pH-independent disruption
of the membrane [140]. Another important function of this protein is to
facilitate the nuclear import of hexon proteins as determined by the inability
of a pVI mutant to import hexons. The precursor of pVI acts as a shuttle,
facilitating the hexon transfer into the nucleus. pVI contains two nuclear-
localization signals (NLS) and two nuclear export signals (NES), which are
proteolytically removed during maturation [141, 142].
Protein VIII (pVIII)
This protein is the least studied of the minor capsid proteins. It is a 15.3 kDa
protein that forms dimers and interacts with nearby hexons facets. The
location of this protein is on the inside of the capsid where it mediates
interactions between GONs and their neighboring hexons and peripentonal
hexons [138]. The protein has also a suggested role in the overall structural
stability of the virion [143].
33
Protein IX (pIX)
pIX is the smallest of the minor capsid proteins with a mass of only 14.3 kDa
and it is also the only one of them that is unique to the mastadenoviruses, it
is absent in all the other genera [144]. The 20 facets of the icosahedral capsid
all contain twelve pIX molecules placed in the cavities between the peaks
formed by the hexons [136, 137]. The pIX monomers are α-helical and form
trimers due to its leucine-zipper domain, which allows the monomers to self-
associate into a coiled-coil structure with three extended arms [105, 144].
Together with nine hexons, pIX has been termed the cement that stabilizes
the GONs [145]. This is shown by Ad5 mutants lacking pIX, which can be
propagated in the same way as wild type virions but they do not form GONs
and they are heat-sensitive [146, 147]. Another potential role of this protein
is to function as a transcriptional activator of the major late genes [148, 149].
pIX has also been proposed to play a role in the packaging of the virion; A
virus defect in pIX can only carry a genome that is approximately 2 kb
shorter than the normal genome, whereas others have demonstrated that
pIX is not involved in the packaging but that lack of pIX renders the virus
non-infectious [150, 151].
The adenovirus core
In contrast to the capsid, the core does not present a well-ordered symmetry
or the coaxial coiled DNA as observed for bacteriophages [152, 153]. The
genome of Ads is a linear double-stranded DNA of approximately 36 kb with
a terminal protein (TP) attached to each 5´end, this protein is involved in
initiation of replication [154, 155]. The core is associated with four other
proteins aside from the TP named protein V, VII, µ and pIVa2. The most
abundant of the core proteins is pVII with ~800 copies per virion. This
protein is together with µ responsible for the packaging of the genome into
nucleosome-like structures [110, 156, 157]. pV is only found in mammalian
mastadenoviruses and interacts with pVI, pVII and/or DNA and thereby
connects the core with the capsid [158, 159]. Like pVII and µ, which interacts
34
with the DNA, pIVa2 also interacts with the DNA but in a sequence specific
way. pIVa2 is also known to activate the major late promoter and is
responsible for the type specific packaging of DNA [160].
Non-structural proteins
There are about 30 non-structural Ad proteins identified. Most of these have
a yet unknown function but are suggested to have catalytic or regulatory
functions [34]. These proteins are produced in very small amounts and they
are thereby difficult to study. However, two of the non-structural proteins
have been structurally defined: the DNA binding protein (DBP) and the viral
cysteine protease [134]. The DBP is a ~50 kDa phosphoprotein involved in
DNA replication and other DNA metabolic processes [161]. It binds to single-
stranded DNA and protects the DNA from digestion; DBP also destabilizes
the DNA helix thus facilitating the replication of DNA [162, 163]. The
adenovirus protease is a 25 kDa protein of which there are 10-30 molecules
present in each virion [164]. Most of the minor capsid proteins and core
proteins are cleaved by this protease and the cleavage product from pVI
binds to the protease and increases its activity [165-167].
35
Picornaviruses
Picornaviruses are small (8.5 MDa) non-enveloped viruses with a positive
sense RNA, which can act directly as an mRNA. They have an icosahedral
symmetry and the size of the capsid ranges between 25-35 nm in diameter.
Due to the lack of a lipid membrane these viruses are insensitive to organic
solvents and the viruses with a tropism for the gastrointestinal tract are acid
stable [168]. The picornavirus particles are composed of approximately 70 %
protein and 30 % RNA [169].
Structural proteins
The icosahedral capsid is composed by 60 heteromeric protomers each
consisting of four structural proteins: viral protein 1 (VP1), VP2, VP3 and
VP4. Five protomers then assemble and form pentamers and twelve
pentamers in turn assemble and form the capsid. Three of the proteins, VP1,
VP2 and VP3 have a molecular weight (Mw) of approximately 30 kDa
whereas the fourth protein VP4 is much smaller and has a Mw of 8 kDa
[169]. The core structure of VP1, VP2 and VP3 is very similar, consisting of
an eight-stranded antiparallel β-barrel and two α-helices. On the other hand,
there is a high degree of diversity in the loops connecting the β-strands. The
five-fold axis is formed by five copies of VP1, while the three-fold axis
consists of a plateau formed by VP2 and VP3 (see figure 4) [22]. The N-
terminal of VP4 and also its precursor VP0 is covalently linked to a myristic
acid. Myristylation has been seen in several of the picornavirus genera, and
this appears to be of importance during capsid assembly but has also been
suggested to play a role in viral entry [170, 171].
An interesting finding in the structure of the first picornavirus studied by X-
ray crystallography, human rhinovirus 14 (HRV-14), was a depression seen
at the five-fold axis. This depression, or canyon, was believed to play a role in
binding to receptors, since this location would be protected against
neutralizing antibodies [172]. This hypothesis was later challenged when it
36
was discovered that neutralizing antibodies against HRV-14 could bind to
residues within the canyon [173]. Below the canyon there is a “pocket” that
contains a host-derived fatty acid. This “pocket-factor” is believed to stabilize
the capsid [174]. Since the receptor-binding canyon and the pocket overlap it
has been suggested that only one component at a time can be bound, this fact
has been exploited when designing antivirals [175, 176]. However, more
recent findings have questioned the role of “pocket-factors” for capsid
stability, suggesting that it could be the cavity itself rather than the “pocket-
factor” that is contributing to the stability [177].
Non-structural proteins
The non-structural proteins of the picornaviruses are encoded from the
3´end of the genome. There are seven to ten known non-structural proteins:
2A, 2B, (2BC), 2C, 3A, (3AB), 3B, 3C, (3CD) and 3D. These proteins are
much more conserved than the capsid proteins, probably due to the lack of
immune pressure and because of their importance [178]. The 2A protein is
known to differ in function between different genera of the picornaviruses, in
enteroviruses it functions as a cysteine proteinase that cleaves its own N-
terminus to release the capsid protein precursor and it also cleaves cellular
proteins that affects viral growth [179]. Proteins 2B, 2C and its precursor
2BC are involved in the remodeling of intracellular membranes to support
viral replication [180, 181]. The EV protein 2B has been called a viroporin, a
protein that oligomerize and inserts itself into membranes to create pores or
channels leading to an increased diffusion of small molecules [182]. The
function of this pore formation is not clear. Protein 2C is one of the most
conserved picornavirus proteins but the exact functions of the protein
remains unclear. It is suggested to e.g. have ATPase activities and RNA-
binding activities [183, 184]. Protein 3A has the highest degree of variability
between the different genera, but they all contain a hydrophobic domain that
anchors the protein to the membranes where the replication occurs. This
protein has been suggested to play a role in the membrane remodeling and
also to be able to disrupt the endoplasmatic reticulum (ER)-to-Golgi
37
secretory transport and disassemble the Golgi complex [185, 186]. The 3B
polypeptide is also called VPg (viral protein genome-linked), and it is
attached to the 5´end of the genome where it serves as a primer to initiate
RNA synthesis [187, 188]. The 3AB precursor protein seems to be directly
involved in the RNA synthesis, either by donating VPg to the initiation of the
RNA chain elongation or as a cofactor for the polymerase [189, 190]. The 3C
polypeptide encodes the proteinase responsible for the majority of the
cleavage of both viral and cellular proteins. 3C and 3CD also contain an
RNA-binding motif and may therefore play a role in the replication process
[191-193]. The most important (and therefore the most studied) protein is
3D, which is an RNA-dependent RNA polymerase (RdRP). The RdRP is the
main subunit of the replication complex, which is responsible for the
synthesis of viral RNAs [191].
Figure 4. Structure of human rhinovirus 16 (HRV-16). (a) Subunit
organization at 3 Å resolution showing capsid proteins VP1-3 (blue, green
and red), VP4 is not exposed on the capsid surface, (b) Close-up of the star-
shaped dome at the fivefold axis and the surrounding canyon and, (c)
Arrangement of VP1–VP3 on the icosahedral lattice. Fivefold, threefold and
twofold symmetry axes are indicated. Images are taken from the Virus
Particle Explorer database (VIPERdb; http://viperdb.scripps.edu) [194].
38
Viral life cycle
Receptors
The initial interaction between the virus and the cell takes place through one
or more cellular receptors. This interaction is the starting point of the
infectious cycle. Most Ads bind to host cells via the most distal part, the
“knob” of the fiber protein [195]. In the case of the picornaviruses, many of
the receptor interactions take place through the canyon, which is formed by
the VP1 protein. Canyon binding was first suggested for HRV14 even before
the actual receptor had been identified [23]. The first receptors identified for
picornaviruses were the poliovirus receptor (PVR/CD155) and the major
group rhinovirus receptor ICAM1 [196, 197]. Some picornavirus receptor
binding sites are localized outside the canyon as for FMDV that instead have
an exposed flexible loop on its capsid surface [198]. A number of different
receptors are known today both for the Ads and the picornaviruses, and even
though they are not related in any way they share a great number of
receptors (see figure 5, table 4 and 5).
Figure 5. Schematic picture of cellular receptors that Ads and
picornaviruses have in common. SA (sialic acid), HSPGs (heparan sulphate
proteoglycans), MHC-1 (major histocompatibility complex-1), integrins and
CAR (coxsackievirus and adenovirus receptor).
39
Table 4. Cellular receptors of the human adenoviruses, classified in species.
Species Type Receptors*
A 12, 18, 31 CAR [129]
B:1
B:2
3, 7, 16, 21
11, 14, 34, 35, 50
Desmoglein 2 [199]
CD80, CD86 [200]
CD46 [201]
HS [202]
CD46 [201, 203]
Desmoglein 2 [199]
CD80, CD86 [200]
C 1, 2, 5, 6 CAR [123, 129]
Heparan sulfate [204]
VCAM-1 [205]
MHC-1 [206]
D 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, 42-49, 51
CAR [129]
Sialic acid [207]
GD1a glycan [208]
CD46 [209]
E 4 CAR [129]
F 40, 41 CAR [129]
G 52 N.D.*
*cellular receptors that mediate direct Ad binding.
