By Elise Hovingh
University of Utrecht
The front cover depicts the artistic
presentation of the four different viruses
which will be covered in this thesis.
Additionally the process of autophagy is
depicted where the double membrane vesicle
is derived from the ER and eventually fuses
with the endosomes/lysosome in order to
degrade its content consisting of organelles or
other cellular/viral components.
Images were taken from:
http://www.turbosquid.com/3d-models/nucleus-cellendoplasmic-reticulum-max/609890
http://endosymbiotichypothesis.wordpress.com/evidenc
e-for-the-endosymbiotic-hypothesis/
http://bilbo.bio.purdue.edu/~baker/projects/papova/pa
pilloma/hpv_1L1L2.html
http://www.sciencephoto.com/media/248957/view
http://www.rkm.com.au/virus/coronavirus/coronavirusvirion.html
http://www.newscientist.com/article/dn17049-expertanalysis-mexican-swine-flu--the-story-so-far.html
Special thanks to my supervisor Dr. Fulvio
Reggiori for a critical look at my manuscript
and his guidance.
1|Page
ABBREVIATIONS
4
ABSTRACT
7
1. AUTOPHAGY
8
1.1 INTRODUCTION
1.2 MECHANISM OF THE AUTOPHAGOSOMES FORMATION
1.3 REGULATION
1.3.1 MTOR
1.3.2 BECLIN 1
1.3.3 UNFOLDED PROTEIN RESPONSE
1.4 ANTIGEN PRESENTATION AND AUTOPHAGY
8
8
9
9
11
11
11
2 HERPESVIRUSES
13
2.1 INTRODUCTION
2.1.1 THE DISEASES
2.1.1.1 Alphaherpesviruses
2.1.1.2 Betaherpesviruses
2.1.1.3 Gammaherpesviruses
2.1.2 INFECTION MECHANISM
2.2 ALPHAHERPESVIRUSES AND AUTOPHAGY
2.2.1 HSV-1 AND AUTOPHAGY: ICP34.5 AND US11
2.2.1.1 HSV-1 and Alzheimer’s disease
2.2.2 VZV IS INCAPABLE OF BLOCKING AUTOPHAGY
2.3 BETAHERPESVIRUSES AND AUTOPHAGY
2.3.1 CMV AND TRF1 IN MODULATING AUTOPHAGY
2.3.2 MTORC MODULATION BY CMV
2.3.3 MOUSE CYTOMEGALOVIRUS (MCMV) AND M45
2.4 GAMMEHERPESVIRUSES
2.4.1 MSHV68 INHIBITION OF AUTOPHAGY THROUGH M11
2.4.2 V-FLIP, V-BCL-2 AND RTA OF KSHV
2.4.3 LMP1 AND EBNA1 OF EBV AND THEIR ROLE IN AUTOPHAGY
2.5 FUTURE DIRECTIONS
13
13
13
14
14
14
17
17
20
21
21
21
22
22
23
24
24
25
26
3 VACCINIA VIRUS
29
3.1 INTRODUCTION
3.1.1 DISEASE
3.1.2 INFECTION MECHANISM
3.1.3 ONCOLYTIC VIRUS
3.2 VACCINIA AND AUTOPHAGY
29
29
30
32
32
2|Page
3.2.1 REPLICATION
3.2.2 PKR PATHWAY
3.2.3 BCL-2 LIKE VIRAL PROTEINS
3.2.4 ATG12-ATG3 CONJUGATE
3.3 FUTURE DIRECTIONS
33
33
34
34
36
4. CORONAVIRUSES
39
4.1. INTRODUCTION
4.1.1 DISEASE
4.1.2 INFECTION MECHANISM
4.2 CORONAVIRUS AND AUTOPHAGY
4.2.1 HIGHJACKING LC3-I
4.2.1.1 The ERAD tuning pathway
4.2.1.2 DMVs and EDEMosomes
4.2.1.3 MODELS
4.2.2 INDUCTION OF AUTOPHAGY
4.3 FUTURE DIRECTIONS
39
40
40
42
43
43
43
44
45
46
5. INFLUENZA
49
5.1 INTRODUCTION
5.1.2 DISEASE
5.1.3 INFECTION MECHANISM
5.2 INFLUENZA AND AUTOPHAGY
5.2.1 INDUCTION OF AUTOPHAGY
5.2.2 REPLICATION
4.2.3 TO BLOCK OR NOT TO BLOCK
5.2.4 H5N1-INDUCED AUTOPHAGY-MEDIATED CELL DEATH
5.3 FUTURE DIRECTIONS
49
50
50
52
53
53
54
55
55
6. CONCLUSIONS
59
REFERENCES
62
3|Page
Abbreviations
ACE2
Angiotensin Converting Enzyme 2
AD
Alzheimer Disease
Atg
Autophagy Associated proteins
BBD
Beclin Binding Domain
CEV
Cell associated Enveloped Virus
CMV
Cytomegalovirus
CNS
Central Nervous System
CoV
Coronavirus
DC
Dendritic Cell
DFCP
Double FYVE-Containing Protein
DMVs
Double Membrane Vesicle
EBV
Epstein-Barr Virus
EEV
Extracellular Enveloped Virus
EGFR
Epithelial Growth Factor Receptor
EM
Electron Micropscopy
ERAD
ER Associated degradation
ERGIC
ER-Golgi Intermediated Compartment
EV
Extracellular Virus
GAGs
Glycosaminglycans
gB
Glycoprotein B
gC
Glycoprotein C
gH
Glycoprotein H
gL
Glycoprotein L
HA
Hemagglutinin
HSV
Herpes Simplex Virus
IBV
Infection Bronchitis Virus
ICP34.5
Infected Cell Protein 34.5
IMV
Intracellular Mature Virus
4|Page
IRE1
Inositol Requiring Enzyme 1
KHSV
Kaposi Sarcoma associated Herpes Virus
MCMV
Mouse Cytomegalovirus
MHC
Major Histocompatibility Complex
MHV
Mouse Hepatitis Virus
mTOR
Mammalian Target Of Rapamcyn
MV
Mature Virus
NA
Neuroaminidase
nAbs
Neutralizing Antibodies
NK
Natural Killer
NLS
Nuclear Localisation Signal
NP
Nucleoprotein
OIS
Oncogene Induced Senesence
PBIF2
Polymerase Basic Protein
PDGFR
Platelet Derived Growth Factor Receptor
PE
Phosphatidylethanolamine
PERK
Protein Kinase RNA-like ER Kinase
PIP3K
Phosphatidylinositol 3 phosphate-OH kinase complex
PKR
Protein Kinase R
PtdIns(3)P
Phosphatidylinositol 3 phosphate
ROS
Reactive Oxygen Species
RTA
Transcription Activator
RVN
Reticulovesicular Network
SA
Sialic Acid
SARS
Severe Acute Respiratory Syndrome
TLR
Toll Like Receptor
TSC1/2
Tuberous Sclerosis Complex 1/2
UPR
Unfolded Protein Response
UTR
UntranslatedRegion
5|Page
v-FLIP
viral FLICE protein
vRNP
viral Ribonucleoproteins
VZV
Varicela Zoster Virus
WT
Wild Type
6|Page
Abstract
Autophagy is an evolutionary conserved process for the recycling of cellular contents
through the delivery of the targeted structures to the lysosome for degradation. Research on
autophagy started in the late 1950s but only within the last two decades, the interaction
between viruses and autophagy has become a topic of interest. Autophagy was shown to
play a key role during the immune response against group A Streptococcus bacteria and the
parasite Toxoplasma by preventing their replication. Additionally sinbis virus infection is
compromised by autophagy. Conversely, autophagy was also proven to be pro-viral
stimulating viral yields of both polio and dengue virus, amongst others. These observations
have indicated that specific viruses can manipulate autophagy either positively or negatively
in order to enhance their own replication. In this thesis the interaction between autophagy
and four different viruses, i.e. herpes, vaccinia corona and influenza virus has been
discussed. Herpesviruses can be divided into three categories: alpha, beta and gamma, and
each class of herpesvirus display some similar characteristics regarding the interplay with
autophagy. Differences are, however, also present. These differences cannot only be found
between classes, but also within classes as well as between infections in different cell types.
