Effect of Rhinovirus Infection on the Host Apoptotic · PDF fileEffect of Rhinovirus Infection...
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Effect of Rhinovirus Infection on the Host Apoptotic Response Kylia Merinda Wall B. Med. Sci Centre for Research in Therapeutic Solutions University of Canberra ACT 2601 A thesis submitted in partial fulfilment of the requirements for the degree of Bachelor of Applied Science (Honours) at the University of Canberra October 2013
Transcript of Effect of Rhinovirus Infection on the Host Apoptotic · PDF fileEffect of Rhinovirus Infection...
Effect of Rhinovirus Infection on the Host
Apoptotic Response
Kylia Merinda Wall
B. Med. Sci
Centre for Research in Therapeutic Solutions
University of Canberra ACT 2601
A thesis submitted in partial fulfilment of the requirements
for the degree of Bachelor of Applied Science (Honours) at
the University of Canberra
October 2013
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© Kylia Merinda Wall 2013
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Abstract
The human rhinoviruses (HRV) are the most common viral cause of upper respiratory
tract infections and are known to cause asthma exacerbations which may have serious
consequences. One of the host’s defences against viral infection is the induction of
apoptosis, a form of programmed cell death that acts to eliminate the virus with minimal
impact on surrounding cells. It is thought that HRV may delay the induction of
apoptosis in infected cells in order to facilitate its own replication. Previous studies have
shown that some picornaviruses, including the closely related poliovirus, are capable of
regulating induction of the apoptotic pathway.
This study aimed to identify the effect of HRV infection on the apoptotic pathway of
infected cells. Ohio-HeLa cells infected with HRV16 of varying multiplicity of
infections (MOIs) and incubation periods analysed by western blot showed no evidence
of PARP, caspase 3 or caspase 9 cleavage, demonstrating that apoptosis was not
induced. Immunofluorescence assay showed that cytochrome c was not released from
the mitochondria of HRV16 infected cells suggesting that the intrinsic apoptotic
pathway is not induced, even at an early stage.
Investigation of the effect of HRV16 infection on cells treated with the chemical
inducer of apoptosis, Actinomycin D (Act. D), demonstrated that HRV16 may actively
suppress the host apoptotic pathway. A reduced number of apoptotic cells were
observed in cells infected prior to treatment when compared to cells treated with Act. D
alone. It was found that HRV16 infection resulted in the cleavage of the extrinsic
apoptosis intermediate RIPK1 dissimilar to that seen during regular apoptotic induction.
It was found that interestingly, the HRV16 proteases 2A and 3C do not appear to be
responsible for this cleavage.
These results suggest that not only does HRV16 infection avoid apoptotic death, but
that it is capable of actively suppressing the host apoptotic pathways, potentially
through the inhibition of the extrinsic pathway via the indirect alternate cleavage of
RIPK1.
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Acknowledgements
I would like to express my deep appreciation and gratitude to my supervisor Dr. Reena
Ghildyal. She has provided me with endless support and encouragement throughout and
she has pushed me to develop far beyond my expectations.
I have received generous help, support and encouragement from everyone in CResTS in
particular the RVG team, Dr. Erin Walker, Lora Jensen, Dr. Deborah Heydet and Robert
McCuaig.
I would also like to acknowledge Dr. Scott Bowden of VIDRL Melbourne and Dr. Wai-
Ming Lee of University of Wisconsin for their kind donation of antibodies used
throughout this study.
Lastly I would like to thank my husband Josh for all of his support, patience and
encouragement.
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Table of Contents
Abstract ........................................................................................................................................ iii
Acknowledgements ....................................................................................................................... v
List of figures ............................................................................................................................. viii
List of tables ................................................................................................................................. ix
1 Chapter One - Introduction ................................................................................................... 1
1.1 Introduction ................................................................................................................... 1
1.2 Picornaviruses ............................................................................................................... 1
1.2.1 Structure ................................................................................................................ 3
1.2.2 Genome ................................................................................................................. 3
1.2.3 Polyprotein and Protein Processing ...................................................................... 4
1.2.4 Viral Attachment and Entry .................................................................................. 6
1.2.5 Viral Replication ................................................................................................... 8
1.2.6 Viral Assembly and Exit ....................................................................................... 8
1.2.7 Viral Effect on Cellular Processes ...................................................................... 10
1.3 Rhinovirus ................................................................................................................... 12
1.3.1 Classification ....................................................................................................... 12
1.3.2 Epidemiology ...................................................................................................... 13
1.3.3 Clinical Disease................................................................................................... 14
1.4 Apoptosis .................................................................................................................... 15
1.4.1 General Apoptosis ............................................................................................... 15
1.4.2 Viruses and Apoptosis ........................................................................................ 18
1.4.3 Picornaviruses and Apoptosis ............................................................................. 20
1.4.4 Rhinovirus and Apoptosis ................................................................................... 22
1.5 Hypothesis and Aims of Present Study ....................................................................... 24
1.5.1 Hypothesis ........................................................................................................... 24
1.5.2 Aims .................................................................................................................... 24
2 Chapter Two – Materials and Methods ............................................................................... 26
2.1 Materials ..................................................................................................................... 26
2.1.1 Virus and Cell Culture Lines .............................................................................. 26
2.1.1.3 Media Used for Cell Culture and Viral Infection........................................................ 26
2.1.2 Buffers, Solutions and Bacterial Culture Media ................................................. 27
2.1.3 Antibodies ........................................................................................................... 28
2.1.4 Commercial Kits ................................................................................................. 29
2.2 Methods....................................................................................................................... 29
2.2.1 Cell and Virus Cultures ....................................................................................... 29
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2.2.2 Analysis ............................................................................................................... 32
3 Chapter Three – Results ...................................................................................................... 34
Rhinovirus Infection Does Not Induce Early or Late Intrinsic Apoptosis .................................. 34
3.1 Introduction ................................................................................................................. 34
3.2 HRV16 Infection Does Not Result in Cleavage of PARP .......................................... 35
3.3 Rhinovirus Serotype 16 Infection Does Not Induce Late Apoptosis .......................... 36
3.4 RV16 Infection Does Not Induce Early Intrinsic Apoptosis ...................................... 40
3.5 Taxol Induces Apoptosis in Ohio-HeLa Cells ............................................................ 43
3.6 Actinomycin D Induces Apoptosis in Ohio-HeLa Cells ............................................. 45
3.7 Summary ..................................................................................................................... 49
4 Chapter Four – Results ........................................................................................................ 50
Effect of HRV16 Infection on Chemically Induced Apoptosis .................................................. 50
4.1 Introduction ................................................................................................................. 50
4.2 Effect of HRV16 Infection on Taxol Induced Apoptosis ........................................... 50
4.3 Effect of HRV16 Infection on Act. D Induced Apoptosis .......................................... 53
4.4 Summary ..................................................................................................................... 57
5 Chapter Five – Results ........................................................................................................ 58
Rhinovirus Infection Leads to the Indirect Cleavage of RIPK1 ................................................. 58
5.1 Introduction ................................................................................................................. 58
5.2 HRV16 Infection and Act. D Treatment Lead to Dissimilar Cleavage of RIPK1 ...... 58
5.3 Cleavage of RIPK1 is Probably Not Carried Out By the 2A or 3C Proteases ............ 60
5.4 Expression of 2A and 3C Proteases Does Not Induce Apoptosis ............................... 61
5.5 Summary ..................................................................................................................... 63
6 Chapter Six – General Discussion ...................................................................................... 64
6.1 Introduction ................................................................................................................. 64
6.2 HRV Infection Does Not Induce Apoptosis................................................................ 64
6.3 HRV16 Inhibits Apoptosis .......................................................................................... 67
6.4 HRV16 Infection Leads to Indirect Cleavage of RIPK1 ............................................ 68
6.5 Conclusion .................................................................................................................. 69
References Cited ......................................................................................................................... 70
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List of figures
Figure 1.1 An example of the picornaviral genome……………………………………………...4
Figure 1.2 Post translational processing of the HRV polyprotein………………………………..5
Figure 1.3 An overview of the picornavirus lifecycle…………………………………………..10
Figure 1.4 Schematic diagram of the extrinsic and intrinsic apoptotic pathways as relevant to
picornavirus infection. ………………………………………………………………………….16
Figure 3.1 HRV16 infection does not lead to cleavage of PARP…………………………..…..36
Figure 3.2 Detection of late apoptosis during RV16 infection…………………………...……..39
Figure 3.3 Effect of varying MOI on the detection of late apoptosis during RV16 infection.….40
Figure 3.4 Cytochrome c is not released during RV16 infection…………………………….....42
Figure 3.5 Taxol treatment induces intrinsic apoptosis in Ohio-HeLa cells…………………....44
Figure 3.6 Taxol treatment leads to cytochrome c release from the mitochondria……………..45
Figure 3.7 Act. D treatment induces apoptosis in Ohio-HeLa cells………………………….…47
Figure 3.8 Act. D treatment leads to cytochrome c release from the mitochondria…………….48
Figure 4.1 Taxol treatment during HRV16 infection may reduce expression of VP2………….52
Figure 4.2 Taxol treatment during HRV16 infection may lead to a reduction in the translation of
the viral polyprotein…………………………………………………………………………….53
Figure 4.3 Act. D treatment during HRV16 infection does not reduce expression of VP2…….55
Figure 4.4 HRV16 infection reduces the induction of apoptosis in Act. D treated cells……….56
Figure 5.1 HRV16 and Act. D treatment leads to cleavage of RIPK1………………………….60
Figure 5.2 HRV16 2A and 3C proteases do not cleave RIPK1 or caspase 3…………………...62
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List of tables
Table 1.1 Cellular receptors utilised by various picornavirus species……………………...……7
Table 2.1 Cell and virus culture media used during this study………………………………....26
Table 2.2 Buffers and solutions used during this study………………………………………...27
Table 2.3 Western blot primary antibodies used during this study………………………..……28
Table 2.4 Western blot secondary antibodies used during this study…………………………...28
Table 2.5 Immunofluorescence primary antibodies used during this study……………….……28
Table 2.6 Immunofluorescence secondary antibodies used during this study………………….29
Table 2.7 Commercial kits used during this study……………………………………………...29
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1 Chapter One - Introduction
1.1 Introduction
Human rhinovirus (HRV), a member of the Enterovirus genus of family Picornaviridae,
is the most common viral cause of upper respiratory infections and is known to be
associated with asthma and chronic obstructive pulmonary disease (COPD)
exacerbations which may result in serious complications, even death (Johnston et al.,
1995; Seemungal et al., 2000). Picornaviruses are small, non-enveloped RNA viruses
which feature a single strand of positive sense RNA surrounded by an outer capsid.
They are responsible for a very wide variety of diseases in humans and other vertebrates
including poliomyelitis, liver disease, cardiomyelitis, respiratory illness and foot-and-
mouth disease. One of the host’s defences against viral infections is the induction of
apoptosis via a range of virus associated triggers. Previous studies investigating the
induction of apoptosis during picornavirus infections have shown evidence that the
induction of apoptosis can be either promoted or suppressed under various conditions or
at different time points post infection. It is hypothesised that HRV may delay the
induction of apoptosis in order to facilitate its own replication. Previous studies have
shown that Enterovirus C (poliovirus), another member of the Enterovirus genus of the
Picornaviridae family which is very closely related to HRV, is capable of delaying the
induction of the apoptotic pathway under optimal viral replication conditions whilst also
showing evidence of promoting apoptosis induction when conditions are not ideal.
These results, and the similarities seen between HRV and poliovirus, support the
hypothesis that the HRV is also capable of altering the host apoptotic response for its
own advantage.
1.2 Picornaviruses
The Picornaviridae are a family of viruses belonging to the Picornavirales order of
vertebrate viruses. The picornavirus family is made up of twelve genera including
Aphthoviruses, Avihepatoviruses, Cardioviruses, Enteroviruses, Erboviruses,
Hepatoviruses, Kobuviruses, Parechoviruses, Sapeloviruses, Senecaviruses,
Teschoviruses and Tremoviruses (King et al., 2012). Currently, 29 species of viruses
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have been identified as belonging within these genera; these are further differentiated
into a large number of viral serotypes (King et al., 2012). There are many medically and
socially important viruses belonging to the Picornaviridae family, including
Enterovirus C (poliovirus (Carstens and Ball, 2009)), Hepatitis A virus (HAV), Foot-
and-mouth disease virus (FMDV), Encephalomyocarditis virus (EMCV), the
Coxsackieviruses (CV) and HRV (Racaniello, 2007; King et al., 2012).
The picornaviral species that have been formally identified to date are responsible for
causing a very broad range of disease and symptoms in vertebrates including humans
and animals such as bovine and swine. The most extensively studied of these,
poliovirus, is a member of the Enterovirus genus of viruses and is responsible for
causing the severely debilitating illness poliomyelitis (Racaniello, 2007; Rhoades et al.,
2011; Minor, 2012). Poliomyelitis is caused by infection of the motor neurons of the
central nervous system and often results in severe disability (Bodian, 1955; Racaniello,
2007; Minor, 2012). Other picornaviruses capable of infecting humans may cause
disease of the liver, as is the case with the Hepatovirus HAV, severe myocarditis, as is
the case with the Enterovirus CV, or in stark contrast, HRV causes a mild respiratory
illness that is most commonly known as the common cold (Racaniello, 2007; Rhoades
et al., 2011). The most notable of the illnesses caused in animals as a result of
picornavirus infection is foot and mouth disease caused by FMDV, which affects
livestock and has had substantial economic impacts worldwide (Racaniello, 2007).
Associated with the highly varied range of illnesses caused by the picornavirus family,
these viruses utilise highly varied modes of infection. Of the human associated viruses,
the respiratory system, digestive system, circulatory and central nervous systems are all
targets, with some viruses capable of targeting multiple systems upon infection
(Racaniello, 2007). For example, poliovirus infection is primarily transmitted via the
faecal-oral route, particularly in developing countries with poor hygiene and sanitation
facilities where it can be transmitted through person to person contact or through
ingestion of contaminated food and water (Bodian, 1955; Pallansch & Roos, 2007;
Minor, 2012). Poliovirus replicates very efficiently within the intestinal tract and is shed
via faeces (Bodian, 1955; Pallansch & Roos, 2007; Minor, 2012). Similarly, HAV is
also transmitted via the faecal-oral route through person to person contact due to poor
hygiene or through ingestion of contaminated food and water (Hollinger & Emerson,
2007). As their name suggests, the HRV’s infect the nasopharyngeal region of the
respiratory system. Human rhinoviruses are acid labile and are therefore unable to
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penetrate and infect via the digestive system, their primary route of infection is via
direct person to person contact with nasal secretions containing very high levels of virus
particles (Racaniello, 2007).
1.2.1 Structure
Picornavirus virions are simple, spherical structures with a diameter of approximately
30nm (Racaniello, 2007). The picornavirus capsids are made up of four structural viral
proteins, VP1, VP2, VP3 and VP4, with the exception of the Parechovirus genus of
viruses, whose capsids contain the VP1 and VP3 proteins as well as VP0, the precursor
protein of VP2 and VP4 (Stanway & Hyypia, 1999; Racaniello, 2007; Minor, 2012).
These four structural capsid proteins fit together through the formation of protomers to
form an icosahedral shaped sphere of approximately 60 proteins, where VP1, VP2 and
VP3 are present on the surface of the capsid whilst VP4 is present on the inner surface
(Racaniello, 2007; Minor, 2012). The capsids of picornaviruses differ slightly in their
surface shape dependant on the cellular receptor they utilise to allow for optimal
receptor-virion interaction (Racaniello, 2007).
1.2.2 Genome
The genomic RNA of picornaviruses consists of a single strand of uncapped positive
sense RNA which is linked at its 5’ end to a VPg (virion protein, genome linked)
protein, as illustrated in Figure 1.1. (Nomoto et al., 1976; Novak & Kirkegaard, 1999;
Racaniello, 2007; Minor, 2012). VPg proteins, which vary from around 22 to 24 amino
acid residues in length, are essential for the synthesis of both positive and negative
stranded RNA, with the protein found to be linked to both forms (Pettersson et al.,
1978; Rhoades et al., 2011). It is thought that VPg is cleaved upon entry of the genomic
RNA into the cytoplasm by the host cell unlinking enzyme and is not required for viral
translation (Racaniello, 2007).