*N.D; not determined.
40
Table 5. Cellular receptors of the human enteroviruses, classified in species.
Species Types Receptors
HEV-A CVA 2-8, 10, 12, 14, 16
EV 71, 76, 89, 90-92
N.D.*
SCARB-2, sialylated glycans, PSGL-1 [210-212].
HEV-B CVB 1-6
CVA 9, 23
EV 69, 73-75, 77-88, 93, 97,
98, 100, 101, 106, 107
E 1-9, 11-21, 24-27, 29-33
CAR, DAF, HS [123, 213, 214]
avβ3, avβ6 , MHC-1,
GRP78 [215-217]
N.D.*
VLA-2 (a2β1), DAF, HS
[218-221]
HEV-C PV 1-3
CVA 1, 11, 13, 17, 19-22, 24, 24v
EV 95, 96, 99, 102, 104, 105, 109
CD155 [196]
ICAM-1, DAF, sialic acid
[222-224]
N.D.*
HEV-D EV 68, 70, 94 DAF and/or sialic acid
[104, 225, 226]
HRV-A HRV 1, 2, 7-13, 15, 16, 18-25, 28-34, 36, 38-41, 43-47, 49-51, 53-68, 71, 73-78, 80-82, 85, 88-90, 94- 96, 98, 100
ICAM-1 [197]
HRV-B HRV 3-6, 14, 17, 26, 27, 35, 37, 42, 48, 52, 69, 70, 72, 79, 83, 84, 86, 91-93, 97, 99
LDLR [227]
HRV-C Types not yet assigned N.D.*
* N.D; not determined.
41
Immunoglobulin superfamily (IgSF)
The immunoglobulin superfamily is a large family of cell surface proteins
with diverse biological functions. These proteins are important for cell
surface recognition and thereby they play a major role in the immune
defense. Members of the IgSF are commonly used as viral receptors and all
of the known interactions take place via the most membrane-distal domain
D1 of the IgSF receptor [228].
Coxsackievirus and adenovirus receptor (CAR)
In 1976, it became evident that CVB3 and Ad2 competed for the same
receptor and that this receptor was different from the already known
poliovirus receptor PVR/CD155 [229]. It was not until 1997 that the receptor
was identified and given its name CAR [123, 230, 231]. CAR belongs to the
immunoglobulin family and it consists of an extracellular domain, an
intracellular domain and a single membrane-spanning domain and it has a
Mw of 46 kDa. The extracellular part has two Ig-like domains, D1 and D2.
The outermost D1 is the domain that binds both CVBs and Ads but they do
not bind to the same part of the domain [124, 232]. CAR function as a
receptor for all CVBs within the picornavirus family and for selected Ad
types in species A, C-F [129, 230, 233, 234]. However, the Ad knob-CAR
interaction function in vitro, which does not necessarily mean that it is a
functional receptor in vivo. This was first indicated in 2001 when CAR was
discovered to be located in tight junctions of polarized epithelial cells were it
associates with ZO-1 [235]. These studies showed that in polarized epithelia,
CAR is not accessible from the apical side and viruses might need a
disruption of the epithelial surface to be able to enter the cells. Recently, an
isoform of CAR (CAREx8) was discovered on the apical side of polarized
airway epithelial cells and this isoform was enough to support viral
attachment [236]. This indicates that CAR could be expressed on the apical
side and thus, accessible as a receptor for Ads. Basolaterally and laterally
expressed CAR has also been suggested to facilitate spread instead of entry,
42
since free Ad fibers can disrupt intercellular CAR-CAR-mediated junctions
and thereby permit intercellular virus transport [237]. The distribution of
CAR is not completely known, but it is present in heart, brain, pancreas,
intestine and also in lung, liver and kidney [230, 238].
Intercellular Adhesion Molecule-1 (ICAM1)
ICAM-1 is present on many cells of the immune system and is involved in
leukocyte adhesion to endothelial cells at sites of infection. ICAM-1 can be
expressed on a wide variety of cells but normally, ICAM-1 expression is
localized to a few cells since the expression have to be induced by the
inflammatory response [239]. The natural receptor for ICAM-1 is a member
of the integrin family called LFA-1 (leukocyte function-associated antigen-1)
[240]. ICAM-1 is highly glycosylated and consists of five extracellular Ig-like
domains, a transmembrane domain and a cytoplasmic domain [241]. ICAM-
1 is one of the major receptors for picornaviruses e.g. the major group of the
rhinoviruses as well as CVA13, 18 and 21 but it is also a receptor for
plasmodium falciparum [197, 223, 242-244].
Poliovirus receptor (PVR or CD155)
The PVR is the receptor for probably the most extensively studied
picornavirus, PV. The receptor is a long, highly glycosylated member of the
IgSF. It is composed of a C-terminal cytoplasmic part, a transmembrane part
and an extracellular part consisting of three Ig-like domains D1, D2 and D3
[196, 245]. PVR is known to activate natural killer cells (NK cells) and it has
a suggested role in cell motility and invasion by tumour cells [246-248]. The
PVR gene is expressed as four different splice variants of which two lack the
cytoplasmic part and therefore are released after expression [249]. The other
two are functional PV receptors. The domain responsible for the interaction
between PVR and the PV canyon is domain D1 [245, 250, 251]. The N-
glycosylation of the D1 domain of the receptor is not involved in viral binding
and PV interacts directly with the protein [252]. This receptor is not known
to function as a receptor for any other virus. The tropism of PV for CNS
43
cannot be explained solely by the expression of the receptor since PVR is
expressed in tissues other than those infected by PV, meaning that other
factors are probably of importance also [196, 253].
Regulators of complement activation (RCA) family
The RCA family has the important role to distinguish “self” from “non-self”
molecules via a number of proteins. They control the activity of the
complement system and thereby prevent the destruction of host tissue and
direct the complement degradation to e.g. harmful microorganisms [254].
These proteins have a common structure consisting of short consensus
repeats (SCRs). Some members of the family are: membrane cofactor protein
(MCP/CD46), factor H, decay accelerating factor (DAF/CD55), C4b-binding
protein and complement receptor types 1 and 2 [255].
Membrane Cofactor Protein (MCP/CD46)
Membrane cofactor protein (MCP/CD46) belongs to the RCA family and its
function is to protect healthy cells from complement-mediated destruction
by binding C3b and C4b to facilitate their degradation by serum factor I. The
protein consists of a cytoplasmic tail, a transmembrane region, an O-
glycosylated STP (serine-threonine-proline) domain and four SCRs (short
consensus repeats). The SCRs are characteristic for proteins of the RCA
family [256]. Several Ads from species B are known to use CD46 as a
receptor: Ad11, Ad16, Ad21, Ad35 and Ad50. From species D, Ad37 have
been reported to recognize CD46 [201, 203, 209, 257, 258]. CD46 is
expressed on all cells except for erythrocytes [259], and in opposite to CAR,
CD46 is available on the apical surface of polarized epithelial cells and not
constricted to the basolateral side or the tight junction [260]. Many
pathogens have been shown to bind to CD46 e.g. Measles virus, bovine viral
diarrhea virus, human herpes virus-6, Streptococcus spp. and Neisseria spp.
[261-265].
44
Decay Accelerating Factor (DAF or CD55)
Like CD46, DAF also protects the cell from autologous lysis by complement.
The protein consists of four SCRs, a heavily O-glycosylated STP region, but
unlike CD46, DAF lacks a cytoplasmic region, and is instead linked to the
plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. Several
EVs have been reported to use DAF as a receptor either for binding only or
for both binding and internalization. However, there is no uniform binding
between these viruses in that they bind to different SCR domains. CVA21 and
EV70 both interact with SCR1; CVB interact with SCR2 (CVB3-RD (adapted
to growth in RD cells)) or SCR3 (CVB1-3 and 6) [224, 266-269]. The only EV
that can use DAF for both binding and internalization is echovirus 7 (E7),
many or most of the other EVs also need another receptor as co-receptor for
internalization [270].
Sialic acid
N-acetyl neuraminic acid (Neu5Ac), which was first discovered in the 1930s
by Klenk and Blix [271], belongs to the family of sialic acids (SA) that
contains more than 50 saccharides with a large diversity. Unlike most of the
vertebrate monosaccharides which have five- or six-carbon backbones, SAs
have a nine-carbon backbone [272]. A characteristic feature of SA is the
carboxyl group at position 1 that gives a negative charge under physiological
conditions, and the amino group at carbon number 5. The SAs are mainly
present on the outermost part of glycan chains of glycoproteins or glycolipids
and thereby easily accessible for interactions with other cells or with
pathogens. They are linked via glycosidic bonds from carbon 2 to either
carbon 3 or carbon 6 on the neighboring galactose or to carbon 8 on a
neighboring SA [273].
The main function of SA is linked to cellular and molecular recognition such
as binding and transport of positively charged molecules (e.g. Ca2+),
protection of glycoproteins or entire cells from degradation by e.g. proteases
45
or macrophages, helping the immune system to distinguish self from non-
self structures, and they are also involved in leukocyte “rolling” [273, 274]. A
number of different microorganisms (virus, bacteria and protozoa) take
advantage of this and use SA as an attachment receptor [272]. Some
examples are; Influenza A virus [275, 276], JC and BK virus [277, 278],
Theiler´s murine encephalitis virus [279], Haemophilus influenza [280] and
Helicobacter pylori [281].
The three EKC-causing types, Ad8, Ad19 and Ad37 belonging to species D
use SA as a receptor [207]. The positive charge of the fiber knobs of these
viruses (9.0-9.1) together with the negative charge of sialic acid (pKa = 2,6)
results in a charge-dependent interaction [282]. The AHC-causing EV70
from the picornavirus family is another virus with ocular tropism that uses
SA as its attachment receptor, but this is only on specific cells [283].