Interaction of vaccinia viruses and coronaviruses are less well defined compared to those of
the herpesviruses. Although evidence exists for autophagy manipulation by vaccinia viruses,
this has not been clarified for coronaviruses. Nonetheless within the coronavirus life cycle an
autophagy protein but not an intact autophagy machinery, was shown to be involved in the
replication of the virus suggesting that autophagy proteins may be involved in other cellular
processes as well. Studies on the influenza virus have generated some conflicting data where
a block in autophagy was shown by some research groups while others revealed the
contrary. Discrepancies are, nevertheless, not only present in literature about the influenza
virus but are also seen in that of herpesviruses. This could be due to the virus behaving
differently when infecting different cell types. Current knowledge on the interaction
between autophagy and these different viruses will be reviewed in this thesis along with the
discussion of the discrepancies and models suggesting possible future lines of research.
7|Page
1. Autophagy
1.1 Introduction
Autophagy is an evolutionary conserved process for recycling cellular contents by the delivery of
targeted structures to the lysosome for degradation. The process requires a series of autophagyassociated genes (Atg), which generate double membrane vesicles (DMVs) called autophagosomes
that sequester cytoplasm as well as cytoplasmic organelles. Autophagosomes have a diameter of
about 0.5-1.5 µm and can subsequently fuse with multivesicular bodies, early endosomes or late
endosomes forming an amphisome. Amphisomes then fuse with lysosomes, forming an
autolysosome, in order to degrade its contents (Figure 1 A) (Crotzer et al. 2010). There are three
different types of autophagy: Microautophagy, chaperone mediated autophagy and macroautophagy
(Pyo et al. 2012). The latter will be discussed in this thesis and be referred to as autophagy.
Autophagy is important in numerous physiological processes such as development, tumor
suppression, prevention of neurodegeneration, T cell homeostasis and survival during starvation or
growth factor withdrawal. Most importantly for this thesis, autophagy has also been shown to be
important in different aspects of the immune system. It can engulf and degrade pathogens limiting
their intracellular proliferation. Furthermore it can present antigens on the major histocompatibility
complex (MHC) recruiting the adaptive immune response. Lastly, it can promote a pro-inflammatory
response by presenting engulfed viral components to endosomal toll like receptors (TLR). The
interaction between autophagy and some pathogens has been experimentally proven. For example,
autophagy prevents group A Streptococci from replicating in the cytosol and targets intracellular
parasite Toxoplasma (Nakagawa et al. 2004; Andrade et al. 2006). Infection with Sinbis virus has
additionally been shown to be compromised by autophagy (Orvedahl et al. 2010). Autophagy can
however also be pro-viral because its stimulation increases, for example, the yields of polio and
Dengue virus (Levine et al. 2011).
1.2 Mechanism of the autophagosomes formation
Autophagosome biogenesis starts with phagophore, a small membranous cistern, formation. These
phagophores are subsequently expanded and close generating the double membrane
autophagosomes (Figure 1 A). This process is initiated by sequential interactions between
mammalian target of rapamcyn (mTOR) and the ULK1 protein complex. This complex consists of
ULK1, mATG13, FIP200 and Atg101. Under normal conditions mTORC is active and interacts with the
UKL1 protein complex phosphorylating ULK1 and mAtg13, which inhibits the ability of the complex to
8|Page
associate with a membrane hence inhibiting autophagy. Inhibition of mTOR, which happens for
example upon starvation , leads to the activation of ULK1 and subsequent translocation of the ULK1
complex from the cytosol to the ER or closely related structures. The next step is nucleation where
the ULK1 protein complex recruits the class III phosphatidylinositol 3 phosphate–OH kinase complex
(PIP3K). This complex contains VSP34, VPS15, Beclin 1 and Atg14. The active complex results in the
production of phosphatidylinositol-3-phosphate [PtdIns(3)P] to initiate the recruitment of autophagy
proteins to the sites of phagophore formation and expansion. Beclin 1 is very important for the
nucleation process. Without Beclin 1 or one of the Atg members autophagy cannot proceed. Beclin
1’s interaction with VPS34 stimulates VPS34’s catalytic activity leading to the local synthesis of
PtdIns(3)P. The phagophore formation and expansion are achieved by generating two protein
conjugates in an ubiquitin-conjugation-like manner. Atg12 is activated by the E1-like enzyme Atg7
and then conjugated to Atg5 by E2-like protein Atg10. This complex subsequently assembles with
Atg16L
and
associates
with
the
elongating
phagophore.
The
second
conjugate
is
phosphatidylethanolamine (PE)-lipidated LC3 also called LC3-II. This conjugate is produced by Atg7
and Atg3, the E1- and E2-like enzymes, respectively (Figure 1 B). The Beclin1/VPS34/p150 unit is
involved in several stages of autophagy depending on Beclin 1’s interactions. Binding to Atg14, as
mentioned, above leads to recruitment onto the autophagosomal membrane however when bound
to UVRAG the complex becomes involved in maturation of autophagosomes. Simultaneously binding
both UVRAG and RUBICON, in contrast, makes the complex inhibitory as it prevents maturation of
the autophagosomes (Pyo et al. 2012; Shi et al. 2012). The lipids that generate the autophagosomal
membrane can either originate from the ER or the mitochondrion, though there have also been
reports that the Golgi, endosomes and the plasma membrane can also be the origin of the
autophagosomal membrane. The ER has been proposed as it targets Atg14, which is required for
autophagy by regulating VPS34. Furthermore activation of autophagy recruits the ER-resident double
FYVE-containing protein (DFCP), which binds PtdIns(3)P suggesting that this is derived from ER lipids.
These ER subdomains containing DFCP are called omegasomes and recruit Ulk1, Atg14, Atg5 and LC3
(Levine et al. 2011). Mitochondria have been suggested as membrane origin as during starvation
Atg5, which is essential for LC3-II recruitment, locates to the outer membrane of the mitochondria
(Hailey et al. 2010).
1.3 Regulation
1.3.1 mTOR
There are several ways through which autophagy can be regulated. The most important and well
known pathway involves mTOR, which not only regulates autophagy but also protein synthesis and
the import of nutrients. Inhibition of mTOR leads to the activation of autophagy through its
9|Page
Figure 1: Autophagosome formation. A. Synthesis of an autophagosome starts with the formation of a phagophore which
is subsequently enlarged and finally encloses the surrounding cytoplasmic content. They could contain protein aggregates,
viral particles or even damaged organelles. The autophagosomes then fuse with endosomes or multivesicular bodies and
eventually fuse with the lysosome, where their contents are degraded. B. The autophagy process can be set in motion by
the mTOR complex. Upon inhibition of this complex by for example starvation, phosphorylation of the ULK1 kinase by
mTOR is prevented. This allows UlK1 to be activated and in turn it phosphorylates FIP200 and mAtg13, two proteins in
complex with ULK1, which leads to recruitment of PIP3K. This is in a complex composed of VSP34, p150, Beclin 1 and Atg14
on the site where the autophagosome will be generated. The complex initiates the elongation of the phagophore by
recruiting the autophagy proteins Atg5, Atg12 and Atg16L. This results in LC3-I lipidation and incorporation into the
phagophore membrane. Adapted from: Pyo et al. 2012 and Orvedahl et al. 2008
downstream targets ULK1 and ULK2. These result in the activation of autophagy via the pathway
described in section 1.2. Treating cells with rapamycin, a natural inhibitor of MTOR, can mimic this
activation of autophagy via MTOR. Upstream mTOR is inhibited by the heterodimer of the tuberous
sclerosis complex 1 and 2 (TSC1/2). The complex of these two components with mTOR and raptor
receives signals from multiple signaling pathways such as the one centered around class I PI3K and
Akt. Akt for example phosphorylates TSC2 catalyzing its dissociation from the TSC1/2 complex and
thus raises the inhibition of mTOR with the consequence of inhibiting autophagy. Additionally, high
levels of amino acids result in the stimulation of Ras-related small GTPases, which activate mTOR
10 | P a g e
(Cavignac et al. 2010). Conversely when the AMP:ATP ratio increases, indicating a lack of energy,
TSC2 is activated by the AMP activated protein kinase (AMPK), which subsequently inhibits mTOR
and activates autophagy (Gwinn et al. 2008). AMPK can also phosphorylate raptor, which results in
the inhibition of mTOR allowing AMPK to directly phosphorylate ULK1 mediating autophagy
activation (Egan et al. 2011).