The single strand of picornavirus RNA contains a long non-coding region at the 5’ end
which is highly structured and contains features such as the internal ribosome entry site
(IRES). The IRES facilitates the binding of cellular ribosomes to the mRNA and allows
for its translation despite the absence of a 5’ cap structure (Racaniello, 2007). The 3’
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non-coding region of both genomic and mRNA is attached to a poly(A) tail. Between
the 5’ and 3’ non-coding regions is a single continuous open reading frame (ORF),
which encodes a single polyprotein that is proteolytically cleaved by virally encoded
proteases to produce both the structural and non-structural viral proteins (see figures 1.1
& 1.2) (Racaniello, 2007; Grubman et al., 2008).
Figure 1.1 An example of the picornaviral genome. The genome consists of a single strand of positive sense RNA with a poly(A) tail at its' 3' end and a VPg
protein linked to its' 5' end. The RNA contains a single ORF flanked by two highly structured non-coding
regions. The ORF encodes a single polyprotein which undergoes proteolytic cleavage in the host cell to
produce the various functional viral proteins. The leader protein (L) is not present in all species but is
present in FMDV and EMCV. The remainder of the proteins encoded as part of the polyprotein can be
divided into structural and non-structural proteins with the VP1, VP2, VP3 and VP4 proteins making up
the structural capsid proteins and the remainder of the proteins included in the non-structural proteins. Of
the non-structural proteins, 2A and 3C have protease capabilities in most picornaviruses and are
responsible for the cleavage of the polyprotein into the various active viral proteins.
1.2.3 Polyprotein and Protein Processing
The picornavirus polyprotein is cleaved via two internally expressed proteases, 2A and
3C. Cleavage starts concurrently with translation and begins with the autocatalytic
cleavage of 2A. The self-cleavage of 2A forms the first of the primary cleavages and
separates the P1 section of the polyprotein, containing the structural capsid proteins,
from the P2/P3 section containing the remainder of the non-structural viral proteins
(refer to figure 1.2)(Ypma-Wong & Semler, 1987). Following the cleavage of 2A, the
remaining polyprotein cleavages are carried out by the 3C protease either on its own, or
whilst in its precursor state as the 3CD protein (Patick & Potts, 1998; Racaniello, 2007).
Similarly to 2A, the 3C protease cleaves itself from the polyprotein, initially forming
the 3CD precursor protein before finally completing its autocatalytic cleavage to result
in the 3C and 3D proteins. Other cleavages carried out by the 3C protease include the
cleavage of P1 into each of the structural capsid proteins as well as the cleavage of the
remaining non-structural proteins (figure 1.2)(Ypma-Wong & Semler, 1987). There is a
high level of conservation across the picornaviral proteins encoded by each of the viral
family members, with the non-structural proteins showing the highest level of
conservation (Buenz & Howe, 2006).
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Figure 1.2 Post translational processing of the HRV polyprotein.
Translation of the HRV RNA results in the production of a single polyprotein which undergoes a series of
cleavages to produce the functional viral proteins. Primary cleavage of the polyprotein results in the
production of P1, P2 and P3 proteins with P1 containing the structural capsid proteins. The first of the
primary cleavages is achieved via the autocatalytic cleavage of the 2A protein releasing the P1 section
from the remaining P2/P3 section. The remainder of the cleavages are carried out by the 3C protein
including the autocatalytic cleavage of itself, the cleavage of P1 to produce the capsid proteins and the
cleavage of each of the remaining proteins to form both the active secondary cleaved proteins and the
further cleaved final protein products.
The picornaviral proteases have been shown to play a role in many aspects of viral
infection from the proteolytic processing of the viral proteins, to the shutting off of host
cell processes, including host RNA transcription. Once cleaved from the polyprotein,
the 2A protease of some picornaviruses, including HRV, has been shown to be
responsible for cleavage of the eukaryotic initiation factors eIF4GI and eIF4GII (Gradi
et al., 1998; Gradi et al., 2003). Cleavage of these factors contributes to the shut-off of
host cell translation, freeing the host machinery to focus on viral mRNA translation. In
a study carried out by Carocci et al, it was found that the 2A protein of EMCV is
required for the inhibition of apoptosis in BHK-21 cells, despite the 2A protein of
cardioviruses and aphthoviruses, including EMCV, not being a protease (Buenz &
Howe, 2006; Carocci et al., 2011). Cells infected with wild type EMCV did not show
signs of apoptosis induction, whereas virus containing a 2A deletion appeared to induce
apoptosis via the activation of caspases. These results suggest that the 2A protein of
EMCV is involved in the inhibition or suppression of apoptosis induction (Buenz &
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Howe, 2006). Cells infected with the altered EMCV were also observed to result in an
accumulation of virus particles within the cytoplasm, indicating that EMCV 2A may
play a role in the release of EMCV from the cell (Buenz & Howe, 2006).
The 3C protease of picornaviruses is responsible for the majority of the processing
cleavages of the viral polyprotein. 3C has also been shown to be responsible for a
number of cellular alterations including alteration of the host cell nucleocytoplasmic
transport pathways (Gustin & Sarnow, 2002). It has been observed that the 3C protease
of a number of picornaviruses is capable of inducing apoptosis when expressed in cell
lines. For example, poliovirus 3C protease induces apoptosis through a caspase
dependant pathway when expressed in HeLa cells (Barco et al., 2000; Buenz & Howe,
2006). Similar results were shown in the human glioblastoma SF268 cells expressing
the 3C protease of the human enterovirus 71 (Li et al., 2002).
1.2.4 Viral Attachment and Entry
The extensive variation of viruses within the picornavirus family requires the utilisation
of a number of different cellular receptors for viral infection (summarised in table 1.1).
Some of the receptors utilised by these viruses include immunoglobulin-like receptors
such as the poliovirus receptor (Pvr) and the major group rhinovirus receptor,
intercellular adhesion molecule 1 (ICAM-1). Others include integrin receptors such as
αvβ3, a receptor used by various CV strains (Racaniello, 2007). This receptor variation
extends to the species level, where for example, HRV serotypes are grouped based on
their cellular receptors, with the major group of rhinoviruses utilising ICAM-1 whilst
the minor group of rhinoviruses use members of the low density lipoprotein receptor
family (Tomassini et al., 1989; Hofer et al., 1994). As well as showing variability in the
selection of receptors required for cellular entry, many viruses within this family also
require the presence of co-receptors for effective infection. A selection of echoviruses,
including echoviruses 3, 6 and 7, not only require the presence of the decay-accelerating
factor (CD55) as their receptor, but also the presence of a β2-microglobulin co-receptor
for entry into the host cell (Racaniello, 2007).
As a result of the variation in cellular receptors, each picornavirus species shows slight
variation in the surface of their capsids. For example, poliovirus and HRV have a
groove within each of the protomers (see section 1.2.6) that make up their capsids. It is
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within this groove that the virus-receptor interactions take place. In contrast, the
aphthoviruses and cardioviruses do not exhibit such groove structures on their capsid
surfaces (Racaniello, 2007).
Table 1.1 Cellular receptors utilised by various picornavirus species
Virus Receptor
Human enterovirus C Poliovirus receptor (Pvr)
Human rhinovirus – major
group
Intercellular adhesion molecule 1 (ICAM-1)
Human rhinovirus – minor
group
Low density lipoprotein receptor family (LDLR)
Coxsackievirus αvβ3 receptor, CD55, ICAM-1
Foot and mouth disease virus αvβ3 receptor
Human hepatitis A virus Human hepatitis A virus cellular receptor (HAVcr-1)
After attachment has occurred, viral RNA is released from within the capsid and enters
the cytoplasm of the host (illustrated in figure 1.3). Whilst it is not fully understood
exactly how this occurs, two possible methods have been proposed. One proposed
method of picornaviral RNA entry into the cytoplasm is through the formation of a pore
within the plasma membrane of the host cell (Racaniello, 2007). In this method,
interactions between the virus particle and the cellular receptor result in conformational
changes to the viral capsid and lead to the formation of a pore within the plasma
membrane (Racaniello, 2007). This has been identified as being the method through
which poliovirus and the major group HRVs are thought to enter the cell (Perez &
Carrasco, 1993; Schober et al., 1998). Though it is not known if the RNA passes
straight through the pore into the cytoplasm or whether it enters though the host’s
endocytic pathway, it is believed that endocytosis alone is not sufficient for the RNA of
polioviruses to enter the cell (Racaniello, 2007). The other proposed method, through
which EMCV is thought to enter the cell, involves receptor mediated endocytosis,
where the binding of the virus to the receptor simply acts to bring the virus into the
vicinity of the cellular membrane to enable it to enter the endocytic pathway
(Racaniello, 2007). After the initial steps of viral entry, additional variation between the
picornavirus species results from the way in which the viral RNA is released from
within the endosomes into the cytoplasm of the host cell. An example of this can be
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seen with the differences between the major group and the minor group HRVs (refer to
section 1.3.1). The RNA of the major group HRVs is released from endosomes through
via a disruption to the endosomal membrane caused by an increase in hydrophobicity
within the endosome. This increased hydrophobicity is caused by structural changes to
the viral capsid induced by the cellular receptor ICAM-1 (Racaniello, 2007). In contrast,
the minor group of rhinoviruses are released from within the endosome through pores
created within the endosomal membrane as a result of significant reductions of the
internal pH induced by the formation of the viral-cellular receptor complex (Racaniello,
2007).
1.2.5 Viral Replication
Synthesis of picornaviral RNA takes place within the cytoplasm of the host cell. It
occurs through the creation of a negative sense replicative intermediate strand, that is
then used as a template for the synthesis of positive stranded genomic RNA (illustrated
in figure1.3)(Racaniello, 2007). This synthesis pattern is strongly favoured towards the
synthesis of positive stranded RNA, with approximately 30-70 times more positive
sense RNA synthesised than negative sense (Novak & Kirkegaard, 1991; Racaniello,
2007). Viral RNA synthesis is carried out by a virally coded RNA polymerase, 3Dpol
,
which is cleaved from its precursor protein, the 3CD protease (see figure 1.2). Along
with the 3Dpol
, many other viral proteins have been shown to act as accessory proteins
in the synthesis of viral RNA, including the 2A, 2B and VPg proteins. However the
structural capsid proteins contained within the P1 section of the polyprotein are not
required for RNA synthesis (Racaniello, 2007).
1.2.6 Viral Assembly and Exit
Assembly of the viral capsid occurs following cleavage of the P1 protein from the viral
polyprotein, and the subsequent cleavage of the VP0, VP1 and VP3 structural proteins
by the 3CD protease (refer to figure 1.2) (Racaniello, 2007). Assembly of the capsid
starts with the formation of a protomer containing one copy each of the VP0, VP1 and
VP3 proteins. A pentamer is then formed from five protomers, with the pentamers then
self-assembling with the newly synthesized RNA to form a provirion (Reviewed by
Racaniello, 2007). The final step in viral assembly is the cleavage of the VP0 protein
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resulting in the VP2 and VP4 proteins, which follows the packaging of newly
synthesised genomic RNA into the provirion. Packaging of viral RNA is highly specific
and ensures that only positive stranded RNA is packaged (Novak & Kirkegaard, 1991).
Once virions are formed, they exit the cell during cellular lyses or degradation resulting
from cytopathic effect or apoptosis. Interestingly in the case of poliovirus, it is thought
the virus utilises microtubules and cellular vesicles to exit the cell in the absence of
cellular lysis (Racaniello, 2007; Taylor et al., 2009).
The picornaviruses have short growth cycles, averaging approximately 8h in cell culture
(Buenz & Howe, 2006). The shedding of HRV particles peaks at 48-72h post infection
as evidenced by viral titres in nasal samples of infected patients (Harris & Gwaltney,
1996; Hendley & Gwaltney, 2004). This peak in viral shedding coincides with an
increase in the release of nasal secretions helping to facilitate the spread of the virus
beyond the host (Hendley & Gwaltney, 2004). Other picornaviruses including
poliovirus, CV, and HAV are shed via faeces, where their major route of transmission is
via the faecal-oral route (Pallansch & Roos, 2007).
10
Figure 1.3 An overview of the picornavirus lifecycle.
1. Viral particles attach to the relevant cell surface receptor, resulting in conformational changes allowing
the virus to enter the cell. 2. The virus enters the cell after attachment via either the formation of a pore
within the plasma membrane or endocytosis. 3. After entry, the viral RNA is released into the cytoplasm.
It is not yet fully understood how the RNA is released from the viral capsid. 4. RNA is translated within
the cytoplasm using host cell machinery to produce the viral polyprotein. 5. The translated polyprotein is
processed via protease cleavage using virally encoded proteases to produce the various functional viral
proteins. 6-7. After entering the cell, the viral RNA is replicated to form both negative and positive sense
strands with there being a far larger proportion of positive sense strands produced than the negative sense
template strands. 8. Following the translation and processing of the structural capsid proteins, they self-
assemble to form the viral capsid. The freshly replicated positive sense viral RNA is then packaged into
the newly formed capsid to form the viral progeny. 9. Viral progeny exit the cell likely helped by the
death and destruction of the host cell.
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1.2.7 Viral Effect on Cellular Processes
As with most viruses, picornaviruses have developed numerous techniques to control or
disrupt host cellular pathways and machinery in order to enhance their survival and
virulence. Examples have been observed where viruses disrupt major cellular pathways
such as host translation, the secretory pathway and nucleocytoplasmic trafficking.
Various studies have shown that during picornavirus infection, a number of host
proteins associated with translation undergo cleavage. These include the translation
initiation factors eIF4G and eIF4A, the poly(A)-binding protein (PABP), and the
translation initiation factor eIF3 (Belsham et al., 2000; Grubman et al., 2008). Cleavage
of eIF4G and eIF4A leads to the inhibition of cap dependant translation, leaving the
host’s translation machinery free to carry out cap independent translation of the viral
mRNA (Belsham et al., 2000; Grubman et al., 2008). Cleavage of these factors also
contributes to the down regulation of host immune proteins (Grubman et al., 2008).
Proteases of enteroviruses, including poliovirus, cleave PABP, a host protein that plays
a major role in the initiation of cap-dependant translation initiation, potentially
contributing to the shut-off of host protein translation (Kuyumcu-Martinez et al., 2002).
In FMDV infection, it has been shown that the eIF4G proteins are cleaved by the viral
leader protease Lpro
(Buenz & Howe, 2006; Grubman et al., 2008), whereas the 3C
protease has been shown to induce at least a partial cleavage of this factor (Belsham et
al., 2000).
A number of picornaviruses, including poliovirus, coxsackievirus B3 and FMDV, have
been found to cause disruptions to the host cell secretory pathway during infection
(Grubman et al., 2008). FMDV infection has been shown to reduce the number of major
histocompatibility complex (MHC) class I molecules present on the surface of infected
cells, leading to a reduction in the presentation of viral antigens on the cell surface and
consequently a reduction in the induction of the cytotoxic T cell response (Grubman et
al., 2008). Another example of cellular processes being affected include the
identification that picornavirus infection results in the alteration of host
nucleocytoplasmic trafficking. Rhinovirus infection results in the accumulation of a
number of different proteins within the cytoplasm that are normally trafficked across the
nuclear membrane through the classical nuclear import pathway, suggesting this
pathway is inhibited by HRV infection (Gustin & Sarnow, 2002). Another cellular
12
impact of picornaviral infection is the alteration of the calcium homeostasis of the
organelles as demonstrated by Campanella et al (Campanella et al., 2004).