Gangliosides
Gangliosides are glycosphingolipids containing one or more sialic acid
residue linked to a glycan backbone, which is further attached to a ceramide
base [284]. Gangliosides were first discovered in the 1930s by Ernst Klenck,
and they were named due to their association with brain gray matter
(“Ganglionzellen”) [285]. They are present in all vertebrate tissues, which
imply that they play a crucial role in the cell [286, 287], however they are
more concentrated in tissues of neuronal origin [288]. Several different
gangliosides have been reported to play a role during infection, either as a
receptor for a toxin or as a receptor for a microbe. For example, the cholera
toxin from Vibrio cholera binds to the ganglioside GM1 a ganglioside that is
also used as a receptor by the SV40 virus [289, 290]. Members of the
polyomaviruses have been shown to interact with gangliosides: the murine
polyomavirus binds GD1a and Gt1b, BK virus binds the gangliosides GD1b and
GT1b and JC virus recognizes GM3, GD2, GD3, GD1b, GT1b, GQ1b and GD1a
to some extent [289, 291, 292].
46
Heparan sulfate proteoglycans (HSPGs)
Proteoglycans are built by long chains of glucosaminoglycans (GAGs) linked
to a core protein, often membrane proteins such as glypicans or syndecans.
GAGs are linear polysaccharides composed of disaccharide building blocks of
either GlcNAc or GalNAc and uronic acid (glucuronic or iduronic). HSPGs
function as co-receptors by binding via heparan sulfate (HS) to either
insoluble ligands in the extracellular matrix (ECM) or to cells, or to soluble
ligands such as growth factors or cytokines and thereby promoting
interactions and subsequent signaling. HSPGs can also function directly as
internalization receptors for soluble ligands e.g. enzymes [293]. A number of
different bacteria, protozoa and viruses have been shown to interact with HS
for infection, including members of the Borrelia burgdorferi, Chlamydia
trachomatis, cytomegalovirus (CMV), dengue virus and herpes simplex virus
[294-298]. HSPGs have been reported to function as receptors to some
extent for Ad types 2 and 5 and as a probable co-receptor for Ad3 and Ad35
[202, 204]. Also, members of the picornaviruses interact with HSPGs;
echoviruses, coxsackievirus B3 and FMDV [214, 220, 299].
Desmoglein-2
Desmoglein 2 (DSG-2), a member of the cadherin cell adhesion molecule
superfamily, was recently reported as the previously unknown receptor “X”
for species B Ad3, Ad7, Ad11 and Ad14 [199, 202]. DSG-2 is, like CAR, a
member of the intercellular junctions. By binding to DSG-2, species B Ads
open the junctions to access other junctional proteins. Species B Ads are
known to produce large amounts of dodecahedral particles (PtDds) during
infection and these incomplete particles are capable of interacting with DSG-
2 and thereby open the junctions. This overproduction of incomplete
particles may be a way for corresponding virions to spread from or within
the epithelial cell layer [199].
47
Integrins
Integrins are part of the immune system where they play a crucial role in e.g.
leukocyte adhesion and cell polarization. They are heterodimeric molecules
composed of a α and a β subunit. So far, 18 α and 8 β subunits form the 24
αβ pairs known for vertebrates [300]. Several integrins are functioning as
co-receptors for Ads e.g. αvβ1/3/5, αMβ2, α5β1 and α3β1 [301-308]. One of the
known binding motifs of integrins is the RGD (Arg-Gly-Asp) motif, which is
exposed on the Ad penton base protein. Pb-mediated clustering of integrins
leads to cell signaling and subsequent internalization [112, 309]. Several
different Ad Pb proteins interact with integrins [117, 158]. Ad40 and Ad41
however lacks the RGD- motif leading to a delayed uptake [117].
Also picornaviruses are known to utilize integrins as their receptors e.g.
echovirus 1 binds to α2β1 (also known as VLA-2; very late activation antigen-
2), CVA9 use αvβ3 or αvβ6 and FMDV use αvβ1/3/6/8 or α5β1 [218, 310-316].
CVA9 and FMDV both contain RGD-motifs and are dependent on this motif
for binding to integrins on the cell surface as blocking of the RGD-motif
protect the cells from infection [216, 317]. However, the binding between
echovirus and α2β1 takes place in an RGD-independent fashion since α2β1
does not recognize this motif [218].
Other receptors
Two molecules from the IgSF, CD80/CD86 have been reported to function
as receptors for Ads from species B [200]. CD80 is expressed on monocytes
and B cells and CD86 is present on antigen-presenting cells. However, CD80
and 86 have been questioned by others about their role as functional
receptors for species B Ads [318].
Scavenger receptor B2 (SCARB2) was recently identified as a receptor for
EV71 and also for CVA16 [211]. Both these viruses cause the common hand,
foot and mouth disease (HFMD) in children, however, EV71 infection can
also lead to neurological complications and death. SCARB2 is a
48
transmembrane protein usually located in the endosomal-lysosomal
compartment, and is involved in e.g. transportation across the endosomal-
lysosomal membranes [319]. This protein has been reported to be important
for internalization of Salmonella typhimurium, Staphylococcus aureus and
Listeria monocytogenes but also as a receptor for high-density lipoprotein
[320-322].
P-selectin glycoprotein ligand-1 (PSGL-1) has also recently been reported to
function as a receptor for HFMD-causing EV71 and CVA16 [210]. This
protein is expressed on cells of hematopoietic origin, on dendritic cells in
lymph nodes and on macrophages in the intestinal mucosa, and play an
important role for recruitment of leukocytes during inflammation
(stimulated by infection) [323-325].
Low density lipoprotein receptor (LDLR) and very low density lipoprotein
receptor (VLDLR) have been reported to be receptors for the minor group
rhinoviruses [227, 326]. Cryo-EM reconstruction of HRV2 and VLDLR
revealed that the receptor interaction takes place on the capsid surface and
not within the canyon [327]. Virus binding to LDLR does not trigger a
conformational change in the capsid or RNA release, instead, the uncoating
is dependent on acidification and therefore takes place in the endosomal
compartment [328-330].
T-cell immunoglobulin and mucin-1 (TIM-1 or HAVCR) have been identified
as a receptor for Hepatitis A virus (HAV) [331]. This is a class I integral
membrane glycoprotein that is expressed in every human organ including
the liver, small intestine, colon, spleen, kidney and testis. The natural
function of this molecule is not known. Recently, HAVCR was shown to co-
localize with the tight junction protein ZO-1, suggesting that HAVCR is
located to and forms a part of the tight junctions [332].
Major histocompatibility complex-1 (MHC-1) has been suggested to function
as a receptor for the fibers of Ad2 and 5 [206]. An increase of fiber binding
49
and gene transfer was seen when MHC-I was expressed on lymphoid cells as
compared to cells lacking MHC-I expression. Later studies have not been
able to confirm the role of MHC-I as an Ad receptor [333, 334]. From the
picornavirus family, CVA9 has been shown to also interact with MHC-I via a
co-receptor called glucose-regulated protein 78 kDa (GRP78) [215]. In their
work they show that MHC-I molecules interact with GRP78 on the cell
surface and that CVA9 in addition to αvβ3 uses GRP78 as a receptor
molecule. αvβ3 and GRP78 are used for binding, but for uptake MHC-I is
needed.
Vascular cell adhesion molecule-1 (VCAM-1) is a member of the IgSF. An
increase in Ad transduction of NIH T3T cells was observed when VCAM-1
was expressed on the cell surface [205]. The D7 domain of VCAM-1 shares
homology with the D1 domain of CAR thus, possibly explaining the
interaction between Ads and VCAM-1.
Soluble components
Other, alternative pathways have been suggested where Ads use components
of blood or other body fluids to bind and infect cells in a CAR-independent
fashion [335-338]. Lactoferrin (Lf) is a body fluid component known to
enhance binding and infection of species C Ads [336]. By interacting with Lf,
Ads can take advantage of Lf receptors. Lf thereby functions as a bridging
factor. From blood, coagulation factors IX, X and also complement factor
C4bp have been reported to function as bridging factors for Ad5 on epithelial
cells and hepatocytes [337, 338]. This interaction takes place via the cell
surface HSPGs. Ad31 from species A is able to interact with factor IX
although if this interaction takes place via HSPGs is not clear [337].
Dipalmitoyl phosphatidylcholine (DPPC) is a major contributor to the lipid
pool of surfactants. Surfactants are used in the treatment of respiratory
distress symptoms in premature children however; these surfactants have
been shown to promote Ad gene delivery [339, 340]. DPPC interacts with the
hexon protein of Ads [341].
50
The life cycle after binding to cells; adenoviruses
Internalization and trafficking
The main mode of entry for Ads is by receptor-induced endocytosis. The two
most studied types, Ad2 and Ad5, enter cells via clathrin-coated pits, but this
mechanism also triggers a second endocytic process, macropinocytosis [342-
344]. After the initial attachment of the Ad-fiber to its receptor, the RGD-
motif in the Pb interacts with integrins (αvβ1/3/5, α5β1), which mediates viral
entry [301, 303-306, 308]. This is confirmed for Ad2, Ad3, Ad4 and Ad5, but
most Ad types display an RGD motif in their penton base, indicating similar
interactions [301]. Integrins that are unable to recognize the RGD-motif
have also been shown to interact with Ads, αMβ2 and α3β1 [304, 307].
Integrin-clustering caused by Ad binding in turn leads to activation of
phosphatidylinositol-3-OH kinase (PI3K) and Rho GTPases which induces
cytoskeletal rearrangements leading to clathrin-mediated uptake [345b,
346a]. Approximately 15 min after uptake, the fiberless virus escapes from
the endosome into the cytoplasm through a process that involves αvβ5
integrins rather than αvβ3 [347]. It is also known that the virus needs the
acidification of the endosome to lyse the endosomal membrane [348].
The capsid is dismantled step by step in a highly ordered fashion that starts
by release of protein IIIa and the fiber leading to release of the Pb, a crucial
step needed for endosomal escape. Protein VI has also been suggested to
have a membrane lytic activity, thus, potentially aiding the endosomal
escape [140]. The partially dismantled particle interacts with dynein and is
thereby transported on microtubules to the nucleus and the NPC (nuclear
pore complex) [349-351]. After viral binding to the NPC, the viral protease
cleaves pVI and thereby completeing the capsid dismantling. Cellular
proteins suggested to play a role in NPC binding are the nuclear pore protein
CAN/Nup214, the chaperone Hsc70 (heat shock cognate protein) and also
histone H1 [352, 353].
51
Figure 6. Infectious entry pathway for Ad2 and 5. Reprinted from [354]
with permission from the publisher.