1.3.2 Beclin 1
As explained, a decrease in amino acid concentration in the cell activates autophagy. This also occurs
as JNK1 phosphorylates Bcl-2. Bcl-2 has the ability to bind Beclin 1 and thus to repress autophagy
(Pattingre et al. 2005). JNK1 phosphorylation of Bcl-2 interferes with the interaction between Beclin
1 and Bcl-2. Beclin 1 is thus more available for autophagosomes formation. The interaction between
Beclin1 and Bcl-2 can also be disturbed by the DAP kinase, which is activated by death signals, and
TRIF and Myd88 which are activated by pathogens and hypoxia, respectively. The last two achieve
this via the TLRs (Cavignac et al. 2010).
1.3.3 Unfolded protein response
Cell stress induces the eIFα2 kinase signaling pathway, which has additionally been shown to increase
autophagy activity. The exact mechanism via which this happens has not been entirely unraveled.
There are four different kinases which phosphorylate eIFα2: GCNZ, protein kinase R (PKR), protein
kinase RNA-like ER kinase (PERK) and HR1, which are activated upon starvation, viral entry, ER stress
and heme deprivation, respectively (Esclatine et al. 2009). PERK has been shown to upregulate Atg12
and might induce autophagy in this way. PERK is additionally involved in the unfolded protein
response (UPR) (Lai et al. 2007). The UPR is activated upon ER stress and copes with alterations in
protein folding. It reduces the load of unfolded proteins via several mechanisms such as enlarging the
ER membrane and by ensuring selective synthesis of proteins involved in quality control and protein
folding. When the ER stress is irreversible, this pathway will assure that the cell goes into apoptosis.
It is known that upon ER stress autophagy is activated probably to degrade damaged ER as well as
protein aggregates (Hetz, 2012). Hence the UPR most likely, either via PERK or additionally via other
pathways, induces autophagy.
1.4 Antigen presentation and autophagy
Autophagy plays a role in antigen presentation on MHC II. Traditionally MHC II displays epitopes from
extra cellular proteins or pathogens, such as bacteria, and triggers a CD4+ T cell-mediated immune
response (Dengjel et al. 2005). However, 10-30 % of the ligand repertoire, the ligandome, consists of
peptides derived from either cytoplasmic or nuclear proteins. This can partially be explained by
autophagy mediating the loading of MHC II as autophagosomes frequently fuse with the loading
11 | P a g e
compartments of MHC II, the endosomes (Levine et al. 2011). Blocking autophagy decreases the
amount of intracellular epitopes displayed on the cell surface but it does not completely abolish the
loading on MHC II. This indicates that other mechanisms may also play a role in this presentation.
Enhancing this T cell mediated immune surveillance could be important when the cells are under
possible dangerous stress conditions. It has been shown that during starvation, when multiple
autophagosomes are generated, the ligandome of MHC II is altered, which can lead to a defect in the
positive and negative T cell selection in the thymus (Levine et al. 2011). Increased autophagy
decreases the amount of active cathepsins, which are proteases within the endocytic compartments.
Impairment of the activity of these cathepsins leads to a less efficient degradation in the lysosome.
The fact that the proteins are turned over less efficiently might favor their presentation on MHC II as
the remaining epitopes are larger (Dengjel et al. 2005). Some viruses are able to modulate this role of
autophagy, a topic that will be discussed in the following chapters.
In this thesis the interaction of two DNA viruses, Herpesviruses and vaccinia viruses, and two RNA
viruses, Influenza viruses and coronaviruses, with autophagy will be discussed. While some viruses
seem to initiate autophagy and benefit from this induction others have evolved anti-autophagy viral
proteins. Overall, however, the viruses cannot simply be divided in stimulants or inhibitors of
autophagy considering the fact that some viruses behave differently in different cell types. Tropism
seems to, at times, determine the negative or positive influence of the virus on the autophagy
mechanism. Additionally the post infection time point can also play a part in the manipulation of
autophagy. Although research on autophagy started in the late 1950s the link between these viruses
and this pathway has only recently been more elaborated (Yang et al. 2010). There are still many
unsolved issues regarding the interaction between, for example, the four viruses discussed in this
thesis and autophagy. These discrepancies and queries will along with the current evidence be
covered in the coming chapters.
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2 Herpesviruses
2.1 Introduction
Herpesviruses are common viruses characterized by their double stranded DNA genome, which
consists of approximately 124-230 kilo bases. The genome is packed within an icosahedral
nucleocaspid covered with tegument and enclosed in a membrane envelope (Engleberg et al. 4th
edition). The herpesvirus family consists of three different subfamilies classified based upon their
host range, replication cycle and cell tropism: The alphaherpesviruses, the betaherpesviruses and the
gammaherpesviruses (Connolly et al. 2011). Herpes simplex virus (HSV), cytomegalovirus (CMV) and
Epstein-Barr virus (EBV), respectively, are classic examples of these three subfamilies. Additionally
Kaposi sarcoma associated herpes virus (KHSV) and Varicella Zoster virus (VZV) are also a known
gammaherpesvirus and alphaherpesvirus, respectively. These viruses, which share the characteristic
of establishing a life-long infection through latency once they have successfully infected an
individual, will be discussed in this chapter (Murray et al. 5th edition).
2.1.1 The diseases
2.1.1.1 Alphaherpesviruses
HSV-1 was the first herpes virus to be discovered (Pei et al. 2011). There are two different types of
HSV, HSV-1 and HSV-2. The difference lies in the prefered way of transmission and where the
symptoms arise. HSV-1 is spread mainly via oral-oral or via oral-genital contact. HSV-2 is primarily
spread via genital-genital contact and is characterized by genital warts (Engleberg et al. 4th edition).
In this thesis only HSV-1 will be further discussed.HSV-1 can result in corneal infection, which can
lead to blindness but is commonly less severe and phenotypically characterized by cold sores. It is
additionally the most common cause of sporadic encephalitis (Orvedahl et al. 2007). This type of
encephalitis has a high rate of mortality without treatment (70%), which can be reduced to 20-30%
with treatment (Overdahl et al. 2008). VZV, which is the causative agent of the chickenpox, is spread
either via the respiratory route or through direct contact with lesions. VZV can reoccur at a later
point in life and is characterized by chickenpox like lesions which are called zoster. Two thirds of the
world population has HSV-1 antibodies as one fifth of sexually active adults have HSV-2 antibodies.
Although a substantial amount of individuals are thus infected with an alphaherpesvirus, most of
these infections are asymptomatic. Only one out of three infected individuals will display
symptomatic disease. These are often the individuals with a weaker immune system (Engleberg et al.
4th edition).
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2.1.1.2 Betaherpesviruses
CMV infection tends to occur during adulthood. It is spread through direct contact as the virus is
excreted in body fluids such as the urine and the saliva. Approximately 50-60% of all adults are
infected with CMV. As with all herpesviruses, not all infected individuals display symptomatic
disease. Nonetheless the virus will persist for life (Engleberg et al. 4th edition). CMV does, however,
pose a main problem both concerning morbidity and mortality in immunocompromised individuals
such as AIDS patients. In these patients it can cause multi-organ disease. CMV additionally causes
postoperative illnesses as the virus can be spread upon blood transfusion as well as after organ
transplantation. Furthermore, CMV can pass the placental barrier and may lead to congenital birth
defects in the first trimester of a pregnancy (Boehme et al. 2012).
2.1.1.3 Gammaherpesviruses
90% of the world’s population is infected with EBV via oral transfer though, as the other
herpesviruses, this is largely asymptomatic (Saha et al. 2011). EBV is associated with
lymphoproliferative diseases being the causative agent of infectious mononucleosis, which occurs
mostly during adolescence and is also known as the kissing disease. This disease is characterized by
pharyngitis, fatigue, a low grade fever and peripheral blood monocytosis (Engleberg et al. 4th edition).
Additionally EBV can be contracted as a result of transplantation and hence can cause posttransplant lymphoproliferative illnesses due to the suppressed immune system of the patient (Saha
et al. 2011). EBV can furthermore result in neoplastic diseases. It was discovered in 1964 as the first
cancer-related virus in the Burkitt’s lymphoma (Epstein et al. 1964). Moreover it can also lead to the
development of Hodgkins disease (Saha et al. 2011). KSHV like EBV can cause several human cancers.