1.3 Rhinovirus
Common colds have been the subject of scientific studies for many years and were
initially thought to be caused by exposure to cold and damp conditions. The idea that
colds were not the result of exposure to cold and damp environments, but rather from
exposure to other infected persons, was first suggested in the 19th
century. This theory
was further developed in the early 20th
century when swabs of nasal secretions of
persons suffering from a cold were effectively able to infect others (Turner & Couch,
2007). Other early studies identified the significant role families played in the spread of
colds. Whilst preliminary studies had identified that there was a virus known to cause
common colds, it was not until the 1960’s that rhinoviruses were formally identified as
the causative virus (Turner & Couch, 2007). By the early 1970’s more than 100
different serotypes of HRV had been described (Turner & Couch, 2007).
1.3.1 Classification
The human rhinoviruses are members of the enterovirus genus of viruses within the
family Picornaviridae. There are over 100 HRV serotypes identified to date which have
been assigned to three genotypes, Rhinovirus A, Rhinovirus B and Rhinovirus C. The
criteria for differentiation of the HRV species from other Enterovirus species is their
acid lability where HRV is inactivated at low pH whilst other enteroviruses are not
(Turner & Couch, 2007). This difference in acid lability reflects the differences seen in
the locations within which infections occur. Rhinoviruses primarily infect the upper
respiratory tract whist other enteroviruses infect via the more acidic digestive tract.
Unlike other enteroviruses, HRV is highly species specific and only grows effectively in
human and a few primate cells. Chimpanzees and gibbons have both been successfully
infected with particular HRV serotypes, however these infections did not cause any
observable illness (Turner & Couch, 2007). This species specificity is caused by the
presence, or absence, of the cellular surface receptors required for HRV entry into the
cell. Three cellular receptors have been identified as being used by various HRV
13
serotypes, the decay-accelerating factor, low-density lipoprotein receptors (LDLR) and
intercellular adhesion molecule 1 (ICAM-1) (Tomassini et al., 1989; Hofer et al., 1994;
Turner & Couch, 2007). The majority of HRV serotypes can be separated into two
groups based on their cellular receptor usage, minor group HRV serotypes and major
group serotypes. The minor group includes the serotypes 1A, 1B, 2, 29, 30, 31, 44, 47,
49 and 62, all of which utilise the LDLR (Hofer et al., 1994; Turner & Couch, 2007).
The remaining HRV serotypes, with the exception of rhinovirus serotype 87 (HRV87),
make up the major group of HRVs and use ICAM-1 as their cellular receptor
(Tomassini et al., 1989; Turner & Couch, 2007). Unlike other serotypes, HRV87
appears to use the decay-accelerating factor as a receptor, similarly to the closely related
Enterovirus 68 (Turner & Couch, 2007).
1.3.2 Epidemiology
As demonstrated by the early investigations into colds, HRV infection is spread through
person to person contact, although infections via inanimate objects such as railings and
door handles and via respiratory aerosols are thought to be possible alternate routes of
transmission. Nasal secretions of HRV infected persons contain a high number of virus
particles, and it is thought that direct person to person contact with these secretions is
the most likely and most effective method of transmission (Hendley et al., 1973; Turner
& Couch, 2007).
The primary site of HRV infection is within the nasopharynx of the upper respiratory
tract. Experiments studying HRV infection routes have shown that the infectious dose
required for infection via nasal drops is significantly lower than that required for
infection via aerosol particles, indicating that the lower respiratory tract is much less
susceptible to HRV infection than the upper respiratory tract (Turner & Couch, 2007).
For a time it was thought that HRV was unable to infect the lower respiratory tract,
however this has since been shown to be incorrect. Despite the nasopharyngeal region
being the primary site of HRV infection, the virus is capable of infecting, and is often
found in, samples collected from the sinuses, the throat and from the middle ear (Turner
& Couch, 2007). Rhinovirus is rarely found in other sites within the body.
Even from the early days of studying the common cold, there has always been the
implication that being exposed to low temperatures or being chilled will induce a cold.
14
Contrary to this long held belief, exposure to cold and damp environments have not
been shown to have any effect on the susceptibility of individuals to colds, and
particularly to HRV infection (Turner & Couch, 2007). Rhinovirus infections occur
more frequently during autumn and spring in temperate climates, with the highest
frequencies occurring during autumn months, however infections do occur all year
round. It is not known why this pattern of infection occurs, however it is likely to be
multifactorial. Rhinovirus infections are far more prevalent in children and their
incidence reduces with age. Family units play a major role in the spread of HRV
infections due to members being in close proximity to each other and having a greater
level of contact with each other than in the general population.
1.3.3 Clinical Disease
Human rhinovirus infections are associated with a wide range of illnesses in addition to
the common cold. An association with asthma and chronic obstructive pulmonary
disease (COPD) exacerbations during HRV infection is well established (Johnston et al.,
1995; Seemungal et al., 2000; Greenberg, 2003). Rhinoviruses are the most common
causative viruses of the common cold, which results in symptoms such as nasal
congestions, sore throat, coughing, sneezing, headaches and general fatigue (Greenberg,
2003). Whilst these symptoms are often not severe and illness is usually resolved within
a few days, HRV infections are responsible for a significant number of absences from
workplaces and schools each year. They are also noted as being one of the most
common reasons for inappropriate use of antibiotics (Greenberg, 2003). These
consequences result in significant impacts both socially and economically. Respiratory
tract infections, including those caused by HRV, often result in more severe illness in
individuals suffering from asthma (Wark et al., 2005). Asthma attacks often occur after
respiratory infections, including those caused by HRV, and it has been shown that these
infections exacerbate asthma symptoms (Johnston et al., 1995; Greenberg, 2003). The
increased severity and exacerbations seen in asthma sufferers during HRV infection,
appear to be a result of inefficiencies in the asthmatic cell’s interferon and apoptotic
responses (Wark et al., 2005). Human rhinovirus infection has also been observed as
being responsible for illnesses such as acute otitis media and serious illness in those
suffering from COPD (Seemungal et al., 2000; Greenberg, 2003).
15
1.4 Apoptosis
1.4.1 General Apoptosis
Apoptosis is a mechanism of controlled cell death, used to eliminate unrequired or
compromised cells without releasing any of the cellular contents, thereby limiting any
potential damage to surrounding cells or triggering an inflammatory response.
Apoptosis can be induced in response to a wide range of triggers, including the removal
of unnecessary cells during foetal development, DNA damage and viral infection
(Barber, 2001). The apoptotic process allows for cells which have been triggered to
partake in apoptosis, to undergo morphological changes which permit them to be
engulfed by neighbouring cells or phagocytes. These morphological changes include
rounding of the cell, cytoplasmic shrinkage, nuclear fragmentation, chromatin
fragmentation and blebbing of the plasma membrane with the cells eventually breaking
down into apoptotic bodies (Barber, 2001; Galluzzi et al., 2008).
The process of apoptosis can be triggered by two distinct pathways, the intrinsic
apoptotic pathway and the extrinsic apoptotic pathway (briefly summarised in figure
1.4). The intrinsic pathway is mediated from within the cell via the mitochondria
(Galluzzi et al., 2008). The mitochondria receive pro- and anti-apoptotic signals passed
on from other organelles within the cell or from within the cytoplasm, resulting in
changes in the permeability of the mitochondrial membranes. In the event of pro-
apoptotic signals being received, the mitochondrial membrane permeability increases,
releasing pro-apoptotic proteins such as cytochrome c (Galluzzi et al., 2008). These
proteins go on to trigger a cascade of signalling events resulting in apoptosis (Galluzzi
et al., 2008). The extrinsic pathway however, is activated via signals external to the cell.
Specific ligands bind to pro-apoptotic receptors on the cell surface such as the
Fas/CD95 receptor and tumour necrosis factor receptors (TNFR-1 & TNFR-2) (Clement
& Stamenkovic, 1994; Micheau & Tschopp, 2003; Galluzzi et al., 2008). This
ligand/receptor binding event causes the formation of a death-inducing signalling
complex (DISC) which in turn triggers a cascade of events including the cleavage of
RIPK1 and caspase 8, ultimately leading to the controlled death of the cell (Micheau &
Tschopp, 2003; Galluzzi et al., 2008). Whilst these pathways are distinctly different, it
is known that they interact and activate each other and the two pathways converge at the
later stages of the cascades. It is also known that in some cases the mitochondria is able
16
to facilitate the activation of death receptors thus resulting in an internal activation of
the extrinsic pathway (Galluzzi et al., 2008).
Figure 1.4 Schematic diagram of the extrinsic and intrinsic apoptotic pathways as relevant to
picornavirus infection.
The extrinsic apoptotic pathway is initiated via the binding of the respective ligand to a pro-apoptotic
receptor on the surface of the plasma membrane. This triggers a signalling cascade that includes the
cleavage of caspase 8 and RIPK1 before the cleavage of the effector caspase 3 which leads to the
induction of the morphological changes characteristic of apoptosis. Induction of the intrinsic apoptotic
pathway is initiated from within the cell. Internal factors act on the mitochondria, increasing the
mitochondrial membrane permeability leading to the release of cytochrome c from within the
mitochondria into the cytoplasm. This leads to a signalling cascade that includes the cleavage of caspase
9. The intrinsic and extrinsic pathways converge as both include the cleavage of caspase 3 which leads to
the morphological changes.
17
Mechanisms behind the induction of apoptosis are complex and there are numerous
triggers and intermediates involved. It has been identified that interferons (IFNs) play a
major role in apoptosis as well as in triggering both the innate and adaptive immune
responses to viral infection (Barber, 2001). IFNs act in the induction of apoptosis
through their regulation of genes such as dsRNA-dependent protein kinase (PKR),
TNF-related apoptosis-inducing ligand (TRAIL) and the interferon regulatory factor
(IRF) family, which in turn utilise apoptosis as part of their antiviral and tumour
suppression functions (Gil & Esteban, 2000; Barber, 2001). Other important molecules
crucial for the apoptotic response include the caspases, the Bcl-2 family of proteins and
the tumour suppressor protein p53. Caspases are a group of enzymes that play a major
role in the apoptotic signalling cascade. They are constantly produced as procaspases by
the cell and are activated via cleavage, either autocatalytically or by upstream enzymes
as part of the apoptotic signalling cascade (Barber, 2001). Once caspases are cleaved
and activated, they in turn cleave specific downstream proteins through the recognition
of specific cleavage sites (Barber, 2001; Richard & Tulasne, 2012). There are
approximately 50 or more caspase substrates identified which when cleaved, act to
cause the characteristic morphological changes associated with apoptotic cells (Barber,
2001; Richard & Tulasne, 2012). For example the Bcl-2 family of proteins are a family
of proteins which act to regulate apoptosis by changing the membrane permeability of
the mitochondria (Galluzzi et al., 2008). The Bcl-2 family is composed of both
proapoptotic and antiapoptotic members that act on each other in response to various
triggers from within the cell, to provide a balance between the induction and inhibition
of apoptosis. This balance helps to prevent the unnecessary premature induction of cell
death (Galluzzi et al., 2008). The cellular expressed tumour suppressor protein p53 is
known to regulate cellular transcription in healthy cells, however it also has the ability
to induce apoptosis when the cell has been compromised, for example during virus
infection or after DNA damage (Barber, 2001). p53 acts to regulate the genes encoding
the Fas and TRAIL apoptotic receptors, as well as the transcription factor NF-κB, to
promote apoptosis (Barber, 2001). Schwarz et al have suggested that the transcription
factor NF-κB is involved in the suppression or delay of apoptosis during EMCV
infection through their studies of knockout mice (Schwarz et al., 1998). It has not been
determined if NF-κB is directly involved in apoptosis suppression or, in the more likely
scenario, that it is responsible for the expression of other antiapoptotic genes.
18
1.4.2 Viruses and Apoptosis
Apoptosis is one of the first cellular responses to viral infection as the cell aims to
eliminate the virus and restrict its replication, further spread and minimise the damage
caused (Barber, 2001; Richard & Tulasne, 2012). It is due to this fact that many viruses
have developed methods of altering the apoptotic pathways to their own advantage.
Viruses have been shown to not only inhibit or delay apoptosis induction in order to
facilitate the production of viral progeny, but have also been shown to have the ability
to induce apoptosis so as to help facilitate virus dissemination. Methods employed by
viruses to regulate apoptosis vary greatly and can affect all stages of the apoptotic
pathway, from the initiation of the signalling cascade right through to the triggering or
prevention of the morphological changes.
Viruses have found many ways of suppressing the host cell apoptotic pathway so as to
allow sufficient time for viral replication. Some viruses, including the human
adenoviruses (ADVs), encode proteins which are homologs of the cellular antiapoptotic
Bcl-2 family proteins that act to block the intrinsic apoptotic pathway (Galluzzi et al.,
2008). The ADV encoded E1B-19K Bcl-2 homolog has also been found to have the
ability to inhibit other apoptotic initiation pathways, including through the prevention of
ligand binding to the TNFR, Fas and TRAIL receptors on the cellular membrane surface
(Galluzzi et al., 2008). The receptor internalisation and degradation complex of ADVs
is capable of inhibiting the extrinsic apoptotic pathway by promoting the internalisation
and degradation of some of the major pro-apoptotic receptors, including TNFR and Fas,
found on the surface of the plasma membrane (Galluzzi et al., 2008).The BHRF1
product of the Epstein-Barr virus (EBV) is an antiapoptotic Bcl-2 protein homolog
which has been found to localise to the outer membrane of the mitochondria where it is
capable of suppressing the intrinsic induction of apoptosis by preventing the
permeabilisation of the mitochondrial membrane (Galluzzi et al., 2008).
The induction of apoptosis during viral infection may be of benefit to either the host or
the virus depending on the timing of induction. Some viruses have been shown to
induce apoptosis in cells as part of viral pathogenesis, for example a number of proteins
encoded by the Human immunodeficiency virus 1 (HIV-1) have been found to promote
an apoptotic response in CD4+ lymphocytes (Galluzzi et al., 2008). There have been a
number of viral proteins that have been identified as direct inducers of apoptosis, as
well as a number act indirectly to induce apoptosis. Additionally, a number of viral
19
proteins are capable of both directly and indirectly inducing the intrinsic pathway of
apoptosis through interactions with the mitochondria. The HIV-1 viral protein R (Vpr)
has been shown to directly interact with the voltage-dependant anion channels of the
outer mitochondrial membrane in order to trigger mitochondrial membrane permeability
and consequentially apoptosis (Galluzzi et al., 2008). The Influenza A virus (IAV)
encoded protein PB1-F2, has the ability to insert into the mitochondrial membranes
where it forms pores within the membranes similar to the proapoptotic Bax member of
the Bcl-2 family (Galluzzi et al., 2008). This pore formation by PB1-F2 is then thought
to increase mitochondrial membrane permeabilisation and ultimately the induction of
apoptosis (Galluzzi et al., 2008). Another example of a viral protein that has been
shown to have the ability to directly induce apoptosis is the hepatitis C virus (HCV)
encoded non-structural protein 4A (NS4A). This protein localises to the mitochondria
where it results in damage to the membranes and causes the release of cytochrome c
into the cytoplasm (Galluzzi et al., 2008). In contrast, the HIV-1 encoded protease
which is required for the processing of mature viral proteins, is able to indirectly induce
intrinsic apoptosis. It does so by promoting the cleavage of caspase 8 which leads to the
cleavage of Bid, a member of the Bcl-2 family, ultimately resulting in the
permeabilisation of the mitochondrial membranes and induction of intrinsic apoptosis
(Galluzzi et al., 2008).
The IFNs are known to play a role in the antiviral response to infection and appear to
increase the sensitivity of infected cells to the induction of apoptosis (Barber, 2001). It
is thought that IFNs are induced by the presence of double-stranded RNA (dsRNA),
which is present during the replication of RNA viruses. Once activated, they act to
increase the sensitivity of cells to the activation of Fas-associated death domain
(FADD) and caspase 8 dependant apoptosis (Barber, 2001). FADD is a molecule
recruited following activation of the proapoptotic death receptors of the extrinsic
pathway (Barber, 2001). The E7 protein of human papillomavirus (HPV) has been
shown to trigger the p53 mediated apoptotic response. However the virus, like many
others, has developed strategies to avoid this induction, including through the
expression of the E6 protein which inhibits the action of p53. Another example of viral
inhibition of the p53 mediated apoptosis is the production of the LANA protein by
human herpes virus 8 (HHV8) viruses which also acts to inhibit the action of the p53
protein (Barber, 2001).