Structure of the genome
The genome of Ads is a linear double-stranded DNA that ranges between 34-
36 kbp in size and it encodes approximately 40 different proteins (see figure
7). It is characterized by the presence of 36 to 200 bp inverted terminal
repeats (ITRs) at its ends and a TP linked to the 5´end [158]. The genome
organization is highly conserved between the genera, and is divided into
early transcription units (E1A, E1B, E2, E3 and E4), delayed early
transcription units (IX, IVa2 and E2 late) and the late transcription unit
(major late) [158, 355]. The major late transcript is further processed into
five mRNAs (L1 to L5) [356]. All are transcribed by the cellular RNA
polymerase II. The genome also encodes one or two virus associated RNAs
(VA RNAs) depending on the serotype. These are transcribed by the cellular
RNA polymerase III [357].
52
Figure 7. Transcription of the Ad genome. The early genes expressed are
coloured green and the late are blue. The VA RNAs and the major late
promoter (MLP) are also outlined in brown and black. Reprinted from [358]
with permission from the publisher.
Gene expression and replication
Early gene expression
The three main functions of the early proteins are: a) to trigger S-phase entry
of the host cells, b) to protect the infected cell from different types of
antiviral defense mechanisms and c) to synthesize the gene products needed
for the viral replication [355]. The E1A proteins are so called trans-
activators, meaning that they are able to activate transcription of both viral
and cellular genes. The E1A proteins can also interfere with complexes
formed by members of the pRb family and the E2F family and thereby push
the cells into S-phase [359, 360], which creates an optimal environment for
viral replication. E1B encodes two proteins that block apoptosis and the
function of both the E1B proteins is needed for inhibition of the E1A-induced
apoptotic program. E2 encodes three proteins that are all involved in DNA
replication, the polymerase (AdPol), the DBP and the TP. E3 proteins are
53
involved in modulation of host responses to the infection. The E4 proteins
have a number of different functions, including transcriptional and
translational regulation, nuclear export of viral mRNA and inhibition of
apoptosis [355]. The Ad genome encodes one or two VA RNA:s, these small
abundant RNAs inhibits the interferon (IFN) response of the innate
immunity by binding to protein kinase receptors (PKR) [357, 361].
Replication
In cell culture, the replication of the Ad genome starts approximately five to
eight hours after infection and is triggered by the accumulation of E2 gene
products. The replication takes place in two steps (see figure 8) [362]:
Initiation takes place at both ends of the genome and thereafter proceeds
continuously giving rise to one double-stranded complex and one single-
strand of DNA (Type I replication). In the second step, complementary
strands to the single-strands are produced. The single-stranded DNA
circularizes by annealing its self-complementary termini to form a
“panhandle”-structure. This panhandle has the same structure as the double-
stranded genome and is therefore recognized by the same machinery that
function in the first step, resulting in the production of a complementary
strand (Type II replication) [355].
The terminal ITRs of the viral chromosome function as replication origins.
By associating with two E2-encoded proteins; the TP and the AdPol, viral
replication is initiated. The TP functions as a protein primer [363]. However,
for efficient replication a number of cellular proteins are also needed e.g.
nuclear factor I &II (NF-I, NF-II) and Oct-1 [364-366]. The role of the viral
DNA binding protein (DBP) for replication is believed to be to displace the
non-replicated strand during the elongation [367]. The viral proteins can
themselves initiate replication but not as efficient as when the cellular
factors are present. In that case, the replication initiation is approximately
200 times more efficient [368].
54
Figure 8. The replication of the Ad genome, shown in the two steps: type I
replication and type II replication. Reprinted from [362] with permission
from the publisher.
Delayed early gene expression
Expression of the delayed genes takes place immediately after onset of DNA
replication but before expression of the late genes and they encode pIX and
pIVa2. pIX is a capsid component but it also activates transcription. The
IVa2 gene product has two functions, one is to activate transcription from
the MLP (major late promoter) and the other is to facilitate packaging of the
genome into the capsids [355].
Late gene expression
The late genes encode the capsid components, which are expressed after the
onset of DNA replication. These genes encode one single transcript of about
28 kb, which is spliced into twenty different mRNAs. These mRNAs are
grouped into five families, L1 to L5. The expression of these genes is
controlled by the major late promoter (MLP) [355].
55
Assembly and release
The accumulation of large amounts of structural proteins and new genome
copies results in the Ad capsid assembly. Hexon monomers assemble into
trimers in the cytoplasm with the help of the L4-encoded 100K protein. Pb
and fiber monomers assemble independently of each other in the cytoplasm
but later join to form pentons [369, 370]. After this initial assembly, the
hexons and penton capsomers are transported into the nucleus where the
final assembly takes place. Import of hexon to the nucleus relies on the
capsid stabilizing protein VI, which shuttles between nucleus and cytoplasm
[371]. The DNA is packaged from the 5´end of the genome were the 200 bp
packaging sequence is situated [372]. The DNA packaging is promoted by
protein IVa2-, 52/55K- (L1) and 22 kd protein (L4) binding to the packaging
sequence [373, 374]. The complete maturation of the provirion into an
infectious virion requires enzymatic processing of proteins VI, VII, VIII, µ
and TP by the Ad protease [373]. There are at least three different viral
systems for release of progeny virions and escape from the site of infection:
a) Interfering with the cellular intermediate filaments by proteolytic cleavage
of the cellular cytokeratin K18 by the viral L3 protease, which renders the
cell more susceptible to lysis [375], b) Accumulation of the adenovirus death
protein (ADP) late during infection leads to cellular lysis [376], c) As
previously mentioned, extensive production and release of Ad fibers during
infection interferes with CAR at the tight junctions and facilitates the
transport of progeny virions from the infected cell [237].
56
The life cycle after binding to cells; picornaviruses
Internalization and trafficking
Previously, picornaviruses were believed to undergo uncoating already at the
cell surface but now it appears as if they are internalized into vesicles first
[329, 377, 378]. The picornaviruses use a number of different entry
pathways:
a) FMDV use integrins as their receptors and are known to enter cells by
clathrin-mediated endocytosis [379, 380]. This is a pH-dependent pathway
where the acidification of the endosome leads to capsid disassembly [168].
Cell culture adapted FMDV use instead HS as receptors and enter cells in a
caveolin- and lipid raft-dependent way [299, 381].
b) CVB3 use DAF and CAR as their receptors and have been shown to enter
cells in a complex process [382]. The entry mechanism of these viruses is
dependent of caveolin-1 and therefore it was suggested that they enter via
caveoli. However, a more recent study showed that entry over the tight
junction leads to internalization of occludin via macropinocytosis [383].
Therefore CVB3 seems to somehow combine caveolin-mediated and
macropinocytic uptake.
c) PV uptake was recently shown to depend on actin, energy and an
unidentified tyrosine kinase [384]. The uptake was not due to clathrin-
mediated endocytosis, caveoli or macropinocytosis. The release of RNA takes
place just beneath the plasma membrane (100-200 nm) (see figure 9).
For PV, HRV and EV, the virus-receptor interaction triggers a
conformational change in the capsid leading to the formation of A-particles
(135S particles) [385, 386]. In the A-particles, the myristoyl-VP4 and the N-
terminal of VP1 are exposed on the capsid surface. The N-terminal part of
57
VP1 has been suggested to penetrate the plasma membrane and thereby
either disrupting the membrane or forming a pore for RNA injection into the
cytoplasm [387]. However, more recent data have suggested a central role of
VP4, as shown by mutational analysis disrupting RNA release and pore-
formation. Also, bacterially expressed myristoylated VP4 from HRV-14 can
by itself form pores in liposomes [388, 389].
Figure 9. A schematic picture of the entry mechanism of poliovirus. After
viral binding to PVR, the virion is internalized through a clathrin- and
caveolin-independent, but actin- and tyrosine kinase-dependent mechanism.
The release of RNA takes place close to the cell membrane (100-200 nm).
After RNA release the empty (80S) capsids are transported via microtubule
towards the cell center perhaps for degradation in the endosomal or
lysosomal compartments. Reprinted from [384].
58
Structure of the genome
The picornavirus genome consists of a single positive-stranded RNA
molecule that by itself is infectious since it encodes all the proteins needed
for viral replication. A feature of the picornavirus mRNA is the VPg protein
that is attached to the 5´end [390, 391]. The genome size varies from 7209 to
8450 bases and contains one single open reading frame (ORF). Both the
3´end and the 5´end of the genome are flanked by untranslated regions
(UTRs). The long 5´UTR of the genome contain the internal ribosome entry
site (IRES) that bind to the ribosomes to initiate translation [392]. The short
3´UTR contain a poly A tail and a secondary nucleic acid structure called a
pseudoknot, these are involved in controlling viral RNA synthesis [393, 394].
The picornaviral genome is divided into three protein encoding domains: P1,
P2 and P3. The P1 region encodes the capsid proteins (structural proteins),
the P2 region mainly encodes proteins involved in structural membrane
rearrangements and the P3 region encodes proteins involved in RNA
synthesis (see figure 10) [168].
Figure 10. The picornavirus genome is a plus stranded RNA and can
thereby be translated directly into proteins. The three protein encoding
domains P1-P3 are outlined, and the products and their functions are
visualized. Reprinted from [395].
59
Translation and replication
Picornaviruses do not carry any proteins into the host cells and since the
RNA cannot be copied by cellular RNA polymerases it must first be
translated into proteins. Although the cap-dependent cellular mRNA
translation system is shut down by picornaviruses the cap-independent
synthesis of viral proteins is allowed due to the existence of an IRES
sequence in the viral genome [392, 396]. The 40S ribosomal subunit bind to
the IRES sequence by the help of a number of different viral translation
initiation proteins (depending on the picornavirus type), and the mRNA is
translated into a polyprotein. The polyprotein is then processed by the viral
proteases, 2A and 3C or 3CD.
Picornavirus infection triggers a rearrangement of intracellular membranes,
filling the cytoplasm with double-membraned vesicles [397]. The
cytoplasmic surface of these vesicles is the place for viral replication [398].
The positive-sense RNA is then copied by the 3D polymerase, using VPg as
its primer, into negative-sense RNA that carries VPg at its 5´end. This
negative-sense RNA then function as a template for production of new
positive-sense RNAs of which some in turn loses their VPg and are translated
into viral proteins whereas others are packaged into capsids during assembly
(see figure 11).