It is the etiological agent of the Kaposi sarcoma, a cancer often seen in AIDS patients, and hence the
result of a compromised immune system (Wah Wen et al. 2010).
2.1.2 Infection mechanism
Generally the lytic and latent life cycles of the three different subfamilies of the herpesviruses are
similar. There are, however, some differences that allow for classification of the families. The main
differences lie in the cellular tropism as the different viruses infect different cell types as well as in
the mechanism of entry into the cell. Herpesviruses require relatively many proteins during the entry
process compared to other enveloped viruses which often only have a few proteins which mediate
both entry and fusion (Connolly et al. 2011). HIV for example only needs gp120 to mediate the entry
and fusion of the virus (Sun et al. 2006).HSV can infect lymphocytes, epithelial cells, fibroblasts and
neurons. Depending on the cell type, the infection mechanism varies. Sometimes endocytosis is
necessary, the exact reason for this is however unknown (Campadeli-Fiume et al. 2012). Three
conserved viral glycoproteins, i.e. glycoprotein B (gB) and a heterodimer consisting of glycoprotein H
14 | P a g e
(gH) and glycoprotein L (gL) (also known as gH/gL), are necessary for fusion of the virions with all the
cell types. Glycoprotein D (gD) is also crucial for fusion. Furthermore glycoprotein C (gC) is needed to
tether the viral particle with the cell plasma membrane by binding specific surface receptors such as
the heparin sulphate proteoglycan or the TNF receptor facilitating entry (Connolly et al. 2011).
Likewise the three conserved proteins are important for EBV cell entry as well. The binding activity
of glycoprotein 42 (gp42) to HLAII for fusion with B cells is additionally important for entry.
Glycoprotein 350/220 (gp350/220) is the tethering protein for EBV and binds CD21. EBV can also
infect epithelial cells, which do not express HLAII and CD21. In this case entry is mediated via
integrins (Connolly et al. 2011). CMV has the broadest cell tropism as it can infect nearly every cell in
the body. According there are many different receptors to which CMV can bind such as integrins like
αvβ3, platelet derived growth factor receptor (PDGFR), the TNF receptor family and the epithelial
growth factor receptors (EGFR) (Soroceanu et al. 2008; Haspot et al. 2012; Wang et al. 2005; Feire et
al. 2004). Additionally CMV can enter a cell in three different ways: Endocytosis, direct fusion or
macropinocytosis (Haspot et al. 2012).
The conserved protein gB, which is present in all herpesviruses, is the ligand for PDGFR and EGFR.
The latter has been shown to be crucial for entry in some cases, for example in monocytes (Chan et
al. 2009). This relevance, however, has been controversial as in fibroblasts, epithelial and endothelial
cell lines EGFR was not required for entry (Isaacson et al. 2007). It was thus speculated that perhaps
integrin heterodimers were sufficient for entry by interacting with gB and gH in these cells lines
(Lopper et al. 2004). Perhaps EGFR serves to determine cellular tropism (Ryckman et al. 2008).
Once the virus has successfully entered the cell, the capsid is transported to the nucleus releasing its
DNA through the nuclear pores. Identically to all DNA viruses, the viral genes are then transcribed
and the viral genome is replicated. Viral genes are transcribed in several steps starting with the
immediate early genes, which subsequently drive the transcription of the early genes resulting in
proteins needed for the replication of the viral DNA (e.g. viral DNA-dependent polymerase). The early
gene products stimulate the expression of the late genes. These late genes encode the proteins that
are needed to assemble and compose the progeny virions. Subsequently these virions bud through
the nuclear membranes and obtain the final envelope by budding into cytoplasmic vesicles derived
from the Golgi apparatus. Finally the mature virions egress via exocytosis (Engleberg et al. 4th edition;
Mingo et al. 2012) (Figure 2).
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All herpesviruses are post infection
persistently present throughout the
life
of
the
host
through
a
mechanism called latency. HSV and
VZV are latently present in the
peripheral ganglia. They enter nerve
extremities that are proximal to the
epidermis and are subsequently
transported to the
bodies.
During
nerve cell
HSV
latency,
similarly to other herpesviruses, the
viral genome circularizes within the
nucleus with minimal transcription
of viral genes. Reactivation occurs
when
the
immune
system
is
compromised for example upon a
Figure 2: General lytic life cycle of the herpes viruses. The herpesvirus
enters via interactions occurring at the plasma membrane betweeb viral
glycoproteins and host membrane surface proteins, which are different in
between the diverse subfamilies. Subsequently the virion is transported to
the nucleus where three sets of genes are transcribed at different times
during the life cycle (immediate early, early and late genes). Subsequently
these gene products ensure replication of the viral DNA and packaging into
a nucleocapsid. The progeny virions bud out of the nucleus through the
nuclear membranes. The final envelope is obtained by budding into a
cytoplasmic vesicle of Golgi origin after which the virions leave the host cell
through exocytosis.
Modified
from:
http://www.studyblue.com/notes/note/n/lecture-20animal-viruses/deck/1405769
systemic
infection,
emotional
stress
sunburn,
or
ageing.
Especially VZV reactivation causing
Zoster is unpleasant resulting in
vesicular
lesions
along
the
dermatome of the reactive virus
containing
nerve
substantial
amount
causing
of
a
pain
(Engleberg et al. 4th edition). CMV
latency is very poorly understood.
The virus persists in hematopoietic
progenitor cells as well as in macrophages maintaining large subsets of CMV specific T cells.
Persistence could perhaps be better explained as a chronic controlled infection. To this end,
insufficient T cell control leads to reactivation of the virus (Goodrum et al. 2012). For EBV, as
opposed to the other herpesvirus classes, latency is the default pathway (Speck et al. 2010). Several
specific latency proteins such as EBNA-1-6 and LMP1/2 regulate a complex process determined by
alternative splicing maintaining latency in the memory B cells. EBV persistence in latent infected
memory B cells induces proliferation by encoding proteins that are essential for the B cell
16 | P a g e
proliferation. Thus loss of immunological control of EBV could result in hematopoietic malignancies
such as Burkitt’s and Hodgkin’s disease (Saha et al. 2011).
2.2 Alphaherpesviruses and autophagy
2.2.1 HSV-1 and autophagy: ICP34.5 and Us11
The link between autophagy and HSV-1 infection was first made in 1979 when, by using electron
microscopy (EM), an HSV-1 virus was observed inside an autophagosome (Smit et al. 1979). The
ability of HSV-1 to inhibit autophagy has been extensively studied. There seems to be a cell specific
and perhaps even time specific modulation of autophagy by HSV-1 showing distinct regulation in
permissive and non permissive cells. Nevertheless, considering the fact that neurons and dendritic
cells (DCs) are both non permissive for the initial infection but display different autophagy responses
towards, this classification might not be as straightforward (Ramussen et al. 2011).
In fibroblasts and neurons HSV-1 makes use of the infected cell protein 34.5 (ICP34.5), encoded by
the neurovirulence gene gamma134.5, to counteract autophagy. ICP34.5 does this in two distinct
ways (Figure 3). It interferes with the double stranded RNA dependent PKR, which is normally
activated by type 1 interferons and phosphorylates the translation initiation factor eIF2α. This
phosphorylation mediates host cell shut down as eIF2α-P can no longer carry out its function of
recognizing start codons. This pathway is also important for the induction of autophagy. This gives
the virus two exquisite reasons to interact with this pathway: The host cell shut down promotes
replication and the block of autophagy prevents virus degradation (Alexander et al. 2007).