20
1.4.3 Picornaviruses and Apoptosis
In the case of picornaviruses, there is evidence that this family of viruses are capable of
both inducing and inhibiting the apoptotic pathways of the host cells they infect
(reviewed by Buenz & Howe, 2006). Apoptosis triggered by picornaviral infection is
thought to be induced via the intrinsic apoptotic pathway and includes the activation of
the caspases 3 and 9 (Belov et al., 2003). The ability of picornaviruses to control the
balance of apoptosis during infection allows the virus enough time to replicate whilst
suppressing the host apoptotic response. It is thought that the viruses then deliberately
activate the apoptotic pathway to help facilitate the spread of the viral progeny, whilst
avoiding the activation of the host immune response. Despite the fact that some
members of the picornavirus family are capable of altering the apoptotic response, the
exact mechanisms and intermediates involved are yet to be fully understood and this is
an area of research that is becoming of great interest.
The suppression of apoptosis in picornaviral infected cells is thought to benefit the virus
by allowing for maximal replication before cell death occurs. In vitro studies of FMDV
infection have shown no evidence of apoptosis induction, however the mechanisms
behind this apparent suppression of apoptosis are still unknown (Grubman et al., 2008).
Through their studies in knockout mice, Schwarz et al. demonstrated that the
transcription factor NF-κB, may be involved in the suppression or delay of apoptosis
during EMCV infection (Schwarz et al., 1998). Further to this, Carocci et al. found that
the 2A protease also plays a role in the suppression of apoptosis during EMCV infection
(Carocci et al., 2011). Another study investigating apoptosis in EMCV infected cells,
found that apoptosis was not induced in infected HeLa cells, most likely due to actions
of the leader protein L (Romanova et al., 2009). This study also found that EMCV
infection was capable of not only circumventing the induction of host apoptosis, but is
also capable of inhibiting apoptosis induced by chemical apoptotic inducers (Romanova
et al., 2009). Studies investigating apoptosis during poliovirus infection observed that
apoptosis is inhibited during productive poliovirus infection (infection under conditions
optimal for maximum viral replication) of various HeLa sub-line cells, however it was
induced when conditions were not ideal (Tolskaya, et al., 1995; Agol et al., 2000; Belov
et al., 2003). Similarly to that seen in EMCV infection, poliovirus infection is also
capable of inhibiting chemically induced apoptosis (Tolskaya et al., 1995). In a study of
coxsackievirus B3, another member of the enterovirus genus, expression of the 2B
protein led to the inhibition of chemically induced apoptosis in HeLa cells through
21
alterations in the Ca2+
homeostasis of the cellular organelles (Campanella et al., 2004).
Whilst these studies have demonstrated that a number of picornaviruses are capable of
suppressing apoptosis induction, the exact mechanisms involved are yet to be fully
understood. It is possible that the inhibition or suppression of host cell apoptosis by
picornaviruses could be controlled by the virally encoded proteases, 2A and 3C. Their
ability to alter and shut-off other aspects of the host cell machinery, as well as the very
small number of virally encoded proteins, makes these proteases highly plausible targets
when trying to determine the mechanisms responsible for the suppression of apoptosis
during picornavirus infection. However, as demonstrated through the work of
Campanella et al, other viral components cannot be ruled out and it is possible that
multiple processes are involved (Campanella et al., 2004).
Various picornaviral components have been identified as being capable of inducing
apoptosis; however their exact mechanisms of action are yet to be elucidated. Studies
investigating the induction of apoptosis in poliovirus infected cells have shown that
virus infection is capable of both inhibiting and inducing apoptosis (Tolskaya et al.,
1995). It has been found that poliovirus may have the ability to induce apoptosis via
various different triggers which lead to the activation of caspases 3 and 9, following the
efflux of cytochrome c from the mitochondria (Belov et al., 2003). These apoptotic
triggers include the alteration of Bcl-2 family proteins, promotion of mitochondrial
membrane permeability by the viral proteins 2B and 3A and expression of the 2A and
3C proteases (Belov et al., 2003; Buenz & Howe, 2006; Galluzzi et al., 2008). Li et al.
found that the expression of the Enterovirus 71 3C protease in human glioblastoma
SF268 cells results in the induction of apoptosis through the activation of caspases,
however they also found that apoptosis was not induced in cells expressing a
deactivated mutant of the 3C protease (Li et al., 2002). Similar results have been
observed in studies examining poliovirus and HRV infection, where the 2A protease has
been demonstrated to induce a number of cellular alterations associated with the
induction of apoptosis including the fragmentation of DNA (Barco et al., 2000; Buenz
& Howe, 2006) Buenz and Howe 2006). Carthy et al. showed that infection of HeLa
cells with Coxsackievirus B3 (CVB3) resulted in the activation of a range of caspases,
including caspases 3 and 9, as well as the cleavage of the caspase substrate PARP,
indicating the induction of apoptosis (Carthy et al., 2003). In the same study,
significantly more cardiomyocytes were found to be apoptotic in patients infected with
enteroviruses such as coxsackievirus B than in cardiomyocytes from uninfected
22
patients. Another study of CV infection also confirmed that apoptosis is induced during
infections (Gomes et al., 2010). The structural VP3 protein and the non-structural 2C
protein encoded by the avian encephalomyelitis virus (AEV) have been found to
promote the induction of apoptosis via the intrinsic pathway (Galluzzi et al., 2008), with
the VP3 protein shown to localise to the mitochondria where it triggers the activation of
downstream caspases (Galluzzi et al., 2008).
The demonstration that picornaviruses have the ability to both induce and supress
apoptosis at various points post infection suggests that these viruses may have
developed strategies allowing them to alter and regulate the balance of pro-apoptotic
and anti-apoptotic tendencies within the cell in order to support their own replication
and spread.
1.4.4 Rhinovirus and Apoptosis
Similarly to all picornaviruses, the effect of HRV infection on the induction of
apoptosis is not entirely understood. Deszcz et al. have demonstrated that Human
rhinovirus 14 (HRV14) infection triggers apoptosis in both HeLa cells and the human
bronchial epithelial 16HBE14o- cell line, when cells are infected with high
concentrations of virus (Deszcz et al., 2005). They demonstrated that during infection
with HRV14, apoptosis was induced via the intrinsic apoptotic pathway, as
demonstrated by the cleavage of caspase 9 and the release of cytochrome c from within
the mitochondria to the cytoplasm; the universal apoptotic makers, caspase 3 and the
caspase substrate PARP were also found to be cleaved. Similar results were obtained by
Drahos and Racaniello, where they observed that apoptosis was induced in HeLa cells
infected with HRV1a at a multiplicity of infection (MOI) of 10 through the
confirmation that PARP, a known caspase substrate, was cleaved in infected cells
(Drahos & Racaniello, 2009). Taimen et al also found that infection with HRV1b
resulted in apoptosis induction, demonstrated through the cleavage of caspase 3 and
PARP (Taimen et al, 2004). In contrast, Gustin and Sarnow showed that HeLa cells
infected with HRV14 showed no signs of PARP cleavage and only a very small amount
of DNA fragmentation suggesting that apoptosis was inhibited (Gustin & Sarnow,
2002).
23
The effect of HRV infection on the host apoptotic pathways is still not completely
understood. Whilst numerous studies have been performed to investigate apoptosis
during other picornavirus infections, very few have focussed on HRV. The observations
made so far have failed to decisively elucidate the effect of HRV infection on apoptosis,
with a number of contradicting observations described. This remains an area where
further investigations are required to improve our understanding of the processes
involved, and may provide potential future therapeutic targets to be identified.
24
1.5 Hypothesis and Aims of Present Study
1.5.1 Hypothesis
Induction of apoptosis is one of the cells first responses to viral infection. It has been
observed that various viral features are capable of inducing the apoptotic response,
whilst in contrast, it has also been demonstrated that some viruses are capable of
altering the apoptotic pathway in order to improve their chances of survival and spread.
Previous studies investigating the effect of picornaviral infection on the host apoptotic
response, have demonstrated that some members of this family of viruses are capable of
inhibiting apoptosis, while others are capable of inducing apoptosis and some appear to
do both. Poliovirus, a member of the Enterovirus genera of picornaviruses which is very
closely related to HRV, has been shown to both inhibit and induce apoptosis depending
on the availability of optimal viral growth conditions. Few studies have investigated
apoptosis during HRV infection, with those carried out so far demonstrating conflicting
results of both apoptotic induction and inhibition. It is likely that the viral 2A and 3C
proteases may play a role in the regulation of apoptosis, particularly due to their
demonstrated ability alter various host signally pathways.
Based on the results seen in studies investigating the induction of apoptosis during
picornaviral infection, particularly those focusing on poliovirus and the few focussed on
HRV infection, it is hypothesised that HRV infection delays the induction of apoptosis
by the host cell in order to facilitate its own replication.
1.5.2 Aims
The overall objective of this research project is to identify the effect of HRV infection
on the induction of apoptosis. In order to determine the apoptotic response to human
HRV infection, the following aims will be addressed:
Aim 1. To determine if HRV infection inhibits or induces the host’s apoptotic
pathways
The apoptotic response in Ohio-HeLa cells, both uninfected and infected with HRV16,
will be investigated in the presence and absence of known chemical apoptosis inducers
to determine if HRV16 infection alters the host’s apoptotic response. Western blot
25
analysis will be used to detect various apoptotic markers as well as known caspase
substrates to confirm apoptotic activity along with immunofluorescence assays.
Aim 2. To determine if the 3C and 2A proteases have a role in apoptosis
induction/inhibition
In order to determine the role that the HRV proteases 3C and 2A play in the apoptotic
response, Ohio-HeLa cells expressing the viral proteases will be studied to determine if
the expression of the viral proteases alters the induction of apoptosis. The apoptotic
response by the Ohio-HeLa cells will be determined using western blot analysis and
immunofluorescence assays.
It is expected that the results of this study will show that the induction of apoptosis in
Ohio-HeLa cells as a result of HRV16 infection is inhibited or delayed by the virus in
order to improve viral replication. It is also expected that the viral protease, 2A and 3C,
will play a role in this alteration.
26
2 Chapter Two – Materials and Methods
2.1 Materials
2.1.1 Virus and Cell Culture Lines
2.1.1.1 Cell Culture Lines
The Ohio strain of the HeLa human cervical carcinoma cell line was used throughout
this study. Ohio-HeLa cells were maintained in growth media (see table 2.1) at 37°C
with 5% CO2.
COS7 cells were used for transfection of the HRV16 2A and 3C proteases. Cells were
maintained in growth media (see table 2.1) at 37°C with 5% CO2.
2.1.1.2 Virus Lines
Human rhinovirus serotype 16 (HRV16) was used throughout this study.
2.1.1.3 Media Used for Cell Culture and Viral Infection
The following media was used throughout this study during the culture of cell and virus
lines and during viral infections.
Table 2.1 Cell and virus culture media used during this study.
Media Composition
Growth media Dulbecco’s modified Eagle’s Medium (DMEM)
supplemented with 10% foetal calf serum.
Maintenance media Dulbecco’s modified Eagle’s Medium (DMEM)
supplemented with 2% foetal calf serum.
Serum free media Dulbecco’s modified Eagle’s Medium (DMEM)
PBS (1x) 137mM NaCl; 2.7 mM KCl; 8.1mM Na2HPO4; 1.47mM
KH2PO4
0.1% Crystal Violet stain 0.1% crystal violet in ethanol
Trypsin/EDTA Gibco
27
2.1.2 Buffers, Solutions and Bacterial Culture Media
The following buffers and solutions were used throughout this study.
Table 2.2 Buffers and solutions used during this study.
Buffer/Solution Composition
PBS (1x) 137mM NaCl; 2.7 mM KCl; 10mM Na2HPO4; 1.7mM
KH2PO4; pH7.4
RIPA Buffer 150mM NaCl; 1% Triton X 100; 0.5% sodium
deoxycholate; 0.1% SDS; 50mM Tris (pH 8); 1x protease
and phosphatase inhibitors (Roche); H2O
Taxol Sigma-Aldrich 1mg/ml in DMSO
Actinomycin D Sigma-Aldrich 1mg/ml in DMSO
Cyclohexamide 2mg/ml in H2O
z-VAD-fmk 5mM in DMSO
Running Buffer 0.1% SDS; 25mM Tris base; 192mM glycine
Transfer Buffer 25mM Tris base; 192mM glycine
Blocking Solution 3% skim milk powder; PBS
Washing Solution (PBST) 0.1% Tween20; PBS
Ponceau S Stain 2% Ponceau S; 30% trichloroacetic acid; 30%
sulfosalicylic acid
ProLong Gold mounting
media with DAPI
Invitrogen
Kanamycin Sigma 10mg/ml in H2O
Luria-Bertani broth 10g/l bactero-tryptone; 5g/l bacto-yeast extract; 5g/l NaCl
Laemmli buffer 4% SDS; 10% 2-mercaptoethanol; 20% glycerol; 0.004%
bromophenol blue; 0.125M Tris HCl; pH6.8
SDS PAGE stacking gel 5% acrylamide; 0.5M Tris-HCl; 10% SDS; 10% APS;
H2O
SDS PAGE separating gel 12.5% acrylamide; 1.5M Tris-HCl; 10% SDS; 10% APS;
H2O
Western blot stripping
buffer
2% SDS; 62.5 mM Tris-HCl pH 6.8; 114.4 mM β-
mercaptoethanol
28
2.1.3 Antibodies
2.1.3.1 Primary Antibodies – Western blot
The primary antibodies used for western blots throughout this project are listed below.
Table 2.3 Western blot primary antibodies used during this study.
Antibody Dilution Source species Manufacturer
VP2* 1:2000 Mouse Gift from Dr Wai-Ming Lee
Caspase 3 1:500 Mouse Santa Cruz
Caspase 9 1:1000 Mouse Santa Cruz
PARP 1:200 Mouse Santa Cruz
αβ-Tubulin 1:1000 Rabbit Cell Signalling Technology
RIPK1 1:1000 Mouse Thermo Scientific * - gift from Dr Wai-Ming Lee, University of Wisconsin
2.1.3.2 Secondary Antibodies – Western blot
The secondary antibodies used for western blots throughout this study are listed below.
Table 2.4 Western blot secondary antibodies used during this study.
Antibody Dilution Source
species
Manufacturer
Anti-rabbit immunoglobulins conjugated to
horseradish peroxidase (goat-anti-rabbit HRP)
1:5000 Goat Invitrogen
Anti-mouse immunoglobulins conjugated to
horseradish peroxidise (goat-anti-mouse HRP)
1:5000 Goat Invitrogen
2.1.3.3 Primary Antibodies – Immunofluorescence
The primary antibodies used for immunofluorescence are listed below.
Table 2.5 Immunofluorescence primary antibodies used during this study.
Antibody Dilution Source
species
Manufacturer
VP2# 1:2000 Mouse Gift from Dr
Wai-Ming Lee
Cytochrome c 1:200 Mouse Santa Cruz
dsRNA* 1:200 Guinea pig Gift from Dr
Scott Bowden * - gift from Dr Scott Bowden, VIDRL, Melbourne.
# - gift from Dr Wai-Ming Lee, University of Wisconsin
29
2.1.3.4 Secondary Antibodies – Immunofluorescence
The secondary antibodies used for immunofluorescence throughout this study are listed
below.
Table 2.6 Immunofluorescence secondary antibodies used during this study.
Antibody Dilution Source
species
Manufacturer
Goat anti mouse Alexa 568 1:1000 Goat Molecular
Probes
Goat anti mouse Alexa 488 1:1000 Goat Molecular
Probes
Donkey anti goat Alexa 488 1:1000 Donkey Molecular
Probes
Donkey anti mouse Alexa
568
1:1000 Donkey Molecular
Probes
2.1.4 Commercial Kits
The following commercial kits were used during this study to detect the presence of
apoptosis.
Table 2.7 Commercial kits used during this study.