Assembly and release
Very little is known about the encapsidation of picornaviruses. The VPg
protein has been suggested to play a role in the encapsidation of the genome
since VPg-lacking RNAs are not packaged correctly [399, 400]. If this
actually result directly in defect packaging, or if there is a link between RNA
synthesis and packaging is not yet known. Moreover, each of the capsid
proteins interacts with the RNA suggesting that encapsidation takes place at
the pentamer level and that packaging of empty capsids does not occur [401,
402]. The final step of the assembly is the maturation of the capsid by
cleavage of VP0 into VP2 and VP4, which stabilizes the capsid. The
60
mechanism behind the maturation is not yet known [403]. The newly
produced virions normally leave the cells by lysis [404]. However, it has also
been suggested that picornaviruses are able to cause non-lytic, persistent
infections [405-407]. More recently, one of the innate responses to microbial
infections, autophagy, was suggested as a way for non-lytic release of
picornaviruses [408].
Figure 11. The poliovirus life cycle divided into different steps: 1) viral
attachment to the PVR, 2) uncoating of the viral RNA, 3) cleavage of the VPg
and cap-independent translation of the RNA, 4) proteolytic processing of the
polyprotein, 5) the positive-sense RNA serves as template for
complementary negative-strand synthesis, 6) initiation of many positive
strands from a single negative strand, 7) newly synthesized positive-sense
RNAs either lose their VPg and serve as templates for translation or, 8)
associate with capsid precursors to undergo encapsidation, 9) release of
progeny virions. Reprinted from [409].
61
Antivirals
Adenoviruses
Since the infections caused by Ads in otherwise healthy (immunocompetent)
individuals, are self-limiting and rather mild, the development of antivirals
have not been of great interest. Previously, the only type of infection where
antivirals were thought to be needed was for treatment of EKC. Later, an
increased knowledge about the severity of Ad infections in
immunocompr0mized patients has led to a boost in the development of anti-
adenoviral drugs [78, 410, 411]. However, no antivirals have yet been
approved for treatment of Ad infections.
The antivirals that are available today against double-stranded DNA viruses
all target the viral polymerase, and some of them have been evaluated for
treatment of Ad infections also [412]. One of these, cidofovir, which is an
acyclic nucleoside phosphonate seems to be the most effective [413]. The
effect of cidofovir has been tested against ocular infections in New Zeeland
rabbits caused by a number of Ads, but mainly from species C (Ad1, Ad5 &
Ad6) [414]. Unfortunately, the EKC-causing types (Ad8, Ad19 & Ad37) do
not infect these rabbits or any other animal model, which makes it difficult
to test the in vivo effect of anti-Ad drug candidates. Nevertheless, the
positive results from this and subsequent studies lead to a large clinical trial
in the US for the evaluation of cidofovir for treatment of ocular Ad
infections. Unpublished results from this study showed a significant efficacy
both in the clearance of the infection and protection from spread to the other
eye [78]. However, later usage in Europe and Hawaii was associated with
rare cases of lachrimal canalicular blockade. Thus, the narrow efficacy-to-
toxicity ratio lead to the discontinuance of this treatment [78].
CMX-001 is a lipid conjugate of cidofovir that is orally administered [415].
Normally, cidofovir is given intravenously due to the low bioavailability
when administered orally. By adding a lipid moiety the bioavailability of
62
orally administered cidofovir was improved. CMX-001 was shown in cell
culture to be a potent antiviral against all tested DNA viruses and animal
studies showed protection against orthopoxvirus, CMV and Ads [412].
Clinical studies are ongoing.
Zalcitabine, a nucleoside reverse transcriptase inhibitor (NRTI) used in the
treatment of human immunodeficiency virus (HIV) infections have been
reported to have an anti-Ad effect both in vitro and in vivo [416, 417]. The
effect of zalcitabine against ocular Ads indicated a more potent antiviral
effect than cidofovir and clinical evaluation of the ocular toxicity gave similar
result, indicating that zalcitabine would be safer to use than cidofovir [418].
A sialylated lipid called NMSO3 have been suggested to have anti-Ad
activity [419]. This compound is a non-toxic broad-spectrum compound with
an activity against a number of different viruses; respiratory syncytial virus
(RSV), HIV, influenza A virus and rotavirus [420-422]. It was shown to have
an effect against all the tested Ad types (Ad2, Ad4, Ad8 and Ad37), and
compared to cidofovir and zalcitabine, NMSO3 showed almost no cellular
toxicity. The effect was due to the interaction of NMSO3 with the virus and
not the cells, and most probably a charge dependent interaction with regard
to the negative charge of the compound. The charge dependent interaction is
the most likely in the case of Ad8 and Ad37, which normally bind to cellular
sialic acid, but the effect seen on the species C Ads (2 and 4) that normally
bind to CAR is not clear. Somehow the Ad-NMSO3 interaction blocks the
ability of the virus to interact with its receptor [419].
N-chlorotaurine (NCT) is the N-chloro derivative of the amino acid
taurine. This amino acid has previously been shown to have microbiocidal
effects against bacteria, yeast and mold [423, 424]. NCT has also been shown
to have an anti-Ad activity against types from species B, C, D and E (1, 2, 3,
4, 5, 7a, 8, 19 and 37) [425]. The anti-Ad effect in the Ad5/NZW rabbit
ocular model indicated a promising in vivo activity. Clinical trials have been
conducted with positive results, reduction of symptoms was seen especially
63
in the more severe infections but NCT could not prevent the formation of
subepithelial infiltrates [426, 427]. However, larger trials are needed to fully
evaluate the efficiency of NCT as an anti-Ad compound.
The immunosuppressant cyclosporine A (CsA) inhibits the transcription of
interleukine-2 (IL-2) and is used during organ transplantation to prevent
rejection [428, 429]. CsA has been evaluated as a treatment for ocular
infections caused by Ads [430, 431]. Topical treatment with 1% CsA reduced
the regular symptoms but also the corneal opacities and topical treatment
with 2% CsA reduced the subepithelial infiltrates in chronic cases [430, 432].
Povidone-iodine (PVP-I) is a common antiseptic used in general surgery
and ophthalmology. PVP-I is known to be toxic for viruses, parasites, fungi
and bacteria [433]. Dexamethasone is a steroid commonly used as an
ophthalmic anti-inflammatory agent [434]. By combining this steroid and
the antiseptic PVP-I a good inhibitory effect was seen on ocular Ad infections
both in a small clinical trial and in the Ad5/NZW rabbit ocular model [435,
436].
64
Picornaviruses
The development of antivirals against picornaviruses started during the 70s
and received a boost in the 80s when the first crystal structure was
determined for HRV-14, a member of the enterovirus genus, and the capsid
canyon was discovered [23]. This canyon was subject for targeting of
different compounds that disrupted viral binding or uncoating [175, 437].
These first compounds were later named WIN compounds, referring to the
Sterling-Winthrop company that developed the compounds [438, 439].
Modifications to increase the potency of these WIN-compounds led to the
development of a substance called Pleconaril [440]. This substance was
found to have a broad-spectrum activity against the prototype strains of EVs
and also against the most commonly isolated EV types and in 1996 the drug
was used in patients with life-threatening enterovirus infections [441, 442].
However, from 2002 the drug was disapproved by the Food and Drug
Administration because of its unconvincing safety profile [443].
A number of replication inhibitors have also been developed, targeting
protein 2C, 3A and 3D. The protein 2C inhibitor guanidine hydro-
chloride was one of the first reported picornavirus inhibitors [444]. The
effect of this compound is due to inhibition of negative strand RNA synthesis
by disruption of the 2C/2BC association with the cellular membrane during
replication [445, 446].
Two other replication inhibitors called EnvirX (Enviroxime) and EnvirD
(Enviradone) have been evaluated for the treatment of AHC-causing viruses
(EV70 and CVA24v) [447]. These compounds gave a significant block of viral
replication and cytopathic effect (CPE) at concentrations that were at least
10 times lower than the cytotoxic levels. However, none of the replication
inhibitors have been very successful since the effect seen in vitro is not
obtained in vivo.
65
Two different protease inhibitors have also been evaluated; Rupintrivir
and “Compound 1” [448, 449]. Although both of them showed promising
effects in initial studies the clinical trials were unsatisfying and the clinical
development of the drugs was terminated [450].
Another way of interfering with viral infections is by blocking the receptor
with antibodies to hinder viral attachment, or by adding soluble receptor
analogues, thus competing out the viral binding. Antibodies against the
major rhinovirus receptor ICAM-1 has been evaluated but the avidity of
these antibodies was judged to be insufficient [451, 452]. Further
development of these antibodies into recombinant high-avidity multivalent
antibodies is an ongoing and apparently promising project [453].
A new promising strategy of inhibiting virus replication is by means of small
interfering RNAs (siRNAs), which is a strategy used in the eukaryotic cells to
inactivate translation of specific proteins. The mechanism behind this is as
follows; cellular double-stranded RNA is used to produce small interfering
RNAs (siRNAs), these siRNAs are bound by RISC (RNA-induced silencing
complex) and directs the degradation of mRNAs containing sequences
complementary to one of the strands of the siRNA [454]. The development of
siRNAs to target the AHC-causing viruses (EV70 and CVA24v) have shown
some promising results [455]. The target for their siRNA is the homologous
regions within the viral polymerase (3D) gene.
66
Results and Discussion
Paper I
Multivalent sialic acid conjugates inhibit adenovirus type 37 from
binding to and infecting human corneal epithelial cells.
Johansson SM, Nilsson EC, Elofsson M, Ahlskog N, Kihlberg J, Arnberg N.
Antiviral Res. 2007. 73:92-100.
This study was a continuation of a previous study by Johansson et al. 2005
where the effect of multivalent 3´SLA (3´sialyl lactose) on Ad37 binding to
and infection of human corneal epithelial cells (HCE) was evaluated [456].
In that study the conclusions were that Ad binding and infection was
inhibited up to 1000 times more efficient with multivalent SA compounds as
compared to corresponding SA monosaccharide. At approximately the same
time, the Ad37 fk was crystallized together with 3´SLA and one intriguing
finding was that the only part of 3´SLA that interacted with the fk was the SA
monosaccharide [126]. No interactions were seen between the fk and the
neighboring galactose or glucose saccharides of the compound. These two
previous studies were the starting point for paper I.