Interference with the PKR pathway is mediated via direct binding of PPIα to the GADD34 domain of
Figure 3: ICP34.5 blocks autophagy. IPC34.5
can
block
autophagy
through
two
mechanisms. It binds PPIα with its GADD34
domain
activating
counteracts
the
it,
which
function
of
in
PKR
turn
by
dephosphorylation of eIF2α. Furthermore it
binds Beclin 1 with its BBD (depicted in black)
sequestering this protein and consequently
disabling Beclin 1 to functionally induce
autophagy. Us11, which is expressed late
during the infection, also plays a role in
inhibiting autophagy. It blocks PKR by either
binding directly or by interfering with PACT,
an activator of PKR. Adapted from: Orvedahl
et al. 2007
17 | P a g e
ICP34.5. PPIα is subsequently activated and dephoshorylates eIF2α (Orvedahl et al. 2007). In vitro
infection with the ICP34.5-/- virus resutling in the inability to prevent autophagy has shown that this
pathway does not seem to be important for replication. Replication defects in vitro are caused by the
host cell shut off. In vivo, in contrast, this inability to control autophagy is in fact important for
replication perhaps due to the fact that autophagy is a potent anti viral mechanism giving the host
cell the overhand in clearing the virus (Leib et al. 2009). Furthermore Us11 is also involved in blocking
autophagy. Us11 is a double-stranded RNA binding protein expressed late during infection. It
antagonizes the PKR pathway either by direct binding or by interacting with an activator of PKR, PACT
(Peters et al. 2002). Early expression of this protein in an ICP34.5 knock out virus leads to inhibition
of autophagy by allowing the virus to dephosphorylate eIF2α (Alexander et al. 2008). This is,
however, not a physiological situation but does indicate that Us11 has the ability to interact with the
PKR-mediated induction of autophagy and block it. This is backed up by the expression of Us11 in
HeLa cells and fibroblasts which was shown to block autophagosomes formation and hence
autophagy suggesting that Us11 is, apart from ICP34.5, also able to mediate autophagy (Lussignol et
al. 2012).
Additionally ICP34.5 also associates with Beclin 1. Beclin 1 is essential for the formation of
autophagasomes through its interaction with VPS34 as mentioned in the introductory chapter
(Cavignac et al. 2010). ICP34.5 binds Beclin 1 directly with its beclin 1-binding domain (BBD). The
exact site where ICP34.5 binds to Beclin 1 is however unknown. Interfering with Beclin 1 results in
inhibition of autophagy. Indeed HSV-1 viruses without the BDD are unable to block autophagosome
formation and are cleared more efficiently even when the GADD34 domain is still present. Thus PKR
inhibition without Beclin 1 inhibition is not sufficient to block autophagy (Orvedahl et al. 2007).
Additionally there is a difference in efficacy of replication between the BDD -/- virus and the wild
type (wt) virus showing a lower rate of replication by the BDD-/- virus. This difference is principally
displayed at a late infection time point suggesting the BDD lacking viruses are not able to overcome
the adaptive immune system. Upon infecting a mouse without functional B and T cells with the BDD /- virus or wt virus, this difference in replication is no longer seen (Leib et al. 2009), suggesting that
the adaptive immune system is triggered when autophagy is not blocked.
Endogenous viral antigens can be presented on MHC complexes in non-permissive cells such as
macrophages and DCs, through autophagy (i.e. cross-presentation) (see 1.4). In macrophages
autophagy is distinctly regulated depending on the phase of the infection. At the early time points
ICP34.5 inhibits autophagy similarly to what observed in fibroblasts and neurons. Late in the
infection, however, the macrophages display a novel form of autophagy. This form involves the
formation of autophagosomes derived from the coiling process of the nuclear membrane. These
18 | P a g e
Figure 4: Formation of a four-layer membrane autophagosome derived
from the nucleus. Schematic representation of the formation of a fourlayer membrane autophagosome derived from the nucleus. 1) Viral
capsids assemble in the nucleus. 2) HSV-1 capsids acquire their envelope
in the perinuclear space by fusing with the nuclear inner membrane. 3)
Fusion with the outer nuclear membrane releases a naked HSV-1 capsid in
the cytoplasm. Upon translocation through the nuclear membranes and
release into the cytoplasm, the capsids can become trapped by the
emerging four-layer membrane autophagosomse. These can subsequently
detach from the nucleus as shown by the arrow. Adapted from: English et
al. 2009
autophagosomes as opposed to the classical 2 membrane autophagosomes contain a 4-layer
membrane structure containing a large amount of the viral gB protein (Figure 4). This suggests that
inhibition of the classical autophagy route triggers this new form of autophagy, which leads to the
presentation of degraded viral peptides such as pG on MHC complexes, thus triggering an immune
response (English et al. 2009a; English et al. 2009b). The exact molecular mechanism of this novel
type of autophagosomes is not unraveled yet.
DCs are antigen presenting cells that are also manipulated by HSV-1. Unlike in the previously
mentioned cells, HSV-1 is not capable of inhibiting autophagy in these cells. Autophagy is induced
independently of the phosphorylation status of eIF2α but dependent on the presence of the viral
genome in the cytosol, which is detected by STING. STING is an important part of the cytosolic DNA
sensor system that among others induces type 1 IFN responses. The exact sensors through which the
viral DNA induces autophagy remain unknown. Autophagy inhibition leads to a decrease in IFN-β
production unveiling a link between autophagy and the innate immune response. The exact
interaction between IFN, STING and autophagy has, however, not been discovered.
Up to date no 4-layer membrane autophagosomes have been found in DCs (Ramussen et al. 2011).
Nonetheless EM was not used to truly determine presence of these structures. Considering the fact
19 | P a g e
that viral DNA in the cytosol induces autophagy, which cannot be overruled by ICP34.5, this fact
raises the question why this phenomenon does not happen in other (permissive) cells and whether it
can perhaps be connected to the formation of the 4-layer membrane autophagosomes. There is one
report in literature that shows autophagy induction at a very early stage of HSV-1 infection in
fibroblasts. In this work the authors proposed that the reason for the upregulation of autophagy
might be due to the presence of viral DNA in the cytoplasm, which can be sensed by DNA sensors
such as STING which was mentioned before (McFarlane et al. 2011). This hypothesis is in line with
the induction of autophagy in DCs. In fibroblasts, however, autophagy induction does not remain
dominant as autophagy is repressed later during infection. HSV-1 does not block the biogenesis of
autophagosome in DCs but rather prevents their maturation. This is illustrated by an accumulation of
the autophagosome cargo protein p62, which is not degraded due to the decreased flux. This
inhibition of autophagosome maturation depends on the BBD of ICP34.5 and interferes with the
immune system as the viral peptides do not reach the membrane for presentation (Gobell et al.
2011). Hence though HSV-1 seems to stimulate an innate immune response it hampers the adaptive
response necessary to clear the virus.
2.2.1.1 HSV-1 and Alzheimer’s disease
HSV-1 and its ability to bypass autophagy through its inhibition has been speculated to play a role in
the development of Alzheimer’s disease (AD). In the elderly HSV-1 DNA is present in large amounts in
areas of the brain which are often affected in AD. This is not seen in younger people suggesting that
HSV-1 only reaches the brain once the immune system has been slightly compromised due to ageing.
Nonetheless nearly every individual is infected with HSV-1 though not everyone develops AD.
Therefore there must be other host factors that determine the susceptibility to AD. Likewise not all
individuals infected with HSV-1 develop cold sores additionally indicating that host susceptibility is
not only dependent on infection but also on other factors. The ApoE protein is a known factor
increasing susceptibility to AD. How HSV-1 and ApoE interact remains unknown. They could possibly
interact at an immunological level where ApoE would make the infected individual less successful in
fighting the infection or they could perhaps interact at the cell surface as they both bind to the same
receptor, i.e. HSPG (Itzhaki et al. 2008). Be that as it may solely the fact that HSV-1 inhibits autophagy
might already be very important in developing the β-amyloid plaques, which are characteristic for
AD. As autophagy is crucial in clearing aberrant proteins, its inhibition would leave the amyloid
proteins untouched allowing the formation of plaques (Orvedahi et al. 2008.). Nonetheless further
research is necessary to pinpoint the exact role of HSV-1 and autophagy in the development of AD
and other neurodegenerative or auto mmune diseases of the central nervous system (CNS).
20 | P a g e
2.2.2 VZV is incapable of blocking autophagy
VZV has the smallest genome of the alphaherpesviruses and does not contain an ortholog of the
HSV-1 γ134.5 gene or a homolog of viral Bcl-2 present in gammaherpesviruses (see below). It is thus
unable to block autophagy (Cavignac et al. 2010). Instead it appears to stimulate autophagy in the
late stages of infection, both primarily and during reactivation, which is seen in both melanoma cells
and fibroblasts (Takahashi et al. 2009). A different study, however, showed that the induction of
autophagy by VZV in fibroblast is already present early during infection. This induction of autophagy
is the result of increased viral glycoprotein synthesis leading to ER stress. The protein XBPI was
present in VZV infected fibroblasts providing proof that the increased ER stress indeed activates the
UPR, which induces autophagy. XBPI is a protein that is expressed when inositol requiring enzyme 1
(IRE1) processes its specific mRNA. The IRE1 pathway is activated by the UPR. In the case of VZV
infection, autophagy might be proviral considering the fact that it is already active at an early time
point of the infection (Carpenter et al. 2011). The induction of autophagy would prolong the life of
the infected cell by reducing apoptosis which would give rise to the beneficial consequence of
allowing the virus to propagate.