Kit Manufacturer
Plasmid Mini Prep Promega
Western Lighting, ECL Perkin-Elmer
Lipofectamine 2000 Invitrogen
2.2 Methods
2.2.1 Cell and Virus Cultures
2.2.1.1 Cell Culture
Ohio-HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)
media supplemented with 10% foetal bovine serum and were incubated at 37°C with
5% CO2. During each passage, at approximately 90-100% confluence the supernatant
was removed from each flask and the adherent cells were washed with 5ml of PBS.
Cells were detached via treatment with 1ml of Trypsin/EDTA for 2-3mins. Cells were
then suspended in 4ml of 10% FBS DMEM. Cells were split 1:5 with 1ml of cell
30
suspension added into each T25ml flask containing 4ml of 10%FBS DMEM and
incubated at 37°C with 5% CO2 until confluent.
2.2.1.2 Virus Culture
Rhinovirus 16 was cultured in Ohio-HeLa cells. Ohio-HeLa cells were cultured and
incubated overnight as described above to give approximately 70% confluence. The
supernatant was removed and cells were washed with 5ml of PBS. 1ml of 2% FBS
DMEM containing enough virus inoculum to give an MOI of 0.1 was added to each
flask of cells which were then incubated at 37°C with 5% CO2 for one hour with gentle
rocking every 15min. After the hour, an additional 4ml of 2% FBS DMEM media was
added to each flask of cells which were then incubated overnight at 37°C with 5% CO2.
The following day cells were checked for evidence of cytopathic effects (CPE) as
confirmation of viral infection before flasks were sealed and stored at -80°C. The flasks
were later thawed at room temperature and the cultured virus was collected and
transferred into 10ml tubes which were then centrifuged at 4000rpm for 15mins at room
temperature to remove cell debris. The supernatant containing the cultured virus was
then removed and distributed into eppendorf tubes and stored at -80°C until required.
2.2.1.3 Virus Titration
All passages of cultured virus were titrated on Ohio-HeLa cells to determine infectious
virus content. Three rows of a 96 well tissue culture plate were seeded with O-HeLa
cells for each virus isolate. Each well was seeded with approximately 5x103 cells per
well diluted in 100µl of 10% FBS DMEM. Ten-fold serial dilutions of the virus culture
were made in 2% FBS DMEM ; 50µl of each virus dilution was added in triplicate to
wells in decreasing concentration with the last three wells having only media added.
The plate was then incubated for 6 days at 37°C with 5% CO2. Following incubation,
the plate was examined under a microscope for signs of CPE. Cells were stained with
0.1% Crystal Violet stain and the CPE positive wells were counted. Virus titration was
then calculated using the Spearman-Karber equation:
Titre = 10 1 + Z(X-0.5)
Where Z equals the log 10 of the dilution factor and X equals the sum of the CPE
positive wells.
31
Three virus cultures were carried out for this study from an original culture provided by
Dr. Reena Ghildyal at passage 2. The virus titrations of each of passage were as follows:
Passage 3 = 6.3 x 106 particles per ml.
Passage 4 = 13.5 x 106 particles per ml.
Passage 5 = 13.5 x 106 particles per ml.
2.2.1.4 Viral Infection
Ohio-HeLa cells were infected with the indicated MOI of HRV16. Cells were seeded in
6-well plates with 2ml of growth media (see section 2.1.3) and incubated overnight at
37°C with 5% CO2 to approximately 70-80% confluence. The media was removed and
cells were washed with 1ml of PBS before the addition of the virus culture diluted in
maintenance media to the indicated MOI to a total of 1ml. Cells were incubated with the
virus for 1hr at 37°C with 5% CO2 with gentle rocking every 15mins. The excess
unincorporated virus was removed and 2ml of fresh maintenance media was added.
Cells were then incubated and treated as indicated.
2.2.1.5 Transfection
2.2.1.5.1 Plasmids
GFP-2A, GFP-2Ainactive, GFP-3C and GFP-3Cinactive clones were generously
provided by Dr. Erin Walker (Walker et al., 2013). Briefly, the sequences for HRV16
2A and 3C were amplified by PCR from the full length HRV16 genome (Lee et al.,
1993) and recombined into the Gateway compatible vector, pEPI-DESTC, to enable the
production of GFP tagged HRV16 2A and 3C proteases (Ghildyal et al., 2005; Ghildyal
et al., 2009). An inactive form of HRV16 2A was generated by mutating the active
Cysteine (Cys 106 (Racaniello, 2007)) to alanine by site-directed mutagenesis and
recombined into the pEPI-DESTC vector as for the wildtype to generate GFP-2Ainac.
Similarly, an inactive form of HRV16 3C was generated by mutating the active
Cysteine (Cys 147 (Racaniello, 2007)) to alanine by site-directed mutagenesis and
recombined into the pEPI-DESTC vector as for the wildtype to generate GFP-3Cinac.
32
The vector (pEPI-GFP) which expresses GFP alone in mammalian cells (Ghildyal et al,
2009) was used as control in all experiments.
2.2.1.5.2 DNA Plasmid Extraction
The GFP-2A, GFP-2Ainactive, GFP-3C and GFP-3Cinactive glycerol stocks were
inoculated in 5ml of Luria-Bertani broth (LB) with 25µl of Kanamycin (final
concentration of 50ug/ml) and incubated at 37°C with constant shaking for 16hrs.
Plasmids were prepared using the Promega Pure Yield miniprep kit as per the
manufacturer’s instructions. The concentration of plasmid DNA was measured using the
Nano-drop spectrophotometer.
2.2.1.5.3 Transfection
COS7 cells were seeded into a 6-well plate in growth medium and grown overnight at
37°C with 5% CO2 until approximately 95% confluent. Cells were then gently washed
with PBS before 250µl of serum free DMEM, 10µl of lipofectamine and 4µg of the
relevant DNA was added and cells were incubated at 37°C with 5% CO2 for 30mins.
2ml of growth media was added to cells and they were incubated overnight at 37°C with
5% CO2 before being lysed and processed for western blot analysis as outlined in
section 2.2.2.1.
2.2.2 Analysis
2.2.2.1 Western Blot Analysis
For each western blot performed, Ohio-HeLa cells were cultured in 6 well plates or
equivalent sized petri dishes. After relevant treatment, cells were lysed with 150µl of
RIPA buffer gently rocked on ice for 30mins. Cell lysates were then transferred into
labelled eppendorf tubes before being stored at -20°C. The cell lysates were mixed with
a 5x Laemmli buffer and heated at 95°C for 5mins. The denatured lysates were
electrophoresed on a 12.5% SDS PAGE gel before being transferred to a nitrocellulose
membrane. Membranes were incubated in 3% skim milk powder in PBS (blocking
33
solution) for 1hr. Following incubation in the blocking solution, the membranes were
incubated with the relevant primary antibody diluted in 1% skim milk powder in PBST
solution overnight at 4°C on a shaker. Membranes were then washed with PBST, three
times for 5mins and once for 10 mins before being incubated for 2hrs in a species
specific secondary antibody conjugated to HRP, diluted in 1% skim milk powder in
PBST. Membranes were washed in PBST followed by enhanced chemiluminiscence
(ECL), exposure and manual development of X-ray films (Kodak film) in order to
visualise the relevant protein bands.
Where required, blots were stripped of probing antibodies using stripping buffer at 50°C
for 10 min, followed by washing in PBST, blocking in 4% skim milk in PBS and
reprobing using different primary antibodies as required.
2.2.2.2 Immunofluorescence Assay
For immunofluorescence (IFA), Ohio-HeLa cells were seeded onto sterile 15mm2
coverslips within a 12well plate and incubated in 1ml of growth media overnight at
37°C with 5% CO2 to approximately 70-80% confluence. Cells were then treated as
outlined for each experiment. After treatment, the media was removed and cells were
washed with 1ml of PBS. Cells were fixed with 500µl of ice cold (-20°C) methanol for
10 mins. Methanol was then washed from cells with two 5 minute washes of 1ml of
PBS. Fixed cells were incubated with 25µl of the relevant primary antibodies for
30mins and then washed with two 5 minute washes of 1ml of PBS. Cells were
incubated with 25µl of the relevant secondary antibody in the dark for 30mins before
again being washed with two 5 min washes with PBS. The coverslips were mounted
onto glass slides with Prolong Gold antifade mounting medium containing DAPI before
curing overnight in the dark. Slides of treated cells were examined and photographed
using Nikon Ti Eclipse confocal laser scanning microscope with Nikon 60x/1.40 oil
immersion lens (Plan Apo VC OFN25 DIC N2; optical section of 0.5µm).
34
3 Chapter Three – Results Rhinovirus Infection Does Not Induce Early or Late Intrinsic Apoptosis
3.1 Introduction
One of the host cell defences against viral infection and spread is the induction of
apoptosis (Barber, 2001; Richard & Tulasne, 2012). Many viruses have developed
strategies to evade this immune mechanism, including a number of picornaviral species,
which have been shown to have the ability to either induce or inhibit the induction of
host apoptosis (Belov et al., 2003; Tolskaya et al., 1995). A few studies investigating
apoptosis during HRV infection have suggested that infection may lead to the induction
of intrinsic, but not extrinsic apoptosis (Taimen et al., 2004; Deszcz et al., 2005; Drahos
& Racaniello, 2009). Interestingly, a study by Gustin and Sarnow showed that infection
of HeLa cells with HRV14 did not result in cleavage of PARP, a marker for late
apoptosis in both intrinsic and extrinsic pathways (Gustin & Sarnow, 2002).
This chapter aimed to determine whether or not apoptosis is induced during infection of
Ohio-HeLa cells with HRV16 through a careful examination of early and late markers
of the intrinsic pathway under various infection conditions.
Following on from the results shown by Deszcz et al, this study paid particular focus on
the induction of the intrinsic apoptotic pathway during HRV16 infection. The intrinsic
apoptotic markers, caspase 9 and cytochrome c, were examined, as well as the universal
apoptotic marker caspase 3. Cytochrome c is released from the mitochondria into the
cytoplasm as one of the initial steps of the intrinsic pathway (refer to figure 1.4).
Caspase 9 is an initiator caspase of the intrinsic apoptotic pathway that once activated,
can go on to activate other apoptotic intermediates, including the caspase 3 effector
caspase. Caspase 3 is an effector caspase that is activated during both the intrinsic and
extrinsic apoptotic pathways (refer to figure 1.4).
As a control, and to determine whether HRV16 induces or is capable of halting
apoptotic cell death, experiments were performed using chemical apoptotic inducers.
Two chemical agents were examined, Taxol and Actinomycin D (Act. D). Paclitaxel
(Taxol) is an anticancer drug derived from the Taxus brevifolia plant (Wani et al., 1971;
35
McGuire et al., 1989). It acts to promote the polymerisation and stabilisation of
microtubules resulting in cell cycle arrest and eventual apoptosis of treated cells (Schiff
et al., 1979; Rowinsky et al., 1988; Woods et al., 1995). Taxol treatment is known to
induce apoptosis in numerous cancer cell lines through the phosphorylation of the anti-
apoptotic gene Bcl-2 (Bhalla et al., 1993; Haldar et al., 1995; Blagosklonny et al.,
1996). Similarly, Act. D is a bacterially derived anti-cancer antibiotic that is known to
induce apoptosis. Act. D acts to inhibit transcription eventually leading to the apoptotic
death of the cell (Vining & Waksman, 1954; Goldberg et al., 1962; Reich et al., 1962).
3.2 HRV16 Infection Does Not Result in Cleavage of PARP
As a first step to elucidating the effect of HRV16 infection on apoptosis, the cleavage of
PARP during infection was examined. Ohio-HeLa cells were seeded and infected with
HRV16 at a MOI of 3 as per section 2.2.1.4. Cells were then treated with 5µg/ml Act. D
and cycloheximide (CHX) for 6hrs, before being lysed with 150µl of RIPA buffer.
Controls were either left untreated, infected for 6h, treated for 6 hrs with Act. D and
cycloheximide or treated for 6 hrs with Act.D and cycloheximide in the presence of the
apoptosis inhibitor z-VAD-fmk. Lysates were processed for western blot analysis and
probed for PARP. As shown in figure 3.1, chemical induction of apoptosis resulted in
cleavage of PARP (lane 2), but HRV16 infection did not (lane 1); infection with
HRV16 was able to inhibit chemical apoptosis as shown by the reduction in intensity of
the cleaved PARP band (lane 4).
The data in figure 3.1 show that HRV16 infection does not induce late apoptosis.
Further investigations in this chapter focussed on determining the stage of the apoptosis
cascade that is inhibited.
36
Figure 3.1 HRV16 infection does not lead to the cleavage of PARP.
Ohio-HeLa cells were infected with HRV16 at MOI 3 and treated with 5µg/ml Act. D and CHX in the
presence or absence of the caspase inhibitor z-VAD-fmk for 6hrs before being lysed for western blot
analysis. Western blot was probed for the presence of PARP with mouse anti PARP primary antibody and
goat anti mouse HRP secondary antibody. Arrows on the left of the blot indicate the approximate protein
size in kDa while arrows on the right of the blot indicate the expected location of PARP and the cleaved
PARP fragment. Results shown are representative of at least 3 independent experiments.
3.3 Rhinovirus Serotype 16 Infection Does Not Induce Late Apoptosis
Further investigation into the effect of HRV16 infection on the induction of host cell
apoptosis was performed using western blot analysis for the cleavage of caspases 3 and
9. Ohio-HeLa cells were seeded and infected as outlined in section 2.2.1.4 with a MOI
of 1. Infected cells were lysed at regular time points post infection (p.i.) (0, 3, 6, 9 and
24hrs p.i.) with 150µl of RIPA buffer (see section 2.1.2) and the cell lysates subjected to
western blot analysis as outlined in section 2.2.2.1. Cells at 24hrs were observed to have
undergone significant CPE with a number of cells having lifted from the well; these
cells were collected and processed along with the attached cells. The blots were initially
probed to detect cleaved caspase 9, as induction of intrinsic apoptosis results in the
activation of caspase 9 via cleavage resulting in a 10kDa cleavage product. The blot
was incubated with the mouse anti caspase 9 antibody followed by HRP-conjugated
secondary antibodies; the antibodies were subsequently stripped from the blot and the
blot re-probed for αβ tubulin as a loading control. As seen in figure 3.2A, there was no
37
detection of the caspase 9 p10 cleavage fragments, nor was there any indication of a
reduction in the procaspase protein. The even loading, as shown by tubulin, supported
the identification that the procaspase 9 protein was not being cleaved, with no obvious
reduction observed over time. Despite the absence of typical apoptotic cleavage of
caspase 9, extra cleavage bands could be seen after 9 and 24hrs in a number of repeat of
this experiment, as demonstrated in figure 3.2A. Due to time constraints this cleavage
was not further investigated, however it may provide a target for future investigation.
The above procedure was repeated, this time probing for the cleavage of caspase 3, an
effector caspase that is cleaved in both the intrinsic and extrinsic apoptotic pathways,
resulting in two cleavage products of 17kDa and 11kDa. Similarly to the caspase 9
results, figure 3.2B shows that procaspase 3 was not cleaved during HRV16 infection,
with no sign of the caspases 3 p11 or p17 cleavage fragments nor any indication of a
reduction in the levels of procaspases 3. Again in some repeats of this experiment,
however not all, extra cleavage of procaspase 3 could be seen at 9 and 24hrs p.i. as seen
in figure 3.2B. Time constraints prevented this cleavage from being further investigated.
These western blots were lastly probed for the HRV structural protein VP2 to confirm
infection had taken place. Figure 3.2C illustrates that VP2 was present from 3hrs post
infection confirming that apoptosis is not induced during HRV16 infection up to 24hrs
p.i. when infected with an MOI of 1. As the viral polyprotein undergoes a series of
cleavages to produce each of the functional viral proteins, each protein may appear in
various lengths dependant on the level of processing that has occurred. This variation is
demonstrated in figure 3.2C where various cleavage products of the viral polyprotein
containing VP2 can be seen.
The data in Figure 3.2 show that HRV16 infection did not induce apoptosis even at 24h
p.i. when extensive cytopathic effect is observed.