Thus, in this study we wanted to see if the same effect could be accomplished
with the much simpler multivalent compound that was based on SA
monosaccharides coupled to HSA (human serum albumine) instead of
3´SLA-HSA. Initially, a number of SA-containing monovalent compounds
were tested and the results showed that mono-, di-, tri-SA as well as 3´SLA
had a similar effect on Ad37 binding to corneal cells. The IC50 values (50%
inhibition of binding compared to the control) of all compounds were
between 1 to 5 mM. Next, the multivalent SA-HSA compounds were
synthesized in a process similar to the one used for producing the
multivalent 3´SLA-HSA compounds. One major difference however, was the
number of steps in the synthesis, 3´SLA-HSA was produced in eleven steps
67
and SA-HSA synthesis only involved four steps. The synthesis was designed
to result in three compounds with varying multivalency: 3-, 8- and 13- SA
saccharides incorporated per HSA, which were further evaluated. The
inhibitory effect of the compounds was first evaluated in a binding assay
using radiolabeled virions. The results showed that SA-HSA were competent
inhibitors of Ad37 binding to HCE cells. The IC50 values (with respect to SA
monosaccharide) for the different compounds were: 5 mM for monovalent
SA, 0,2 mM for 3-valent, 10 µM for 8-valent and approximately 5 µM for the
13-valent compound. The IC50 values for mono- and 13-valent SA were
comparable with the corresponding results seen for mono- and 19-valent
3´SLA-HSA in the previous study. The effect of these new compounds was
then tested on Ad37 infection of corneal cells and as expected, the results
were similar to the ones from the binding assay. The IC50 value for the 8- and
13-valent SA-HSA was approximately 3 µM whereas monovalent SA however
did not reach 50% inhibition at concentrations up to 5 mM. The 3-valent
compound was not included in this experiment. The results from both the
binding and the infectivity assay showed a clear valency-dependent
inhibition. Based on the previously suggested hypothesis that the effect seen
of 3´SLA-HSA on Ad37 binding and infection was due to the ability of the
compound to aggregate virions we set up a new experimental method to try
to confirm and also measure the level of aggregation. The setup was to
incubate virions with or without (control) compound and then measure the
amount of radioactively labeled virions in the supernatant (top 90 µl) and in
the pellet (bottom 10 µl) after centrifugation at different speeds (g). In
samples with compound, centrifugation at increasing g lead to increased
amounts of virions in the pellet, and to decreased amounts of virions in the
supernatant. This was also seen in the controls (w/o compound), but to a
much smaller extent. For example, at the highest g the majority of virions in
the control were in the supernatant, whereas in samples with compound, the
majority of virions were found in the pellet. Thus, this simple assay
confirmed our hypothesis that the effect seen on Ad37 binding and infection
was in fact due to the aggregating ability of the compound.
68
From these data we concluded that SA-HSA is as efficient as the previously
studied 3´SLA-HSA in inhibiting Ad37 binding and infection. The
concentration needed to reach the IC50 was similar for both compounds and
was approximately 1000 times lower than the concentration required to
reach IC50 with SA monosaccharides. There was a clear correlation between
the order of valency and the effect seen. Using the aggregation assay we
could also conclude that the effect seen on viral binding and infection is
because of the ability of the compound to bind to several viral particles at the
same time and thereby aggregating them, which more efficiently prevent
binding of virions to cells. SA-HSA and 3´SLA-HSA were equally efficient
inhibitors, but since SA-HSA is synthesized in only four steps this molecule
would be easier and cheaper to produce and would therefore be a better
antiviral candidate than 3´SLA-HSA. This smaller SA-containing compound
may also be easier to systematically modify at different positions in order to
increase the affinity for the Ad37 fk as compared to the multivalent 3´SLA,
which is a more complex structure, and would therefore be more difficult to
modify since e.g. there are more groups to protect. Multivalent SA has been
shown to be an effective inhibitor of both viruses and bacteria in vitro [457,
458]. The problem with many of these drugs is that the drug is delivered to a
large organ that is difficult to reach. We present a candidate drug that targets
viruses that infect the eye, which is a small and accessible organ for topical
administration. In conclusion, we present here a new drug candidate type for
treatment of Ad-caused epidemic keratoconjunctivitis (EKC).
69
Paper II
Design, synthesis, and evaluation of N-acyl modified sialic acids
as inhibitors of adenoviruses causing epidemic
keratoconjunctivitis.
Johansson S, Nilsson E, Qian W, Guilligay D, Crepin T, Cusack S, Arnberg N,
Elofsson M.
J Med Chem. 2009. 52:3666-78.
In this paper we set out to evaluate the effect of modified SA on Ad37
binding to and infection of human corneal epithelial cells. The aim was to see
if the binding between the Ad37 fk and modified SA could be enhanced as
compared to native SA. If so, such results could be used for design and
development of more efficient antiviral drug candidates for treatment of
EKC. Structure-based design and docking studies (based on the known
crystal structure of Ad37 fk in complex with 3´SLA) were used to design a
small library of ten different SA-derivatives. In these derivatives, the N-
acetyl group was replaced by a number of different groups. According to the
structural data of the Ad37 fk-3´SLA complex, the N-acetyl group of SA was
directed into a deep hydrophobic pocket, and thus we hypothesized that the
inhibitory effect of SA could be enhanced by substituting this group with a
larger, more hydrophobic group.
The inhibitory effect of the compounds was evaluated in biological assays.
Three of the compounds inhibited binding of Ad37 virions to corneal cells.
These three compounds were 17a, which was predicted to be a good inhibitor
according to docking data, and 17e and 17g. 17a was equally efficient as SA
and the other two were slightly less efficient. All of the other SA-derivatives
with large N-acetyl substituents (17b, 17c, 17d, 17h and 18) were unable to
inhibit Ad37 binding. Five of the compounds, 17a, 17b, 17c, 17e and 17g were
then evaluated by their ability to inhibit Ad37 infection (17b and c were
70
included as negative controls). Once again, compounds 17a and 17g showed a
similar activity as SA, but compound 17e on the other hand had almost no
effect at all. Compounds 17b and 17c had no effect either in the binding or
infectivity assays. Next, compounds 17a-c was coupled to HSA in a similar
way as in the previous studies with SA-HSA, with a varying valency (14-17-
valent). These multivalent compounds were then evaluated for their ability
to block Ad37 infection and the results showed that the N-acyl modified SAs
all behaved as their monovalent counterparts. The only multivalent
compound that actively blocked Ad37 infection was compound 22a (which
was based on compound 17a) however, the inhibitory effect was not as good
as the effect seen with SA-HSA. The final step was then to co-crystallize the
fk together with the three active compounds 17a, 17e and 17g to get a detailed
picture of the exact interaction, and then compare this interaction with that
of SA-Ad37 fk. In the docking studies compound 17e was suggested to
interact with the fk in a different way as compared to SA. This was confirmed
by X-ray crystallography data, showing that 17e was not detected in the
known SA binding site or anywhere else in the fk protein. The effect seen by
this compound is most likely due to unspecific binding, or, due to binding to
another capsid protein. The structural data showed that compound 17a and
17g interacted with the fk in a similar way as SA. However, no additional
interactions were obtained due to the substitutions made.
In this study we evaluated the possibility to improve the binding between SA
and the Ad37 fk by substituting the N-acyl group of SA. Of the ten initial
compounds, three were shown to be as effective as SA in inhibiting binding
of Ad37 virions to HCE cells. Of these three compounds, only two had an
inhibitory effect comparable to SA in the infectivity assay. The
corresponding, multivalent conjugates showed a valency-dependent
inhibition of Ad37 infection, although not as efficient as SA-HSA. Finally, co-
crystallization data confirmed that the modified compounds that displayed
an inhibitory effect interacted with the fk in the same way as unmodified SA.
The conclusions were however, that the most promising anti-Ad compound
was the previously tested SA-HSA. In order to obtain a more efficient
71
inhibitor of Ad37, we conclude that more extensive structure-activity studies
are required. An alternative strategy for development of successful inhibitors
might be needed due to the complicated synthesis of modifying SA. A
possible strategy could be the use of non-carbohydrate sialyl mimetics,
which would require less complicated synthesis and a greater possibility of
varying the functional groups, or, a trivalent SA with three spacers designed
to fit into the three known SA binding sites.
Paper III
Sialic acid is a cellular receptor for coxsackievirus A24 variant, an
emerging virus with pandemic potential.
Nilsson EC, Jamshidi F, Johansson SM, Oberste MS, Arnberg N.
J Virol. 2008. 82:3061-8.
Acute hemorrhagic conjunctivitis (AHC) is a severe ocular infection caused
primarily by two members of the picornavirus family: Coxsackievirus A24
variant (CVA24v) and enterovirus 70 (EV70). These viruses have since they
were first discovered in 1969 caused three pandemics affecting probably
more than 100 million people [25, 104]. Previously, EV70 was the main
causative agent but during the past 10 to 15 years most of the outbreaks have
been caused by CVA24v. Two receptors for EV70 have previously been
identified, a) SA on human corneal epithelial cells and on human monocytes
(U937) and b) CD55/DAF on Hela cells [104, 225]. In this study we set out to
identify the so far unknown receptor for the other AHC-causing
picornavirus, CVA24v. First we evaluated the ability of CVA24v to bind and
infect different CHO cells that express specific, known picornavirus and Ad
receptors. CVA24v bound almost equally well to all CHO-cells expressing
CAR, CD55/DAF, CD46/MCP, ICAM-1 and also GAG deficient (pgsB-618)
cells. Lec2 cells however, which are deficient in SA expression showed a
72
much weaker binding of CVA24v. When SA was enzymatically removed from
the CHO cells, a strong reduction in binding was observed for all cells, except
for Lec2 where only a 10 % reduction of the originally low binding was
observed. An infectivity assay was then performed to investigate if SA was
important also for the infection and again, CVA24v infected all CHO-cells to
an equal extent except for the Lec2 cells, which were more or less resistant to
infection. These initial results suggested that SA play a role in CVA24v
binding and infection.
To find suitable cell lines for further tests we first evaluated a number of
epithelial cells that had been used previously for isolation of CVA24v, for
their susceptibility to virus binding and entry and also expression of known
picornavirus receptors: Hep2, Hela, A549 and green monkey kidney cells
(GMK). Also, two cell lines that represent the ocular tropism of this virus
were included: human corneal epithelial cells (HCE) and normal human
conjunctival cells (NHC). Here we found that the cells most susceptible to
infection were the NHC and HCE cells together with the larynx epithelial cell
line Hep2. CVA24v also showed the highest binding to two of these, NHC
and Hep2. Expression of cell surface receptors by flow cytometry showed
that NHC cells had, in comparison to the other epithelial cells, the highest
expression of the following known picornavirus receptors: SA, CAR,
CD55/DAF, CD46/MCP and ICAM-1. The ocular cell lines NHC and HCE
had SA expression levels approximately three to four times higher than the
other cell lines. The receptor identified for other Coxsackie A viruses, ICAM-
1, was almost only expressed on NHC cells. Based on these findings HCE,
NHC and Hep2 cells, representing the ocular and respiratory tropism of
CVA24v, were used in further studies.