2.3 Betaherpesviruses and autophagy
2.3.1 CMV and TRF1 in modulating autophagy
The interplay between CMV and autophagy appears to be more complex than with the
alphaherpesviruses (Fig. 5). CMV infection, like HSV-1, also induces autophagy at an early phase of
infection (McFarlane et al. 2011). It has been proposed that this could be due to presence of viral
DNA in the cytoplasm or perhaps the induction lies in the virus binding to HSGPs, which are
necessary for the viral cell entry. Nonetheless 24 hours post infection the tide turns and autophagy is
blocked. This block depends on de novo synthesis of viral proteins as UV inactivated virus is not able
to inhibit autophagasome formation. CMV does not contain an ortholog for either γ134.5, viral Bcl-2
or v-FLIP (see below) (Chaumorcel et al. 2012). It does however have a homolog of ICP34.5 namely
TSR1. TSR1 has been shown to bind PKR directly inhibiting its phosphorylation of eIf2α (Hakki et al.
2006). Thus like HSV-1, this results in inhibition of host shut off and perhaps it is also involved in a
block of autophagy. The N-terminal domain of TSR1 can also bind Beclin 1. To determine whether
both binding sites were crucial for autophagy inhibition, mutant version of TSR1 lacking the binding
domains have been made. The mutant that did not contain the PKR binding site still managed to
inhibit autophagy and, therefore, like in HSV-1, the interaction with Beclin 1 is essential. Surprisingly
infection with a TSR1 defective mutant does not lead to a stimulation of autophagy although
inhibition is lost. This might be due to the fact that CMV has another highly homologous protein,
IRS1, which could be functionally redundant with TSR1. These proteins have identical amino termini
21 | P a g e
suggesting that IRS1 should also be able to bind Beclin 1, be it probably less efficiently than TSR1
(Chaumorcel et al. 2012).
2.3.2 mTORC modulation by CMV
Another path via which CMV modulates autophagy is by acting on the mTORC pathway. Activation of
mTORC kinase activity leads to inhibition of autophagy. mTORC activation leads to, amongst others,
phosphorylation of 4EBP1 and p70S6K. These two proteins are involved in protein synthesis. Cells
infected with CMV show an increased phosphorylation of both proteins suggesting activation of
mTORC. Infected cells are even resistant to rapamycin, an inhibitor of mTORC (Chaumorcel et al.
2008). CMV influences mTORC by for example regulating TSC1/2 through the viral protein Ul38.
TSC1/2 is a negative regulator of mTORC which is activated by CMV’s Ul38 (Esclatine et al. 2009). In
CMV infected cells even starvation conditions which normally inactivate mTORC are unable to
counteract the action of CMV and thus mTORC remains active and perinulclear keeping autophagy
inhibited (Clippinger et al. 2011). This indicates that the interaction of CMV with the mTORC pathway
is very potent at inhibiting autophagy.
2.3.3 Mouse cytomegalovirus (MCMV) and M45
CMV inhibits autophagy 24 hours post infection but also seems to make use of autophagy to
overcome the immune system. The viral protein M45 in MCMV inhibits activation of TLR2, TLR3,
TLR4, TRL7, TLR9, TNFR and NFκB. M45 was previously known to bind RIP1, a protein downstream of
TNFR, TLR3 and TLR4. This, however, does not explain the inhibition of TLR2, TLR7 and TLR9, which
are not controlled by RIP1. The C-terminal RNR R1 homology domain of M45 has been shown to
interact with NEMO. NEMO is the scaffold of the IKK complex downstream of RIP1 (Fliss et al. 2012).
Upon activation of the IKK complex, NEMO degrades the inhibitor of NFκB resulting in the activation
of NFκB. Activation of NFκB is thought to be necessary at the start of infection as it mediates the
transcription of the immediate early genes (Israel, 2010). CMV infection results in NEMO degradation
through the targeting of NEMO by M45 to the autophagosome (Fliss et al. 2012). By degrading NEMO
the IKK complex is unable to activate hence NFκB will remain bound to its inhibitor. This block on
NFκB already starts at an early time point post infection but is only fully established later during the
infection (Montag et al. 2006). Considering the fact that autophagy is inhibited 24 hours post
infection, the degradation of NEMO via autophagy will be less efficient from this time point onwards.
This might be the reason why M45 targets both NEMO and RIP1 as once the NEMO route becomes
less efficient, the virus can subvert to the RIP1 pathway, which is still intact.
22 | P a g e
Figure 5: CMV interacts with autophagy via TRF1 and M45. CMV induces autophagy early in infection but inhibits
autophagy 24 hours post infection. This is achieved by activating mTORC through for example Ul38 interacting with TSC1/2,
a negative regulator of the mTORC complex, which inhibits autophagy as well as activates protein synthesis. mTORC
activation can be determined by phosphorylation of 4EBP1 and p70S6K. Furthermore the viral protein TSR1 and its
homologue IRS1 which are the homologues of HSV-1‘s ICP34.5 play a role. TRF1 binds PKR inhibiting phosphorylation of
eIF2α. This could also inhibit autophagy even if the binding to Beclin 1 of TRF1 has been shown to be the critical interaction
to block autophagy. MCMV also contains the protein M45 which can bind the scaffold of the IKK complex, NEMO, and
target it to the autophagosome thus making use of autophagy. Degradation of NEMO results in the inability of the complex
to degrade the inhibitor of NFκB and hence NFκB will not be able to function as a transcription factor.
2.4 Gammeherpesviruses
Gammaherpesviruses promote or suppress autophagy depending on the virus, cell context and
whether the virus is in the lytic or latent stage (Figure 6). EBV, KSHV and MHV68 will be discussed in
this section. All these viruses express Bcl-2 orthologues. Bcl-2 has an anti apoptotic function in the
cell but it has also been shown to be an inhibitor of autophagy by binding Beclin 1 indicating that
these two pathways are closely regulated. V-Bcl-2 also known as orf16 and M11 in KSHV and MHV68,
respectively, are both able to bind Beclin 1 and thus inhibit autophagy (Cavignac et al. 2010. V-Blc-2
binds Beclin 1 with its BH3 binding domain, which consists of a hydrophobic pocket (Sinha et al.
2008). The binding between Beclin 1 and M11 is much stronger than the binding of Bcl-2 to for
example bax or bak, which are pro-apoptotic proteins. This bound complex is not easily displaced
suggesting that is likely very potent in inhibiting autophagy (Ku et al. 2008).
23 | P a g e
2.4.1 MSHV68 inhibition of autophagy through M11
Two distinct domains of MHV68’s M11 perform its anti-apoptotic and the anti-autophagy roles.
Beclin 1 is bound by the α1 helix of the hydrophobic groove while bax and bak are bound by the BH3
domain. The anti autophagy property of M11 was shown to be dispensable during the lytic stage but
crucial during latency. The anti-apoptotic property, in contrast, is important during reactivation
(Xiaofel et al. 2009). There are, however, contradicting studies where a M11 defective mutant had no
effects on latency and another where there was some initial reduction in latency induction was found
but that this resolved 6 months post infection (Lima et al. 2005; Ganappa et al. 2002). MHV68
inhibition of autophagy during the lytic stage has not been described yet though an anti apoptotic
protein important in the lytic phase, vMAP, had been discovered (Xiaofel et al. 2009). Considering the
fact that autophagy and apoptosis are both inhibited by M11, perhaps this protein is able to
negatively regulate autophagy in the lytic phase. Conflicting with the idea that autophagy might be
inhibited during the lytic phase, autophagy induction has been related to survival of detached
epithelial cells infected with MHV68. Inhibition of autophagy in these neoplastic cells causes cell
death (Suarez et al. 2011). In this case viral persistence was aided by inducing autophagy again
hinting to a divergence in autophagy modulation depending on the cell type and context. The exact
mechanism is still subject of ongoing research.