To confirm that the lack of caspase cleavage was not due to suboptimal HRV16
infection, Ohio-HeLa cells were infected with HRV16 of varying MOI (0.1, 1 and 10)
for 3, 6 and 24hrs before being lysed for western blot analysis. The cellular lysates were
analysed by western blot analysis as per the methodology used above. As illustrated in
figure 3.3, no p11 cleavage fragment of caspases 3 could be seen, nor is there any
evidence of a reduction in the procaspase 3 protein. As with the results of the previous
experiment, cells at 24hrs were observed to have undergone significant CPE with a
38
number of cells rounding and lifting from the well, these cells were collected and
processed along with the attached cells.
These results suggest that the caspase 9 dependant intrinsic apoptotic pathway is not
induced during HRV16 infection. The identification that caspase 3 is not activated
during infection suggests that neither the intrinsic nor extrinsic pathways are activated,
as caspase 3 is an effector caspase located at the point beyond which both the intrinsic
and extrinsic apoptotic cascades converge (see figure 1.4). These results also suggest
that the absence of apoptotic induction is not dependant on time or MOI.
39
Figure 3.2 Detection of late apoptosis during RV16 infection.
Ohio-HeLa cells were infected with HRV16 at MOI of 1 before being lysed at various time points for
western blot analysis for the detection of markers of late apoptosis. Western blot analysis was carried out
as outlined in section 2.2.2.1. Numbers on the left of each blot indicate the approximate protein size in
kDa. Each blot was incubated with anti-β tubulin to show equal loading in all lanes and is shown below
each blot. A. Blot was incubated with mouse anti caspase 9 p10 primary antibody and goat anti mouse
HRP secondary antibody. Numbers on the right of the blot show the procaspase 9 protein as well where it
would be expected to see the p10 cleaved caspase 9 fragment. B. Western blot was incubated with the
mouse anti caspase 3 primary antibody and the goat anti mouse HRP secondary antibody. Arrows on the
right show the procaspase 3 protein as well as the expected location of the p17 and p11 cleaved caspase 3
fragments. C. Western blot was incubated with mouse anti-VP2 primary antibody and goat anti mouse
HRP secondary antibody to confirm that infection had taken place. Markers on the right side of the blot
demonstrate the presence of the viral VP2 protein in its various stages of cleavage from the polyprotein.
Results shown are representative of at least 3 independent experiments.
40
Figure 3.3 Effect of varying MOI on the detection of late apoptosis during RV16 infection.
Ohio-HeLa cells were infected with RV16 with varying MOI (0.1, 1.0, and 10) before being lysed at
various time points for western blot analysis. Western blot analysis was carried out for the detection of
caspase 3 cleavage, a marker of late apoptosis as outlined in 2.2.2.1. The blot was incubated with mouse
anti caspase 3 p11 primary antibody followed by the goat anti mouse HRP secondary antibody. Arrows
on the left of the blot indicate the approximate protein size in kDa. The arrows on the right of the image
show the procaspase 3 protein as well as the expected location of the p11 and p17 cleaved caspase 3
fragments. Results shown are representative of at least 3 independent experiments.
3.4 RV16 Infection Does Not Induce Early Intrinsic Apoptosis
The apoptotic pathways include a complex cascade of events that ultimately result in the
characteristic apoptotic cell death. This complex cascade provides numerous targets for
viral inhibition of apoptotic death. The finding that the executioner caspase 3 is not
cleaved during HRV16 infection suggests that HRV could possibly interfere with the
pathway further upstream. To identify whether HRV16 may be halting the intrinsic
apoptotic cascade upstream of caspases 3, immunofluorescence assay was performed as
per the methodology outlined in section 2.2.2.2 followed by confocal microscopy. Ohio-
HeLa cells were seeded onto sterile coverslips to approximately 80% confluence. Cells
were then infected with HRV16 with an MOI of 5 and incubated at 37°C with 5% CO2
for 9hrs before being fixed and probed with the mouse anti cytochrome c and guinea pig
anti dsRNA primary antibodies followed by Alexa 568 conjugated goat anti mouse and
Alexa 488 conjugated goat anti guinea pig secondary antibodies. Figure 3.4A
demonstrates the cytochrome c (in red) concentrated within the mitochondria of mock
41
uninfected and untreated cells. Figure 3.4B shows both infected and uninfected cells, all
showing cytochrome c concentrated within the mitochondria. Infected cells are
demonstrated by the presence of dsRNA shown in green. Despite various physical
differences that can be seen between infected and uninfected cells, including
cytoplasmic shrinkage and rounding of the cell, there does not appear to be any change
in the appearance of cytochrome c, with it appearing in concentrated areas rather than
diffuse throughout the cytosol.
These results further confirm that the intrinsic apoptotic pathway is not induced during
HRV16 infection.
42
B.
A.
Figure 3.4 Cytochrome c is not released during RV16 infection.
Ohio-HeLa cells were left uninfected (A) or infected with HRV16 with an MOI of 5 (B) for 9 hrs before
being fixed and incubated with guinea pig anti dsRNA and mouse anti cytochrome c primary antibodies
followed by Alexa 488 conjugated goat anti guinea pig and Alexa 568 conjugated goat anti mouse
secondary antibodies. Coverslips were mounted onto slides using antifade mounting medium containing
DAPI stain A. Uninfected cells show no sign of dsRNA. Arrows indicate the cytochrome c accumulated
within the mitochondria. B. Infected cells are shown by the presence of dsRNA antibody shown in green.
Infected cells show cytochrome c still accumulated within the mitochondria as demonstrated by the
arrows. Results shown are representative of at least 3 independent experiments.
43
3.5 Taxol Induces Apoptosis in Ohio-HeLa Cells
Ohio-HeLa cells were treated with the chemical inducer of apoptosis, Taxol, to
demonstrate that apoptosis could be effectively induced in this cell line. The aim was to
confirm that the results observed during HRV16 infection in sections 3.2 and 3.3 were a
consequence of infection itself rather than the cells’ inherent resistance to apoptosis.
Western blot analysis for the cleavage of procaspase 3 and immunofluorescence assay
for the release of cytochrome c from the mitochondria were used to detect the induction
of apoptosis.
Initially, Ohio-HeLa cells were grown in a 6-well plate in 2ml of growth media
overnight at 37°C with 5% CO2 to approximately 80% confluence as outlined in section
2.2.1.1. Cells were then treated with varying concentrations of Taxol (0.25µg/ml,
0.5µg/ml and 1 µg/ml diluted in maintenance media) or DMSO (Taxol was dissolved in
DMSO before dilution) before being lysed for western blot analysis after 2 or 24 hrs of
treatment as per the method outlined in section 2.2.2.1. The results suggested that the 2
hour incubation was not enough to allow the Taxol to induce apoptosis as there was no
evidence of caspase 3 cleavage (figure 3.5A). With 24h of treatment, cleaved caspase 3
could be seen with all three concentrations of Taxol; 0.5 µg/ml Taxol demonstrated the
strongest level of cleavage at 24hrs treatment (figure 3.5B). Whilst it would be expected
that the level of caspase 3 cleavage would increase with increased Taxol concentration,
the results seen in figure 3.5B do not demonstrate this. It is likely that the lower level of
p11 cleaved fragment seen in the 1 µg/ml cell lysates compared to that seen in the 0.5
µg/ml lysates is a result of the concentration combined with the long incubation leading
to the further degradation of the cleaved fragment or resulting in the cells deteriorating
too far for the cleaved fragment to be recovered.
To further demonstrate that Taxol induced detectable apoptosis in Ohio-HeLa cells,
immunofluorescence assay was carried out for the detection of cytochrome c. Cells
were seeded onto sterile coverslips as outlined in section 2.2.2.2 and grown to
approximately 80% confluence. Cells were either treated with 5µg/ml of Taxol for 3hrs
or left untreated before being washed and fixed as outlined in section 2.2.2.2 and
incubated with the mouse anti cytochome c primary antibody followed by Alexa 488
conjugated goat anti mouse secondary antibody. Coverslips were mounted onto slides
using antifade mounting medium containing DAPI. Slides were then imaged and
analysed using confocal microscopy. Figure 3.6A shows untreated cells demonstrating
44
cytochrome c concentrated within the mitochondria as indicated by the arrows. Figure
3.6B shows a number of cells demonstrating cytochrome c having been released from
the mitochondria into the cytosol, revealing a diffusion of cytochrome c throughout the
cytosol. These cells are most likely to be at the very early stages of apoptosis with none
of the characteristic physical changes associated with apoptosis being observed, which
is expected given the short incubation period of only 3hrs.
The above results show that Ohio-HeLa cells undergo the induction of apoptosis after
treatment with Taxol, a known chemical inducer of apoptosis. The data presented
clearly show that Ohio-HeLa cells respond to Taxol treatment by induction of the
intrinsic apoptotic pathway as shown by release of cytochrome c from mitochondria
into the cytoplasm at 3h post treatment and cleavage of the effector caspase 3 at 24h
post treatment.
Figure 3.5 Taxol treatment induces intrinsic apoptosis in Ohio-HeLa cells.
Ohio-HeLa cells were treated with varying concentrations of Taxol and lysed after 2 or 24hrs. Western
blots were incubated with the mouse anti caspase 3 p11 primary antibody followed by the goat anti mouse
secondary antibody. Arrows on the left of each blot indicate the approximate protein length in kDa. The
arrows on the right of each blot demonstrate the location or expected location of the procaspase 3 protein
or the p11 and p17 cleavage fragments. A. cells were incubated with Taxol for 2hrs. As indicated by the
arrows on the right, no cleavage of the procaspase 3 was observed with no sign of p11 or p17 fragments
nor an obvious reduction in the level of the procaspase protein. B. Cells were treated with Taxol of
various concentrations as indicated. As indicated by the arrows on the right, after 24hrs treatment with 0.5
and 1.0µg/ml of Taxol, both p11 and p17 cleavage fragments can be seen demonstrating that Taxol
treatment induces apoptosis in Ohio-HeLa cells. Results shown are representative of at least 3
independent experiments.
45
Figure 3.6 Taxol treatment leads to cytochrome c release from the mitochondria.
Ohio-HeLa cells were seeded onto sterile coverslips and either left untreated (A) or treated with 5µg/ml
of Taxol (B) for 3 hrs before being fixed and incubated with mouse anti cytochrome c primary and Alexa
488 conjugated goat anti mouse secondary antibodies. Coverslips were mounted onto slides using
antifade mounting medium containing DAPI stain. A. Untreated cells show cytochrome c accumulated
within the mitochondria demonstrated by the arrows. B. Cells treated with Taxol are shown with a
majority of cells having released cytochrome c demonstrated by the absence of concentrated regions of
cytochrome c. The arrow indicates an unaffected cell where cytochrome c has not been released. Results
shown are representative of at least 3 independent experiments.
B.
A.
46
Actinomycin D (Act. D) is an antibiotic used in cancer therapy and is known to induce
apoptosis in treated cells. Similarly to studies with Taxol, western blot analysis for the
cleavage of caspases 3 as well as immunofluorescence assay for the detection of the
release of cytochrome c from the mitochondria was used to demonstrate the induction of
apoptosis in Ohio-HeLa cells treated with Act D.
For western blot analysis, Ohio-HeLa cells were seeded into a 6-well plate in growth
media and incubated overnight at 37°C with 5% CO2 to approximately 90% confluence.
Cells were then either treated with 1 µg/ml Act. D diluted in maintenance media or left
untreated before being lysed after 3 or 5hrs and processed for western blot analysis
(section 2.2.2.1). The western blot was incubated with the mouse anti caspase 3 p11
antibody as per the method outlined in section 2.2.2.1. Figure 3.7 shows some caspase 3
cleavage after 3hrs treatment and an increased level of cleavage in the cells treated for
5hrs. Only partial, low level cleavage was observed at the longest treatment times (5hrs)
in this experiment.
Cells treated with Act. D were also analysed by immunofluorescence for the
mitochondrial release of cytochrome c. Cells were seeded onto sterile coverslips and
incubated overnight at 37°C with 5% CO2 to approximately 80-90% confluent. Cells
were treated with 5µg/ml Act. D for 9hrs before being fixed and incubated with mouse
anti cytochrome c primary antibody followed by Alexa 488 conjugated goat anti mouse
secondary antibody as per the method outlined in section 2.2.2.2. Figure 3.8
demonstrates representative images of both untreated and treated cells. Figure 3.8A
shows untreated cells with the arrows demonstrating cytochrome c accumulated within
the mitochondria. Cells treated with Act. D were seen to undergo apoptosis with figure
3.8B demonstrating cells at various stages of apoptotic death. The arrows indicate
cytochrome c still contained within the mitochondria of cells that are either unaffected
or in the initial stages of apoptosis. The boxed and enlarged cell shows an example of a
cell in the later stages of apoptosis showing cytoplasmic shrinkage and blebbing of the
plasma membrane, changes characteristic of apoptotic death. This cell shows
cytochrome c having been released from the mitochondria and localised diffusely
throughout the cytoplasm rather than concentrated within the mitochondria.
These results confirm that apoptosis can be effectively induced in Ohio-HeLa cells
treated with Act. D demonstrated by the cleavage of caspase 3 and release of
47
cytochrome c from the mitochondria, which was observed after treatment of Ohio-HeLa
cells with 5µg/ml of Act. D.
Figure 3.7 Act. D treatment induces apoptosis in Ohio-HeLa cells.
Ohio-HeLa cells were treated with 1µg/ml Act. D before being lysed at 3 or 5hrs and analysed by western
blot for the identification of cleavage of procaspase 3. The blot was incubated with mouse anti caspase 3
p11 primary antibody followed by goat anti mouse HRP secondary antibody. Arrows on the left indicate
the approximate protein length in kDa. The arrows on the right indicate the location of the procaspase
and cleaved caspase 3 locations. The evidence of both p11 and p17 cleavage fragments in both the 3 and
5hr samples demonstrates that Act. D cleavage induces apoptosis in Ohio-HeLa cells. Results shown are
representative of at least 3 independent experiments.
48
Figure 3.8 Act. D treatment leads to cytochrome c release from the mitochondria.
Ohio-HeLa cells were seeded onto sterile coverslips and either left untreated (A) or treated with 5µg/ml
of Act. D (B) for 9hrs before being fixed and incubated with mouse anti cytochrome c primary and Alexa
488 conjugated goat anti mouse secondary antibodies. Coverslips were mounted onto slides using
antifade mounting medium containing DAPI stain. A. Untreated cells show cytochrome c accumulated
within the mitochondria demonstrated by the arrows. B. Image shows cells at various stages of apoptosis
following treatment with Act. D. Arrows demonstrate cytochrome c not yet released from within the
mitochondria. Inset image shows close up of later stage apoptotic cell with the cytochrome c having been
released from the mitochondria and seen diffuse throughout the cytosol. Results shown are representative
of at least 3 independent experiments.
B.
A.
49
3.6 Summary
The results presented in this chapter indicate that infection of Ohio-HeLa cells with
HRV16 does not induce apoptosis and in particular, does not induce apoptosis through
the intrinsic pathway. It was confirmed that apoptosis could be reliably induced in
Ohio-HeLa cells after treatment with Taxol and Act. D. The lack of apoptosis induction
during HRV16 infection was not limited by the amount of virus used in the experiments
nor the time at which the samples were analysed. This was shown by the use of a high
MOI (10) and analysis through to 24hrs p.i. when significant cell death was observed.
50
4 Chapter Four – Results Effect of HRV16 Infection on Chemically Induced Apoptosis
4.1 Introduction
It has previously been observed that some picornaviral species are capable of avoiding
the apoptotic response of the host cell. Previous studies investigating poliovirus have
found that apoptosis is inhibited during productive poliovirus infection (infection under
conditions optimal for maximum viral replication) within HeLa cells suggesting that
poliovirus is capable of actively inhibiting the induction of apoptosis (Tolskaya et al.,
1995; Agol et al., 2000; Belov et al., 2003). The observation that HRV16 infection did
not induce apoptosis suggests the possibility that HRV16, like poliovirus, may be
capable of actively suppressing the host apoptotic pathways.