To test if SA was a functional attachment receptor for CVA24v, NHC, HCE
and Hep2 cells were treated with neuraminidase to remove cell surface SA,
and then incubated with isotope-labeled CVA24v virions. A substantial
reduction in virus binding was seen on CVA24v binding to HCE cells (85%)
and Hep2 cells (58%) but only a modest decrease was seen with the NHC
73
cells (28%). Further, incubation of cells with the SA-binding lectin wheat
germ agglutinin (WGA) resulted in a strong reduction in CVA24v binding to
HCE (93%), but not to NHC or Hep2 cells (46% and 30% respectively). The
effect of neuraminidase and WGA was also evaluated in an infectivity assay
showing a strong inhibitory effect with both neuraminidase and WGA using
HCE cells (90% in both cases), but only minor inhibitory effects were
observed with the other two cell types. The previously discussed 13-valent
SA-HSA, which is a very potent inhibitor of Ad37 binding to and infection of
HCE cells, was also evaluated for its ability to inhibit CVA24v binding to
ocular cells. The binding to NHC cells was not affected at all but the binding
to HCE cells was reduced by more than 50% at the highest concentration
(0,5 mM with respect to SA). This effect was however not at all in the same
range as the effect seen for Ad37 binding where the IC50 was at a
concentration of approximately 5 µM. Thus, SA-HSA was thereby considered
not to be a suitable candidate for treatment of CVA24v-caused AHC. A
possible explanation for the lack of effect of SA-HSA on CVA24v binding
could be that SA, when coupled to HSA, is less able to access the binding site
on the viral capsid.
Since other viruses with an ocular tropism have been shown to have a
preference for SA linked via α2,3 glycosidic bonds to neighboring galactose
(SAα2,3Gal) [459], we set out to investigate if this was also the case for
CVA24v. We first used two types of lectins, maackia amurensis lectin type II
(MALII), which recognizes SAα2,3Gal-containing glycans, and the sambucus
nigra (SNA) lectin, which recognizes SAα2,6Gal-containing glycans.
SAα2,3Gal was the dominant type present on the surface of NHC and HCE
cells, according to flow cytometry analysis. When trying to inhibit viral
binding to these cells, MAA only displayed a small inhibitory effect on
binding to HCE cells and the opposite was seen for the NHC cells, were only
SNA exerted a small inhibitory effect. A pronounced inhibitory effect on
CVA24v binding was however seen when the two lectins were mixed: 67%
reduction for NHC and 66% for HCE. Finally, in order to test whether the
SA-containing receptor(s) were linked to either a protein or to a lipid, we
74
first treated cells with proteases prior to the addition of isotope-labeled
virions. The use of three proteases resulted in a significant reduction of
CVA24v binding, thus suggesting that CVA24v uses a sialylated protein
rather than a lipid as a cellular receptor.
In this work we investigated whether any of the previously known
picornavirus or Ad receptors were used by CVA24v. Using receptor-
expressing CHO cells we found that CVA24v interacted with SA and not with
any of the other known receptors. From experiments where we i)
enzymatically removed cell surface SA, ii) blocked cell surface SA with SA-
binding lectins, or iii) pre-incubated virions with soluble SA-containing
molecules, we concluded that the major receptor for CVA24v on CHO cells
and on human corneal epithelial cells contain SA. Regarding cells originating
from conjunctiva or larynx, SA contributes to some extent to binding and
infection but on these cells SA does not seem to be a main receptor. From
experiments where cells were treated with proteases, we also concluded that
the receptor used by CVA24v on corneal and conjunctival cells most likely
contain one or more protein component(s), and gangliosides are less likely to
be used as receptors for CVA24v. It has been demonstrated that SA-binding
viruses have a tendency to use either SAα2,3Gal or SAα2,6Gal as cellular
receptors, and the general idea has been that respiratory viruses like human
influenza A uses SAα2,6Gal whereas ocular viruses such as EV70 and Ad37
prefer SAα2,3Gal. The distribution of SA can to some extent explain the
tropism for different SA-binding viruses since it is known that the most
frequent SA type found on corneal and conjunctival cells is SAα2,3Gal, and
in the respiratory tract the most frequently found SA type is SAα2,6Gal
[460]. For CVA24v a pronounced linkage could not be established since the
virus seems to be able to interact with both types of SA, which we found a bit
puzzling in the beginning. However, whereas the other AHC-causing virus,
EV70, is not associated with respiratory disease, CVA24v is more often
coupled to respiratory symptoms [81, 83, 84, 461]. Therefore, our results
indicating a possible interaction with both SA2,3Gal and SA2,6Gal actually
matches the tropism of CVA24v.
75
Paper IV
The GD1a glycan is a cellular receptor for adenoviruses that cause
epidemic keratoconjunctivitis
Emma C. Nilsson, Rickard Storm, Johannes Bauer, Susanne M. C.
Johansson, Aivar Lookene, Jonas Ångström, Mattias Hedenström, Lars
Frängsmyr, Hugh Willison, Fatima Pedrosa Domelöf, Thilo Stehle and
Niklas Arnberg.
Nat Med. 2011. 17:105-9.
Ad37 is one of the main causative agents of the ocular infection called
epidemic keratoconjunctivitis (EKC) and has previously been reported to
interact with two different receptors: SA and CD46 [207, 209]. However, so
far only SA has been confirmed by others to be a functional receptor [462-
464]. The main goal with this study was to identify the exact structure of the
SA-containing glycan that function as a receptor for Ad37. The starting point
was a glycan array performed by the Consortium for Functional Glycomics
(CFG), where Ad37 fk interaction with a library of 260 different glycans was
investigated. The array resulted in a number of hits, which represented
sialylated plasma proteins (that were not investigated further), sulfated
glycans (that were found to not prevent Ad37 binding) and one interesting
hit corresponding to the disialylated glycan found in ganglioside GD1a. To
test if the GD1a glycan was a functional receptor for Ad37, a number of
binding and infection assays were performed. We found that soluble GD1a
glycans and monoclonal antibodies against GD1a (clone EM9) each inhibited
viral binding to and infection of HCE cells. Soluble GD1a glycans also
blocked Ad37 fk binding to cells and in situ staining of corneal tissue showed
binding of both Ad37 fk and EM9. These results together suggested that
Ad37 binds to cell surface molecules that contained the GD1a glycan motif.
To find out if the receptor actually was the GD1a ganglioside we performed a
number of assays: a) a combinatorial glycolipid array was performed to see if
76
there was any interaction between the Ad37 fk and GD1a or any other
gangliosides; b) ELISA with immobilized GD1a and soluble fk; c) viral
binding to Lec2-cells pre-incubated with or w/o GD1a ganglioside; d) viral
binding to HCE cells in which the de novo ganglioside synthesis was
inhibited; e) pre-incubation of virions with GD1a-containing liposomes prior
to binding experiments. However, none of the above experiments indicated
that ganglioside GD1a would be a cellular receptor for Ad37. On the other
hand, protease treatment of cells as well as cellular inhibition of de novo O-
linked glycosylation significantly reduced viral binding, leading to the
conclusion that the receptor used by Ad37 is not the ganglioside GD1a but
rather one or more proteins that carry the GD1a glycan motif, or, a very
similar motif.
To better understand the Ad37 fk-GD1a interaction we first modeled the
glycan onto the X-ray crystallography-based Ad37 fk model obtained
previously [126]. This led to a preliminary model indicating that both SAs of
the GD1a glycan interacted with two out of the three known SA binding sites
in the Ad37 fk. To further confirm this model, a saturation transfer
difference (STD) NMR was performed. Large methyl peaks were seen,
corresponding to the acetamide groups of the two SAs (Neu5AcA and
Neu5AcB), which were not seen with fk alone or with fk incubated with
galactose, thus indicating a close contact between both SAs in GD1a and
Ad37 fk. This was also confirmed by resolving the protons H3eq and H3ax
for Neu5AcA and Neu5AcB. The large methyl peak probably resulted from an
overlap of the two Neu5Ac peaks. Thus, all peaks in the STD spectrum of the
Ad37 fk-GD1a were directly related to an interaction between these
molecules.
To verify the results from the modeling and the STD-NMR we also solved
the crystal structure for Ad37 fk in complex with GD1a at 1.65 Å resolution.
This confirmed our previous results that the fk engages one single GD1a
glycan and that both SAs bind similarly to two of the three known binding
sites. The amino acids directly involved in the binding are: Tyr312, Pro317
77
and Lys345. In silico substitutions suggested that Lys345 played a major role
in the interaction between GD1a and the Ad37 fk protein. This was later
confirmed by flow cytometry analysis with wt Ad37 fk and Lys345Ala
mutants. By substituting Lys345 with Ala the SA-dependent binding was
lost.
The SA-Ad37 fk interaction between the highly positively charged fk (pI:9,1)
and the negatively charged SA (pKa:2,6) is charge-dependent [282]. As
described previously, this interaction is quite weak, with a Kd of 5 mM, which
can be compared to the affinities known for the interactions between CAR
and many other Ad fk:s (nM range) [465]. The affinity between the Ad37 fk
and the GD1a glycan was analyzed by surface plasmon resonance (SPR). The
results showed that the association rate constant was different at high and
low glycan concentrations, one interaction was of high affinity (19 µM:
measured at low glycan concentrations) and one interaction was of low
affinity (265 µM: measured at high glycan concentrations), suggesting that
the GD1a glycan binds to two different parts of the fk protein. The fact that
the high affinity binding was more than 250-fold higher than the SA-Ad37 fk
interaction is probably due to the fact that both GD1a SAs interact with the fk
at the same time. The low affinity binding on the other hand needs to be
investigated further. The fact that only one GD1a molecule interacts with one
fk at a time (1:1; according to structural data) suggested that the
internalization of Ad37 is not dependent on receptor clustering as could be
the case for CAR- and CD46-binding Ads, which are able to interact with
three receptor molecules/fk (3:1). GD1a binding capacity seems to be a
conserved feature among the species D Ads as shown by flow cytometry
analysis with fk proteins from Ad8, 9, 19p, 19a and 37. Soluble GD1a
prevented all these fk:s but not species C Ad5 fk from binding to HCE cells.