2.4.2 v-FLIP, v-Bcl-2 and RTA of KSHV
KSHV’s v-Bcl-2 actively suppresses autophagy during lytic replication of the virus also by binding to
Beclin 1 (Liang et al. 2008). Additionally KSHV encodes the viral FLICE inhibitory protein (v-FLIP),
which inhibits autophagy by binding Atg3 and thus preventing autophagosome elongation via
inhibition of LC3 lipidation. V-FLIP is important in the inhibition of oncogene-induced senescence
(OIS). OIS is the result of v-cyclin. This viral protein leads to the activation of a DNA damage-sensing
pathway followed by mTOR inhibition, which leads to autophagy activation. Autophagy is in this
sense the Achilles heel of the cellular senescence response targeted by v-FLIP (Leidal et al. 2012).
Preventing the infected cell to stop proliferating is important for viral persistence and explains why
KSHV can cause cancer. The v-FLIP has also been shown to protect epithelial cells from anoikis and
thus contributes to the oncogenic characteristics of KSHV. KSHV is therefore able to target autophagy
at two different phases, v-Bcl-2 which completely stops the formation of autophagosomes and v-FLIP
which counteracts autophagosome elongation. The previously mentioned M11 protein lies in the
genomic locus that v-FLIP is expected to occupy in MSHV68. Hence perhaps M11 does not only
inhibit autophagy by binding Beclin 1 but also acquires the ability to function as v-FLIP by blocking
the hosts’ antiproliferative response (Leidal et al. 2012).
24 | P a g e
On the other hand KSHV has also been shown to induce autophagy during its lytic reactivation via the
transcription activator (RTA). RTA, which is present in the nucleus, indirectly degrades the repressors
of lytic reactivation by modulating the ubiquitin-proteasome pathway and autophagy. The
mechanisms employed by RTA for the modulation of autophagy are unknown. It could potentially be
due to an induction of the expression of autophagy-related proteins. This degradation process,
however, needs to be tightly regulated limiting the replication in order to bring the virus back to its
default latent stage since (Wen et al. 2010). To this end the v-Bcl-2 could bring the RTA-driven lytic
reactivation back to latency by inhibiting autophagy. This nicely portrays the different requirements
for autophagy in different phases of the infection cycle.
2.4.3 LMP1 and EBNA1 of EBV and their role in autophagy
The proteins BALF1 and BHRF1 are two v-Bcl-2 proteins produced by EBV. Both are expressed early
during the lytic cycle and the latter is also present during latency. It is, however, not known whether
these proteins are able to bind Beclin 1 and whether they have an effect on autophagy (Taylor et al.
2011). Nonetheless, it has been shown that LMP1 regulates autophagy during latency. The amount of
LMP1 expression has an effect on the level of autophagy induction. LMP1 is a transmembrane
protein and is one of the 9 EBV proteins still transcribed during latency. LMP1 regulates its own
synthesis and degradation via interplay with the UPR and autophagy. An intermediate level of LMP1
is needed to drive efficient B cell proliferation which is beneficial for the virus (Lee et al, 2008). LMP1
is an ambiguous molecule as it stimulates UPR and autophagy eventually inducing apoptosis through
its 6 transmembrane domain so that it can regulate its own levels. It subsequently blocks apoptosis
by signaling via the C-terminus. Apoptosis is inhibited by inducing expression of the cellular Bcl-2
homolog, Bcl-2a1. Cellular Bcl-2 has been shown to be able to bind Beclin 1 so perhaps the homolog
could also inhibit autophagy when LMP1 should no longer be degraded. Furthermore the two v-Bcl-2
proteins might also play a part if they indeed have the ability to inhibit autophagy. This
overexpression of the cellular Bcl-2 homolog, however, does not seem to fully inhibit autophagy. The
exact manner how LMP1 triggers autophagy, directly or indirectly, is unclear (Pratt et al. 2012).
Another latency protein, EBNA1, also interacts with autophagy. In this case, however, the interaction
with autophagy does not appear to be beneficial for the virus. EBNA1 is presented on MHCII via
autophagy stimulating a response mediated by CD4+ T cells. Normally EBNA1 localizes to the nucleus,
which partially protects it from being displayed on the cell surface. Indeed when EBNA1 is artificially
modified to be cytoplasm-bound, the amount of EBNA1 epitopes on MHCII significantly increases.
25 | P a g e
Figure 6: Gammaherpesviruses interact with autophagy in divergent ways. Beclin 1 is a protein necessary for
autophagosome formation. Viral Bcl-2 proteins, MSV68’s M11 and the v-Bcl-2 of KSHV, bind to Beclin via their
hydrophobic groove and inhibit autophagosome formation. KSHV additionally contains another protein that
inhibits autophagy during latency, i.e. v-FLIP. This protein associates with Atg3, an important component of the
autophagy machinery, and inhibits autophagy. During lytic reactivation, however, KSHV promotes autophagy
indirectly via RTA. EBV also has two v-Bcl-2 proteins but these have not been proven to bind Beclin 1. EBV does
induce autophagy via a yet undetermined mechanism triggered by the latent membrane protein LMP1.
Question marks indicate that the exact mechanism is still unknown.
2.5 Future Directions
It is clear that autophagy plays an important role during the latent and lytic phases of the life cycle of
the herpesviruses. Not all herpesviruses interact with autophagy through the same mechanisms and
for the same reasons. Some viruses even seem to have developed different subversion strategies in
different types of cells. The most important protein that herpesviruses exploit to inhibit autophagy is
Beclin 1 as all of the viruses discussed, with the exception of VZV, have developed a way to interact
with this protein. This protein will come back in the coming chapters as well as it seems to be a
protein which is universally targeted by viruses. HSV-1 and CMV achieve this through the ICP34.5 and
its homolog TSR1 respectively. The gammaherpesviruses do this with their v-Bcl-2 proteins, with a
remaining question mark concerning the v-Bcl-2’s of EBV (Chaumorcel et al. 2012; Cavignac et al.
2010).
For the v-Bcl-2 of MSHV68, M11, contradicting results have been obtained regarding its importance
during latency (Lima et al. 2005; Ganappa et al. 2002; Xiaofel et al. 2009). This is due to differences in
the design and execution of the studies as different doses and administration routes were used. In
order to obtain a solid conclusion concerning the role of M11, additional investigations will need to
be carried out under the same experimental conditions. The fact that during TSR1 deficient CMV
26 | P a g e
infection autophagy is neither inhibited nor further induced was suggested to be due to the presence
of the homolog IRS1 (Chaumorcel et al. 2012). To prove this redundancy, it might be interesting to
look at the impact on CMV’s life cycle and autophagy when removing both proteins, as well as
whether production of IRS1 increases to compensate when TRF1 is no longer present. Additionally it
has not been shown whether IRS1 truly binds Beclin 1 hence a co-immunoprecipitation assay could
be used to determine whether IRS1 and Beclin 1 are binding partners. In macrophages HSV-1’s
negative regulation of autophagy results in the induction of a different kind of autophagy that gives
rise to 4-layer nucleus-derived autophagosomes (English et al. 2009a; English et al. 2009b). Looking
at whether this is a cellular response to this block or whether this is induced by the virus itself would
be a very interesting next step in the investigation of these unique autophagosomes. Here one could
for example look at whether gene expression is different between the early and late infection phases
or whether deleting parts of the virus proteins will abolish the formation of these structures.
Autophagy is activated by a few herpesviruses as well. VZV seems to induce it to keep the host cell
alive and permits maintenance of the optimal conditions for replication (Takahashi et al. 2009). If this
is indeed the case for VZV, these data somehow contradict data obtained for HSV-1, which show that
this virus greatly benefits from inhibiting autophagy to block viral degradation and antigen
presentation at the plasma membrane. Accordingly, activation of autophagy in HSV-1-infected cells
leads to a decrease of viral potency. It might be interesting to look at the effect of inhibiting
autophagy in cells infected with VZV, which should reduce VZV fitness. KSHV also stimulates
autophagy through RTA upon reactivation of the virus. The exact mechanism how RTA induces
autophagy remains speculative but might be found by analyzing gene expression pre and post RTA
activation of autophagy. EBNA1, protein involved in EBV latency, is presented by MHCII via
autophagy though as it resides in the nucleus, it is not entirely clear how it is exposed at the plasma
membrane. Perhaps EBV also triggers the formation the 4-layer nuclear membrane derived
autophagosomes, which could explain why nuclear antigens are transported on the cells surface
through MHCII. This is something interesting to be investigated. Although the process is probably not
very efficient because the antigens are only displayed in small quantities, the mechanism of antigen
presentation would be interesting for further investigation.