This chapter aimed to determine if HRV16 infection is capable of actively suppressing
the induction of chemically induced apoptosis.
Ohio-HeLa cells were infected with HRV16 prior to induction of apoptosis via
treatment with Taxol and Act. D. The efficacy of this chemically induced apoptosis was
then investigated.
4.2 Effect of HRV16 Infection on Taxol Induced Apoptosis
Data presented in Chapter three have shown that HRV16 infection does not induce the
intrinsic apoptotic pathway, raising the possibility that HRV16 actively suppresses
apoptosis. Therefore, the ability of HRV16 infection to inhibit chemically induced
apoptosis was investigated. Ohio-HeLa cells were seeded onto sterile coverslips and
incubated overnight to approximately 80% confluence as outlined in section 2.2.1.1
followed by infection with HRV16 with an MOI of 5 as outlined in section 2.2.1.4.
Following infection, cells were either treated with 5µg/ml of Taxol and incubated for
3hrs before being washed and fixed as per the method in section 2.2.2.2, or control cells
were left untreated. Cells were processed for immunofluorescence assay where cells
were incubated with mouse anti VP2 primary followed by Alexa 568 conjugated goat
51
anti mouse secondary antibodies before being imaged and analysed using confocal
microscopy. Interestingly, it was observed that the level of the viral structural protein
VP2 appeared to be lower in cells both infected with HRV16 and treated with Taxol
(see figure 4.1A) when compared with cells that were infected but not treated with
Taxol (figure 4.1B).
To confirm whether or not VP2 was in fact reduced during Taxol treatment, a similar
experiment was carried out, this time utilising western blot analysis following the same
infection and chemical treatment as that used in the previous experiment. Cells were
lysed and processed for western blot before being incubated with the mouse anti VP2
primary followed by the relevant HRP conjugated secondary antibodies as per section
2.2.2.1, with the results shown in figure 4.2. The results shown in figure 4.2 confirm the
observations in immunofluorescence assay (figure 4.1) that VP2 expression is reduced
in HRV16 infected cells treated with Taxol compared to cells infected only,
demonstrating that cells treated with Taxol had reduced levels of VP2. In particular, the
levels of the larger VP2 containing fragments of the viral polyprotein were lower than
in infected cells that were not treated. As the viral polyprotein undergoes a series of
cleavages to produce each of the functional viral proteins, each protein may appear in
various sizes dependant on the level of processing that has occurred. This variety is
demonstrated in figure 4.2 (as well as that in figure 3.2C) where the cells infected with
HRV16 show VP2 present in protein moieties of various sizes. The reduction in the
level of larger polyprotein fragments in Taxol treated cells suggests that the translation
of new polyproteins may be inhibited by Taxol treatment. This observed reduction was
not due to unequal sample loading as even loading is demonstrated by tubulin.
52
Figure 4.1 Taxol treatment during HRV16 infection may reduce expression of VP2.
Ohio-HeLa cells were seeded onto sterile coverslips and infected with HRV16 with MOI of 5 before
being either treated with 5µg/ml of Taxol or left untreated for 3hrs p.i.. Cells were then fixed and
incubated with mouse anti VP2 primary and Alexa 568 conjugated goat anti mouse secondary antibodies.
Coverslips were mounted onto slides using antifade mounting medium containing DAPI stain. A. Cells
were infected but not treated with Taxol. Infected cells are shown as indicated by the arrows with the
presence of the viral VP2 protein shown in red. B. Cells were infected and treated with Taxol. Infected
cells are shown by the arrows with the viral VP2 protein indicated in red. As compared to infected only
cells seen in A., cells treated with Taxol show lower levels of VP2. Results shown are representative of at
least 3 independent experiments.
B.
A.
53
Figure 4.2 Taxol treatment during HRV16 infection may lead to a reduction in the translation of
the viral polyprotein.
Ohio-HeLa cells were infected with HRV16 with an MOI of 5 before being treated with 5µg/ml Taxol for
3hrs. Western blot was incubated with mouse anti VP2 primary and goat anti mouse HRP secondary
antibodies. The first lane shows lysate from uninfected and untreated cells; second lane shows lysate from
infected cells that were not treated; the third lane shows lysate from cells treated with Taxol and infected;
the fourth lane shows lysate from uninfected cells that were treated with Taxol. Results shown are
representative of at least 3 independent experiments.
4.3 Effect of HRV16 Infection on Act. D Induced Apoptosis
Following on from the results demonstrating that infected cells treated with Taxol
resulted in a reduction in the viral VP2 protein, this study focused on the use of Act. D
to determine if HRV16 had the ability to inhibit or disrupt the apoptotic signalling
cascade.
Initially, the effect of Act. D treatment on the expression of VP2 was investigated to
ensure that Act. D treatment did not interfere with the translation of the viral
polyprotein as was the case with Taxol treatment. Ohio-HeLa cells were seeded onto
sterile coverslips and grown to approximately 80% confluence before being infected
with HRV16 at MOI 5 and treated with 5µg/ml of Act. D for 9hrs. Cells were then fixed
and probed with mouse anti VP2 primary antibody followed by Alexa 568 conjugated
goat anti mouse secondary antibody. Figure 4.3 demonstrates that no difference in the
level of VP2 expression could be seen between infected cells with (figure 4.3A) and
54
without (figure 4.3B) treatment with Act. D. This suggests the Act.D treatment does not
interfere with the translation of the viral polyprotein.
To examine if HRV16 infection had the ability to inhibit or reduce the induction of
apoptosis in cells treated with Act. D, cells were seeded onto sterile coverslips and
grown overnight to approximately 80% confluence (see section 2.2.2.2.). Cells were
then washed and infected with HRV16 with MOI of 5 as outlined in section 2.2.1.4.
Cells were then treated with 5µg/ml of Act. D for 9hrs before being washed and fixed
(see section 2.2.2.2) Coverslips were mounted onto slides using antifade mounting
medium containing a DAPI stain before being photographed by confocal microscopy.
The effect of HRV16 infection on Act. D induced apoptosis was measured by
quantitative analysis. A series of representative widefield images were taken of each
sample and the number of apoptotic and healthy cells were counted. Results in figure
4.4 show pie-charts depicting the percentage of apoptotic and healthy cells counted after
each treatment. The results show a marked reduction in the proportion of apoptotic cells
in cells infected with HRV16 prior to treatment with Act. D in comparison to those cells
treated with Act. D alone. It was observed that 73% of cells treated with Act. D were
apoptotic compared to 45% of cells infected with HRV16 prior to Act. D treatment.
These results suggest that HRV16 infection may have to ability to actively inhibit the
induction of apoptosis of the host cell.
55
Figure 4.3 Act. D treatment during HRV16 infection does not reduce expression of VP2.
Ohio-HeLa cells were seeded onto sterile coverslips and infected with HRV16 with MOI of 5 before
being either treated with 5µg/ml of Act. D or left untreated for 9hrs p.i.. Cells were then fixed and
incubated with mouse anti VP2 primary and Alexa 568 conjugated goat anti mouse secondary antibodies.
Coverslips were mounted onto slides using antifade mounting medium containing DAPI. A. Cells were
infected but not treated with Act. D. Infected cells are shown as indicated by the arrows with the presence
of the viral VP2 protein shown in red. B. Cells were infected and treated with Act. D. Infected cells are
shown by the arrows with the viral VP2 protein indicated in red. As compared to infected only cells seen
in A., cells treated with Act. D show no difference in the levels of VP2. Results shown are representative
of at least 3 independent experiments.
B.
A.
56
Figure 4.4 HRV16 infection reduces the induction of apoptosis in Act. D treated cells.
Ohio-HeLa cells were grown on sterile coverslips and either left untreated (mock), infected with HRV16
with MOI of 5, treated with 5µl/ml of Act. D or a combination of both. Cells were then fixed and the
coverslips were mounted onto slide with antifade mounting medium containing DAPI stain. Cells were
imaged with the Nikon Ti Eclipse confocal microscope as described in section 2.2.2.2. Both apoptotic and
healthy cells were counted from a series of representative images taken of each treatment type as
exemplified by the one healthy and two apoptotic cells shown (B). The percentage of each was then
calculated and represented as pie charts above (A). Results shown are representative of at least 3
independent experiments.
57
4.4 Summary
The results presented in this chapter, together with those presented in Chapter 3, suggest
that HRV16 infection does not result in the induction of apoptosis, probably through its
ability to actively suppress apoptotic pathways. Initially it was found that treatment of
HVR16 infected cells with Taxol may inhibit the translation of the viral polyprotein due
to a reduction seen in the levels of VP2 expression. Since Taxol acts to cause cell cycle
arrest by stabilising the microtubules of the cell, it is likely that this stabilisation of the
microtubules may be the cause of the inhibition of the translation of the viral
polyprotein. Experiments investigating apoptosis in infected cells treated with Act. D
demonstrated a reduction in the number of apoptotic cells when compared to uninfected
cells treated with Act. D, suggesting that HRV16 infection may actively inhibit the
apoptotic pathway.
58
5 Chapter Five – Results Rhinovirus Infection Leads to the Indirect Cleavage of RIPK1
5.1 Introduction
Receptor interacting protein kinase 1 (RIPK1, also known as RIP1) is a 74kDa protein
that interacts with Fas and TNRF1 receptors, crucial to the regulation of programmed
cell death (Clement & Stamenkovic, 1994; Stanger et al., 1995; Ting et al., 1996).
Cleavage activation of RIPK1 has been identified as being crucial to TNF-induced
apoptosis, where it has been demonstrated that RIPK1 cleavage is carried out by
activated caspase 8 (Lin et al., 1999). Cleavage of RIPK1 has also been identified in
other apoptotic pathways including TRAIL and Fas induced apoptosis (Lin et al., 1999).
This makes RIPK1 an effective target in the investigation of extrinsic apoptosis during
HRV16 infection.
The picornaviral proteases are known to play a role in the alteration or inhibition of a
number of host cell signalling pathways including translation initiation and
nucleocytoplasmic trafficking (Gustin & Sarnow, 2002; Grubman et al., 2008). This
ability to interact and alter the host signalling pathways make the viral proteases prime
candidates when investigating the possible mechanisms of HRV16 inhibition of
apoptosis. This chapter aimed to investigate the effect of HRV16 infection on extrinsic
apoptosis, using RIPK1 as a marker, and begins to elucidate the possible mechanisms
through which HRV16 infection may interact with the host cell apoptotic pathways,
with a particular focus on the 2A and 3C proteases.
5.2 HRV16 Infection and Act. D Treatment Lead to Dissimilar Cleavage of RIPK1
After the results in chapter three demonstrated that HRV16 infection did not induce
apoptosis, and in particular did not appear to induce intrinsic apoptosis, the involvement
of the extrinsic apoptotic pathway was investigated in an attempt to elucidate the
possible mechanisms of HRV16 suppression of apoptosis as found in chapter four.
RIPK1 is a key component of the TNFR, TRAIL and Fas receptor mediated apoptotic
59
pathways. The binding of TNFα, Fas and other ligands to their receptors promotes
various signalling cascades, including extrinsic apoptosis within which one of the
crucial steps is the cleavage of RIPK1.
Western blot analysis for the cleavage of RIPK1 was carried out as per the methods
outlined in section 2.2.2.1. Briefly, Ohio-HeLa cells were seeded in a 6-well plate in
growth medium overnight at 37°C with 5% CO2 until approximately 80% confluent.
Cells were then either left untreated, infected with HRV16 with an MOI of 5, treated
with 5µg/ml Act. D or a combination of both. Cells were lysed after 9hrs with RIPA
buffer and processed for western blot analysis. The western blot was incubated with the
mouse anti RIPK1 primary and goat anti mouse HRP secondary antibodies. As shown
in figure 5.1, RIPK1 is cleaved during both HRV16 infection and Act. D treatment. The
cleavage patterns observed differ between the treatments such that cleavage of RIPK1
by Act. D treatment results in a cleavage fragment of approximately 40kDa while
cleavage due to HRV16 infection results in a cleavage fragment of approximately
21kDa. Both cleavage fragments can be seen in samples that were infected with HRV16
and treated with Act.D indicating that mechanisms behind each cleavage are most likely
independent of each other. The cleavage observed during Act. D treatment is consistent
with that seen in studies by Lin et al. (Lin et al., 1999) where they described a 42kDa
cleavage fragment resultant from TNF-induced apoptosis, confirming that the cleavage
observed in this study during Act. D treatment is consistent with the induction of
apoptosis.
The demonstration that HRV16 infection induces RIPK1 cleavage demonstrates the
involvement of the extrinsic apoptotic pathway in the host’s response to HRV16
infection. The observation that the cleavage fragments resulting from HRV16 infection
differ from those observed during Act. D treatment, suggests that this alternate cleavage
may play a role in the inhibition of apoptosis by HRV16.
60
Figure 5.1 HRV16 and Act. D treatment leads to cleavage of RIPK1.
Ohio-HeLa cells were either left untreated, infected with HRV16 with an MOI of 5, treated with 5µg/ml
Act. D or both for 9hrs. Western blot was incubated with mouse anti RIPK1 primary and goat anti mouse
HRP secondary antibodies. The arrows on the left of the blot indicate the approximate protein size in
kDA while the arrows on the right demonstrate the RIPK1 protein and the RIPK1 cleavage fragments
resultant from HRV16 infection and Act. D treatment. Results shown are representative of at least 3
independent experiments.
5.3 Cleavage of RIPK1 is Probably Not Carried Out By the 2A or 3C Proteases
The picornaviral proteases 2A and 3C are known to play a role in the alteration of a
number of host cell pathways including translation initiation and nucleocytoplasmic
trafficking via cleavage of specific host cell proteins (Grubman et al., 2008; Gustin &
Sarnow, 2002). Following on from the identification that HRV16 infection leads to
alternate cleavage of RIPK1 compared to Act. D treatment, this study aimed to
determine if the 2A or 3C proteases play a role in this alternate cleavage.
Active and inactivated 2A and 3C proteases were transfected into COS7 cells as
outlined in section 2.2.1.5. Cells were lysed 20h after transfection, and processed for
western blot analysis (as per section 2.2.2.1) where they were initially probed for the
detection of GFP to confirm the efficacy of the transfection. Figure 5.2A shows that the
GFP-3C, GFP-3C inactive and GFP-2A inactive fusion proteins were effectively
61
transfected into the COS7 cells as demonstrated by the larger protein bands in lanes 1, 2
and 4, with GFP alone being approximately 27kDa. It can be seen that the GFP-2A
fusion protein was not detected; this is most likely the result of the active 2A protease
cleaving itself from GFP. The GFP-2A cells were not discounted from this study as it is
known that the 2A protease is highly active and may therefore have had the opportunity
to act within the host cell despite its presence not being confirmed by western blot.
Following confirmation that the transfections were successful, the blot was incubated
with the mouse anti RIPK1 primary antibody followed by the goat anti mouse HRP
secondary antibody. Figure 5.2B demonstrates that no cleavage of RIPK1 had occurred.
The blot was over developed in order to visualise very low levels of cleaved protein.
This resulted in a large amount of background non-specific bands however after
comparison with the results in figure 5.1, no specific cleavage was seen, particularly not
at the points where cleavage was seen during HRV16 infection and Act. D treatment.
These results suggest that the viral 3C protease is not responsible for the cleavage of
RIPK1 seen during HRV16 infection. It is also suggested that the viral 2A protease is
not likely to be responsible for these cleavages either. It can be confirmed that
inactivated 2A protease does not result in RIPK1 cleavage however this study cannot
rule out that active 2A protease may play a role. Due to time restraints, possible
mechanisms behind the cleavage of RIPK1 in HRV16 infected cells could not be further
investigated.