However, Ad9 and 19p are not associated with EKC, indicating that the
ability to bind GD1a is not a determinant of the ability to cause EKC. To
confirm this, the ability of GD1a to inhibit infection of these species D types
would need to be evaluated.
78
In this work we have identified the GD1a glycan motif as a receptor for Ad37
using a number of different methods. We concluded that the cellular
receptor for Ad37 is not the ganglioside GD1a but rather one or more
glycoproteins carrying the GD1a motif, or, a highly similar motif. We could
also confirm that both SAs of the glycan interacted with the knob, leading to
a relatively high affinity binding (µM range). The detailed structural and
functional analysis of the interaction presented here might be useful for the
design and development of antiviral drugs for treatment of EKC. A small
(drug-like) compound occupying all of the three SA binding sites could
perhaps be an even more potent inhibitor than the previously discussed SA-
HSA. A smaller compound may also be easier to synthesize and therefore
cheaper to produce than the large and complex SA-HSA.
79
Concluding remarks
Virus-caused ocular infections are problematic in many parts of the world,
especially in densely populated areas. EKC and AHC are highly contagious
and severe ocular infections caused by members of the adenovirus and
picornavirus families, respectively. Millions of people fall ill every year due to
these viruses, resulting in substantial economic losses. Despite this, there are
no antiviral drugs available to treat these infections.
I have evaluated the inhibitory effect of multivalent sialic acid-containing
compounds on Ad37 binding and infection and could conclude that these are
potent inhibitors. The effect seen of these compounds were highly dependent
on the order of valency of sialic acid. We also concluded that the mechanism
of action of these compounds were due to aggregation of virions.
By structural modifications of native sialic acid we tried to improve the
binding capacity between Ad37 and sialic acid. Ten different compounds
were biologically evaluated but only two of them had an inhibitory effect on
Ad37 infection similar to the effect seen with native sialic acid. These
compounds were then coupled to human serum albumin in a multivalent
fashion but none of them were as potent inhibitor as the previously
evaluated multivalent compound based on native sialic acid.
The picornavirus member coxsackievirus A24v (CVA24v) is one of the main
causative agents of AHC. We identified sialic acid as a cellular receptor for
this ocular virus and, also found that CVA24v is, unlike many of the other
sialic acid-binding viruses, able to use both SAα2,3Gal- and SAα2,6Gal-
containing glycans as receptors. This ability is in agreement with the tissue
distribution of these glycans as well as the tropism of CVA24v.
Finally, we have in detail characterized the sialic acid-containing receptor
used by Ad37. We show that the branched, di-sialic acid containing glycan
motif of ganglioside GD1a is a functional receptor for Ad37 in vitro. By using
80
a number of methods we concluded that the receptor is not the GD1a
ganglioside, but rather one or more proteins containing the GD1a glycan
motif, or, a highly similar motif. Based on structural analysis, STD-NMR and
X-ray crystallography we also showed that both sialic acids of the GD1a
glycan dock into the binding cavity of the Ad37 fk, thus occupying two of the
three known sialic acid-binding sites. This would also explain the relatively
high affinity binding, for being a protein-carbohydrate interaction, that we
see (19 µM).
These results show that there seem to be a common receptor for these EKC-
and AHC-causing viruses, namely, sialic acid. By studying the sialic acid-
virus interaction in detail we can gain knowledge that will help to design and
develop antivirals for treatment of ocular infections.
81
Acknowledgements
Tack till alla underbara människor som jag har haft möjligheten att lära
känna under resans gång samt alla nära och kära som har stöttat mig.
Först av allt ett stort tack till min fantastiska handledare Niklas. Din
positiva anda och ditt brinnande intresse för forskningen har smittat av sig.
Jag är otroligt imponerad över allt som du har åstadkommit. Jag är glad och
tacksam för de möjligheter som du har gett mig samt allt det som jag har fått
uppleva under alla år på virologen. Tack!
Göran, det var du som väckte intresset för virus hos mig genom någon av
dina inlevelsefulla föreläsningar. Få personer har den omfattande kunskap
om virus, i synnerhet adenovirus, som du. Tack för dina uppmuntrande ord,
kluriga frågor och tänkvärda kommentarer.
Mina biträdande handledare, Magnus du finns alltid där för att svara på
frågor om det mesta. Tack för att du uppmärksammar mig på lämpliga
kurser att gå. Johan, du brinner verkligen för forskningen och vi visste nog
redan för länge sedan att du skulle lyckas med något stort.
Ett stort tack till dig Katrine för allt du ordnar, det är papper som ska fyllas
i, saker som ska bokas och inte minst svar på alla frågor som man har. Du
gör dessutom alltid detta med ett leende på läpparna.
Kickan och Karin för att ni alltid finns där och svarar på de frågor som
man kommer med. Ni vet allt som man behöver veta om vad som ska göras
på lab. En extra kram till dig Kickan, som har blivit en del av vår numera
stora grupp, för allt jobb du har gjort när det gäller Nature-peket.
Mina fantastiska vänner, Cissi och Emma, ni har varit saknade under
vintern även om vi har träffats lite då och då. Ni har bidragit till att jag alltid
har haft roligt på jobbet, även om själva ”jobbet” inte alltid har gått som man
har velat. Cissi, jag tror aldrig att jag har träffat på en sådan glädjespridare
som du. Det gör att du kommer att lyckas med allt du tar dig för och du
kommer att bli en mycket omtyckt läkare. Jag bokar in mig redan nu
Emma, en mer träningstokig och påhittig tjej får man nog leta efter. Du har
en sådan energi och härlig inställning till det mesta. Jag funderar fortfarande
ibland på vad han tänkte när det dök upp två små tjejer (Emma och Emma)
med släpvagn (som vi dessutom inte kunde köra med ) för att hämta det
82
ganska så tunga skåpet. Jag hoppas verkligen att du snart hittar tillbaka till
ditt rätta element, virus!
Carin, Annasara, Maria och Anna, mitt ”nya” kaffe-gäng. Stort tack till
er, ni förgyller min vardag. Annasara, för alla pratstunder under den
”gemensamma” graviditeten samt trevligt resesällskap och barnlek. Carin,
för ditt glada humör och för att du ställer upp vid alla tillfällen. Maria, för
att du orkar med allt prat om barn och vabbande, och Anna för att du har
fört in lite ”nytt blod” här på virologen.
Rickard, vilken tur att du dök upp när du gjorde, precis när jag skulle vara
mammaledig. Du tog över där jag lämnade och drev projektet framåt fastän
jag inte var där. Fantastiskt att Ad37 projektet får fortsätta några år till nu
när jag slutar.
Nitte, det är tur att någon har tagit hand om mitt kära coxsakievirus så att
det lever vidare ett tag till. Tack också för att du emellanåt förgyller vår
vardag med dina härligt spydiga mail.
Fariba, för all den tid och arbete som du lade ner på coxsackievirus-
projektet. Stort tack även till Ya-Fang, Fredrik, Mari, Therese, Lars,
Marko, Sharvani, Johanna, Jim, Mårten, Jonas, Karin, Janne,
Lisa och Dan för den trevliga stämning som ni alla bidrar till.
Ett stort tack till alla på landstinget, trevliga luncher, vinlotteri med
vinprovningar och framförallt, det efterlängtade fredagsfikat. Tack även till
alla de studenter som har hjälpt mig under åren.
Tack Sussi och Mikael Elofsson för allt som har med kemi att göra. Extra
stort tack till dig Sussi för allt samarbete, jag insåg att ditt namn finns med
på alla mina publikationer
Soffe och Hasse, vi ses alldeles för sällan men ändå så känns det som igår
när vi ses. Soffe, du är min äldsta och bästa vän. Minns du alla våra
omvägar hem från skolan, eller vår gemensamma partygarderob på Nydala
. Tänk att även vi har blivit vuxna, det trodde jag nog aldrig. Hasse, jag
har nog aldrig träffat någon som är så härligt tankspridd som du. Ni är
underbara!
Nicke och Anneli, tack för att ni har varit vårt trevliga rese-sällskap under
alla år. Vi börjar ha gjort några resor tillsammans, även om det nu är mer än
två år sen senast. Det lilla uppehållet kan ju ha berott på att det har
tillkommit två underbara och fantastiska små killar.
83
Elisabeth, Rebecca, Lars, Andreas, Elisabeth, Vickan och Ida min
fina ”extra familj”. Jag minns fortfarande den varma och välkomnande
känslan som infann sig första gången jag hälsade på hos er för snart 13 år
sedan. Rebecca, min vän du är saknad här i Umeå. Tack för alla trevliga
middagar, pratstunder och träningspass. Kom bara ihåg, du är underbar och
unik och kommer att lyckas med allt du vill här i livet.
Många kramar och tack till mormor Margit och farmor Gunnel. Ni har
under åren snällt frågat vad det är jag håller på med och förklarat för mig
hur viktigt det är för forskningen det just jag håller på med. För att göra min
vardag lite lättare så har ni slitit i köket för att skicka påsar med alla sorters
kakor och även världens godaste palt.
Petra, min kära syster. Jag sörjer att vi bor så långt ifrån varandra, tänk så
mycket shopping vi skulle kunna göra om vi sågs lite oftare, och tänk vad
roligt det hade varit för våra finaste pojkar att kunna leka med varandra. Jag
skulle önska att jag hade fått en gnutta av din energi och spontanitet, du är
underbar! Glöm aldrig det!
Kerstin och Michael, mina älskade föräldrar, tack för allt ert stöd. Utan er
hade detta inte varit möjligt. Ni har under alla år gjort allt för mig, mer än
vad jag någonsin förväntat mig. Det är en fantastisk trygghet att veta att vad
som än händer så finns ni där och ställer upp för mig och min lilla familj.
Min lilla glädjespridare Hjalmar, du är meningen med livet. Hur trött jag
än är så glömmer jag det när jag ser ditt leende och den kärlek som du
utstrålar. Mitt livs kärlek David, du är underbar, fantastisk, stöttande och
uppmuntrande, du är min bästa vän. Jag har dig att tacka för allt. Du är min
trygghet här i livet. Jag älskar dig! Kaan Naan!
84
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