5.4 Expression of 2A and 3C Proteases Does Not Induce Apoptosis
Previous experiments with various picornaviruses have found that the expression of the
viral proteases alone can induce apoptosis of the host cell. The ability of 2A and 3C
protease to induce apoptosis was examined by western blot following the expression of
the active and inactive forms, in transfected cells. The western blot was incubated with
the mouse anti caspase 3 primary antibody followed by the goat anti mouse HRP
secondary antibody. Figure 5.2C shows that neither the expression of the active nor
inactive proteases induced apoptosis as demonstrated by the absence of cleavage of
caspase 3. As with the previous experiment, it cannot be ruled out that active 2A may
have the ability to individually induce apoptosis, time constraints prevented further
investigation during this study.
62
Figure 5.2 HRV16 2A and 3C proteases do not cleave RIPK1 or caspase 3.
COS7 cells were transfected to express active or inactive 2A or 3C proteases as GFP-fusion proteins.
Western blots of the cell lysates are shown above. Arrows on the left of the blots indicate the approximate
protein size in kDa while arrows on the right indicate the position or predicted position of each of the
proteins being analysed. A. Western blot was incubated with the mouse anti GFP primary antibody
followed by the goat anti mouse HRP secondary antibody to determine the efficacy of the transfection. It
can be seen that the GFP-3C, GFP-3C inactive and GFP-2A inactive were effectively transfected. GFP-
2A appears to have been cleaved, with GFP appearing only alone in the GFP-2A transfected cells. B.
Western blot was incubated with mouse anti RIPK1 primary and goat anti mouse HRP secondary
antibodies. As demonstrated by the arrows on the right, there is no evidence of cleavage of RIPK1 as was
seen during HRV16 or Act. D treatment outside of the background bands seen in all treatments. Blot was
over developed to detect any low level cleavages. Background bands seen are consistent with those seen
in figure 5.1 C. Western blot was incubated with the mouse anti caspase 3 primary and goat anti mouse
HRP secondary antibodies. As demonstrated by the arrows on the right, there is no evidence of cleavage
of caspase 3. Results shown are representative of at least 3 independent experiments.
63
5.5 Summary
This study aimed to elucidate the mechanisms with which HRV16 infection may
interact with the host cell apoptotic pathways. Following on from the findings in the
previous chapter that HRV16 infection did not induce early or late intrinsic apoptosis
and that late stage apoptosis was not induce by either pathway, the possibility of the
involvement of the extrinsic apoptotic pathway was investigated.
A wide range of different initiators of extrinsic apoptosis have been identified, all
resulting in differing signalling pathways that ultimately converge to trigger the effector
caspase 3 and the characteristic physical changes unique to apoptotic cell death. In this
study, the cleavage of RIPK1 during HRV16 infection was investigated. RIPK1 is a
protein that is known to be cleaved during TNFR, TRAIL and Fas induced extrinsic
apoptosis. RIPK1 was found to be cleaved during treatment with Act. D resulting in the
detection of an approximately 40kDa sized cleavage fragment, consistent with results
shown during apoptosis by Lin et al (Lin et al., 1999). HRV16 infection also induced
cleavage of RIPK1, however it resulted in a cleavage fragment of approximately 21kDa.
It was investigated whether the viral 2A or 3C proteases may have been responsible for
this alternate cleavage. Transfection with both active and inactive forms of the 2A and
3C proteases did not result in cleavage of RIPK1 nor cleavage of caspase 3, indicating
that infection by HRV16 leads to the indirect cleavage of RIPK1 with neither of the
HRV16 proteases being responsible. The identification that RIPK1 is cleaved during
HRV16 infection alternately to that demonstrated during apoptosis suggests that the
virus may have the ability to disrupt the induction of the extrinsic apoptotic pathway
and thus may have the ability to inhibit the induction of host apoptosis.
Due to time restraints, the mechanisms behind HRV16 cleavage of RIPK1 could not be
elucidated but provides opportunity for future research.
64
6 Chapter Six – General Discussion
6.1 Introduction
HRV is the most common viral cause of upper respiratory tract infections and causes
significant physical and financial burden throughout the world. Whilst illness caused
directly by HRV is mild and self-limiting, the association of HRV infection with severe
asthma and COPD exacerbations, in combination with its high prevalence throughout
all populations, makes it a virus of significant medical and social interest (Johnston et
al., 1995; Seemungal et al., 2000; Greenberg, 2003).
One of the body’s first responses to the onslaught of viral infection is the cellular
induction of apoptosis, triggered to eliminate viruses in order to limit their effect and
minimise their spread. Just as cells have developed immune responses against viral
pathogens, viruses have developed strategies of their own to circumvent or disrupt these
responses. A number of picornavirus species have been found capable of suppressing
the induction of the host apoptotic response. Of particular interest, poliovirus has been
observed as being capable of suppressing the apoptotic response when viral growth
conditions are ideal in order to help facilitate viral growth (Tolskaya, Romanova et al.,
1995; Agol, Belov et al., 2000; Belov, Romanova et al., 2003). There have been very
few studies investigating the effect of HRV infection on host cell apoptosis. Those that
have been performed have reported somewhat contradictory results.
The ability of viruses to circumvent or suppress the apoptotic pathways of host cells
significantly increases their virulence and can have serious health and societal
consequences. This study aimed to identify and provide further clarification of the effect
of HRV16 infection on the host apoptotic pathways so as to provide opportunities for
the identification of potential novel therapeutic targets.
6.2 HRV Infection Does Not Induce Apoptosis
HRV is an RNA virus that undergoes replication and translation of its genome within
the cytoplasm of the host cell. Whilst the HRV genome, like all picornaviruses, consists
of a single strand of RNA, double stranded RNA (dsRNA) is formed as an intermediate
65
of the viral replication process. The presence of dsRNA is known to induce the
apoptotic response in cells as it is not present under normal cellular conditions
(Iordanov et al., 2005). Cells are capable of recognising viral dsRNA through a range of
cellular dsRNA interacting intermediates. One of the pathways through which
exogenous dsRNA can be detected by the cell is through its interaction with the toll-like
receptor 3 (TLR3) (Alexopoulou et al., 2001). TLR3 is activated by the binding of
dsRNA following its entry into the cell via the endocytic pathway (Akira et al., 2006;
Jiang et al., 2008). Following its activation, TLR3 leads to the activation of NF-κB via
RIPK1 which goes on to induce an apoptotic signalling cascade ultimately leading to
cell death (Alexopoulou et al., 2001; Robbins et al., 2003; Koyama et al., 2008).
Cytosolic sensors of dsRNA include the dsRNA dependant kinase (PKR), melanoma
differentiation-associated gene 5 (MDA-5) and retinoic acid-inducible gene I (RIG-I)
(Akira et al, 2006; Koyama et al, 2008). PKR is activated through the binding of
dsRNA and goes on to induce apoptosis through the subsequent activation of eIF-2,
FADD and NF-κB (Levin & London, 1978; Der et al., 1997; Balachandran et al., 1998;
Srivastava et al., 1998; Williams, 1999; Gil et al., 1999). Similarly, MDA-5 and RIG-I
are activated by binding of dsRNA (Koyama et al, 2008). MDA-5 and RIG-I induced
apoptosis is mediated by the interferon-β promoter stimulator 1 (IPS-1) through
activation of FADD and NF-κB (Kawai et al., 2005; Koyama et al., 2008; Lei et al.,
2009). Thus, several cellular pathways activated by the presence of dsRNA lead to the
apoptotic death of the cell.
Various studies have observed that infection with picornaviruses, including HRV,
induces the recognition of viral dsRNA and subsequent activation of the TLR3, PKR,
MDA-5 and RIG-I mediated pathways (Yeung et al., 1999; Kato et al., 2006; Slater et
al., 2010). These findings are consistent with the knowledge that HRV produces dsRNA
as an intermediate during viral replication and the confirmation of its presence during
HRV16 infection demonstrated in this study (see figure 3.4). With the well-established
recognition of dsRNA by various cellular intermediates, which result in a series of
antiviral responses including the induction of apoptosis, it is expected that HRV, like all
RNA viruses, would induce this response. Previous studies have shown apoptosis to be
both induced and suppressed during picornavirus infection, depending on the viral
species (Tolskaya et al., 1995; Buenz & Howe, 2006). Interestingly, it has been
observed that apoptosis is not induced during EMCV and some poliovirus infections
and that these infections are capable of actively suppressing the induction of chemically
66
induced apoptosis (Tolskaya et al., 1995; Romanova et al., 2009). These findings that a
number of picornaviruses are capable of avoiding host cell apoptosis, despite the
presence of their dsRNA replication intermediates, suggest that they have developed
strategies to actively suppress the apoptotic pathways, however the mechanisms behind
this active suppression are yet to be elucidated.
This study found that HRV16 infection did not lead to the induction of apoptosis in
Ohio-HeLa cells (see chapter 3). These results differ from those observed previously
during HRV 1a, 1b and 14 infections, where it was observed that apoptosis was induced
as demonstrated by the cleavage of caspase 3, the caspase substrate PARP, and release
of cytochrome c from the mitochondria (Taimen et al 2004; Deszcz et al., 2005; Drahos
& Racaniello, 2009). The different results found in chapter 3, in comparison to those
found with HRV1a and HRV1b, may be explained by the fact that these viruses are
minor group viruses whilst HRV16 used in this study belongs to the major group. The
main difference between the major and minor groups is their use of cellular receptors
during entry into the host cell (Tomassini et al., 1989; Hofer et al., 1994). This
difference may play a role in the alternate results seen however further investigations
are required where minor and major group viruses are analysed in parallel for their
apoptosis inducing ability. Studies investigating the major group HRV14 have found
contradicting results, with one study observing cleavage of the caspase substrate PARP
and DNA fragmentation characteristic of apoptosis, whilst the other observed these not
to have occurred (Gustin & Sarnow, 2002; Deszcz et al., 2005). The differences
observed in these studies may be explained by the varying levels of virus used during
infection. Deszcz et al used an MOI of 100 during infection, which may have been
responsible for the induction of apoptosis that was observed due to the saturation of
virus particles present (Deszcz et al., 2005). In contrast, Gustin and Sarnow used an
MOI of 10 during their infections, a level that is more comparable with natural infection
doses (Gustin & Sarnow, 2002). In this study, viral infections were carried out with an
MOI of between 0.1 and 10, comparable to that used by Gustin and Sarnow, as were the
results observed (Gustin & Sarnow, 2002).
This study also demonstrated that not only was apoptosis not observed during HRV16
infection, but that it does not appear as though the intrinsic apoptotic pathway is
induced. This was demonstrated by caspase 9 not being cleaved and cytochrome c not
being released from the mitochondria (see chapter 3). This is in contrast to results
observed by Deszcz et al., who suggested that the intrinsic apoptotic pathway was
67
induced during HRV14 infection (Deszcz et al., 2005). As mentioned above, very high
levels of virus were used during the study carried out by Deszcz et al. particularly in
comparison to those used in this study, which may have had an indirect role in inducing
the intrinsic apoptotic pathway (Deszcz et al., 2005).
6.3 HRV16 Inhibits Apoptosis
The results in chapter 4 suggest that HRV16 infection not only avoids the induction of
host apoptosis, but that it may actively suppress the apoptotic pathways of the host cell.
Ohio-HeLa cells infected with HRV16 prior to treatment with Act. D resulted in a
reduced number of apoptotic cells compared to those treated with Act. D alone. The
observation that both very early and late intrinsic apoptosis were not induced during
infection (chapter 3), raises the possibility that the extrinsic apoptotic pathways may be
associated with HRV infection, and potentially other picornaviruses. The fact that
caspase 3 was not cleaved during HRV16 infection suggests that if extrinsic apoptosis
was induced, it was halted at an early stage of the pathway (refer to figure 1.4). This
study demonstrated that RIPK1, an intermediate of the extrinsic apoptotic pathway, was
cleaved during HRV16 infection differently to that seen during apoptosis induced by
Act. D (chapter 5). RIPK1 is a protein of 74kDa, that interacts with the intracellular
domains of the extrinsic apoptotic receptors Fas and TNFR1, resulting in an apoptotic
response following cleavage by caspase 8 (Stanger et al., 1995; Grimm et al., 1996; Lin
et al., 1999). It has previously been observed that cleavage of RIPK1 occurred during
TRAIL and Fas induced apoptosis resulting in a 42kDa cleavage fragment similar to
that observed in this study in cells treated with Act. D (Lin et al., 1999). The cleavage
fragment observed during HRV16 infection was smaller, at approximately 21kDa
(figure 5.1). This alternate cleavage of RIPK1 may play a role in the potential
suppression of apoptosis by HRV16 infection. The involvement of RIPK1 also suggests
that the extrinsic apoptotic pathway may be implicated in the response of Ohio-HeLa
cells to HRV16 infection.
Interestingly, the cleavage of RIPK1 by Act. D occurs after a short length of treatment,
whist induction by the intrinsic pathway, demonstrated in chapter 3 by the release of
cytochrome c from the mitochondria, occurs only after long treatment times (9hrs
incubation was used in this study). This suggests that Act. D treatment directly induces
68
the extrinsic apoptotic pathway whilst the effect of Act. D on the cellular transcription
may, indirectly, be the cause of intrinsic apoptotic induction.
During this study, it was observed that treatment with Taxol may inhibit the translation
of the HRV polyprotein (chapter 4). Results shown in chapter 4 demonstrate a reduction
in the expression of VP2, with western blot analysis demonstrating a reduction in the
amount of larger VP2 containing polyprotein lengths present in cells treated with Taxol
after infection. These results suggest that processing of the polyprotein was not affected
but that new polyprotein synthesis was inhibited. Taxol acts to stabilise the
microtubules of the cell (Schiff et al., 1979; Rowinsky et al., 1988; Woods et al., 1995).
The results shown in this study suggest that HRV16 infection may utilise the
microtubules of the host cell during translation. It has previously been identified that the
microtubules are utilised by poliovirus for release of the virus from the cell (Taylor et
al., 2009), whilst it has also been observed that HRV utilises the microtubules for
transport of proteins throughout the cell (Grassme et al, 2005). This supports the
possibility that there is an important role microtubules may play in HRV16 polyprotein
translation.
6.4 HRV16 Infection Leads to Indirect Cleavage of RIPK1
Based on previous evidence demonstrating the ability of picornavirus proteases 2A and
3C to cleave or alter a number of cellular components, it was thought likely that these
proteases may be responsible for the alternate cleavage of RIPK1 observed during HRV
infection. Results shown in chapter five demonstrate that cells expressing the HRV16
2A and 3C proteases do not result in cleavage of RIPK1. These results confirmed that
the 3C viral protease is not responsible for the alternate RIPK1 cleavage, however the
potential role of 2A could not be ruled out as the presence of GFP-2A could not be
confirmed by western blot analysis due to the highly active nature of the protease
resulting in it cleaving itself from GFP. Cleavage of RIPK1 by 2A is unlikely however,
as when the translation sequence for the RIPK1 protein was analysed through a
picornaviral protease cleavage site predictor (NetPicoRNA 1.0), no potential 2A
cleavage sites were identified. Two potential 3C cleavage sites were identified at the
89th
and 525th
amino acids, neither of which aligned with the cleavage of RIPK1 seen
during HRV16 infection. Whilst it has been shown here that the HRV proteases are not
responsible for the cleavage of RIPK1, further investigation is required to determine the
69
exact mechanisms behind this cleavage and its potential role in the suppression of
apoptosis during HRV infection.
6.5 Conclusion
Despite a number of studies performed to investigate the induction of apoptosis during
infection with a variety picornaviruses, very little is known about the effect of HRV on
host cell apoptosis. The limited number of studies carried out to date have shown mixed
results, but a majority have suggested that HRV infection induces host cell apoptosis.
The results presented in this study show that host cell apoptosis is not only avoided
during HRV16 infection, but that the virus is capable of actively suppressing it. The
identification that HRV16 infection did not induce the intrinsic apoptotic pathway, but
that it led to the cleavage of RIPK1 not consistent with apoptotic cleavage, suggests that
the virus acts to inhibit the extrinsic apoptotic pathway. Whilst further investigation of
the exact mechanisms behind the cleavage of RIPK1 and suppression of extrinsic
apoptosis are required, these results provide a basis for future studies and an opportunity
for potential new therapeutic targets to be identified.
70
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