Additionally EBV encodes for two v-Bcl-2 proteins of which it is not known whether they bind Beclin
1 (Taylor et al. 2011) to inhibit autophagy for example during the lytic phase as activation of this
pathway seems to be important during latency. Whether the v-Bcl-2 proteins of EBV are able to bind
Beclin 1 will need to be further studied by coimmunoprecipitation for example. The fact that like
KSHV, EBV can cause lymphoproliferative disease and even cancer suggests that EBV will also need to
overcome OIS (Leidal et al. 2012). To this end EBV might contain a functional counterpart of the v27 | P a g e
FLIP protein. However it seems that intermediate LMP1 levels cause proliferation of B cells during
EBV infection so could also lead to lymphoproliferation. Perhaps there is a v-FLIP like protein that
aids LMP1 in regulating autophagy when autophagy activity needs to be decreased thus inhibited.
In conclusion, while herpesvirus infection progression has extensively been linked to autophagy
there are many questions that will need to be addressed by further research; starting from what
determines the different outcomes by the same virus in different cells to whether we can extend the
information gained on one virus to a sub- or even an entire virus family. The route to full
understanding of herpesvirus-autophagy interaction is nowhere near the end.
28 | P a g e
3 Vaccinia virus
3.1 Introduction
Vaccinia virus is the virus that was used in the smallpox vaccine which lead to the eradication of
smallpox in 1977. It belongs to the same genus, Orthopoxviruses, as the variola pox virus, which is the
causative agent of the smallpox. Vaccinia, however, displays much milder symptoms. The exact
origin of vaccinia is not known; it has been cultured in labs for years but there are no records of its
original isolation. One hypothesis is that it might be a hybrid of the variola and the cowpox virus. It is,
however, difficult to determine the origin because there are many strains of vaccinia in different labs
which have different biological properties (Henderson et al. 1999). Vaccinia, a brick shaped virus, is
one of the largest DNA viruses. It contains a 190 kB linear double stranded DNA genome that codes
for 200- 250 genes. Half of these genes encode immediate and delayed early genes. Vaccinia is a
unique virus as it produces two different kinds of virions which can spread the infection (Roberts et
al. 2009). Furthermore vaccinia causes lysis of the host cell via necrosis. Though smallpox has been
eradicated the potential role for variola, of which there are still stocks present, in the light of
bioterrorism has kept an interest in vaccinia research (Ensirink et al 2002). Additionally, due to the
fact that vaccinia is able to accept up to 25 kB of foreign DNA, it is popular in genetic engineering. In
this regard, vaccinia is mostly used in oncolytic virus research (Kirn et al. 2009).
3.1.1 Disease
Vaccinia infection occurs upon vaccination with a live vaccine against smallpox, which is nowadays no
longer used as the disease is considered to be eradicated. 95% of the vaccinated individuals
displayed full immunity for 5-10 years. The vaccine does not cause many severe side effects although
there are some complications that could arise depending upon the immune status of the vaccinated
individual (Lee 2012). These complications were in some cases lethal resulting in an average of 1
death per one million vaccinated individuals. In most vaccinated individuals mild pain would occur for
about 7-10 days post vaccination at the spot of the vaccination, where a rash could also appear. The
mild pain might be accompanied by regional lymphadenopathy and a low grade fever (Enserink et al.
2002). Due to these mild symptoms 1/3rd of the vaccinated individuals missed days of school or work
hence the vaccine had an economical impact. The most common adverse effect was the acute
vaccinia syndrome, which is characterized by fever, headache, mylagias and fatigue (Belongia et al.
2003). More severe side-effects included progressive vaccinia that led to metastatic infection of
organs, necrosis of the skin around the vaccinated site and could also be fatal especially in individuals
with a T cell deficiency (Cono et al. 2003). Children younger than 15, with eczema, are predisposed to
post vaccination eczema vaccinatum, which can lead to hospitalization and at times to death. In even
younger children microglial encephalitis and post vaccinial encephalopathy can occur resulting in
29 | P a g e
fever, seizures and coma. Adults
may develop a less severe CNS
complication
namely
demyelination (Lee 2012). The
virus
can
spread
from
the
vaccination site, where it is shed,
up to 21 days post vaccination.
Consequently it is advised to
cover
the
vaccination
site.
Furthermore the virus can drain
from the primary site to the eyes,
eyelids, nose and peritoneum,
where
it
can
cause
inflammation.
mild
Nowadays
laboratory workers and medical
personnel
Figure 7. Life cycle of the vaccinia virus. A. Entry of EV and MV particles is
principally initiated by the induction of macropinocytosis resulting in the
endocytosis of the virions. Here the fusion complex is activated by the
are
at
risk
of
contracting vaccinia due to the
fact that it is increasingly used
progressive acidification of the phagosome, either directly or after the first
for
foreign
gene
transfer
membrane of the EVs has been dissolved, allowing the viral core to be released
research (Cono et al. 2003).
into the cytoplasm. This acidification is not always necessary; it depends on the
the perinuclear region where it replicates and is transcribed within replication
3.1.2 Infection mechanism
Vaccinia has a broad cell type
factories. The core and associated proteins are enclosed in a membrane of
tropism that depends on strain
which the origin has not been elucidated yet and this event leads to the
and more in particular on the
strain. B. Once the core has been released, it travels on microtubule towards
formation of MVs. Here the assembly halts for many particles, which are then
released upon cell lysis. Some particles continue on microtubules towards the
post entry steps rather than the
site where they obtain their second membrane. This has been suggested to
entry
either be of endosomal or trans Golgi origin. Obtaining a second membrane
different vaccinia virions; the
leads to the generation of EV, which can be divided into EEVs and CEVs. The
mature virions (MV) and the
former are propelled towards neighboring cells by an actin tail whereas the
latter are released via exocytosis and allowed to infect more distant cells.
Adapted from: Schmidt et al.2012 and Roberts et al. 2008
itself. There
are two
extracellular virons (EV). These
do not share any epitopes hence
use
different
factors
for
attachment (Bengali et al. 2012). MVs have been shown to bind to glycosaminglycans (GAGs) though
this depends on cell type and strain type. Heparin sulfate and chondriotin sulfate have also been
shown to be bound by MV epitopes. For EVs no cellular attachment factor has been identified. The
30 | P a g e
main entry route of both virions is via endocytosis. To induce endocytosis, MVs and EVs initiate
macropinocytosis. Macropinocytosis is the result of actin rearrangements which cause large amounts
of fluid to be taken up into the cell. This was shown in several cell lines such as epithelial cells as well
as monocyte-derived DCs. In CHO cells, however, the entry of MVs seemed to be independent of
macropinocytosis. For EV, the virions which have a double membrane, the outer membrane needs to
be lost in order to fuse with the endocytic membranes. This fusion is mediated by an entry/fusion
complex consisting of 12 different viral proteins, the exact molecular details and architecture of
which are currently unknown. The complex is, however, solely necessary for the virion fusion and it
does not interfere with the binding process. Several strains require acidification to activate this
complex while others are able to fuse in neutral conditions. Nonetheless an acidic environment as
well as the exposure to GAGs is needed to disrupt the second membrane of the EV virions (Figure 7
A) (Schmidt et al. 2012). There is also a report stating that EVs enter the cell via fusion with the
plasma membrane after they have shed their second membrane outside of the cell (Roberts et al.
2008). Once the core, containing the viral structural proteins, the compact DNA genome and the
transcription enzymes, enters the cytoplasm, it is transported by microtubules to the perinuclear
region where the replication factories are established. The vaccinia virus replicates entirely in the
cytoplasm. Here partial uncoating allows for early mRNA transcription. The immediate and delayed
early genes are the first to be transcribed and play a part in the replication of the viral DNA as well as
in helping the virus to evade the immune system by manipulating the host cell to the advantage of
the virus. Intermediate genes, of which there are relatively few compared to the large amount of
immediate and delayed early genes, are subsequently transcribed. These genes encode regulatory
proteins that drive the late gene expression, which are involved in the assembly of the novel virus
particles as well as enocde the enzymes that are packed within the new virions. Next the virions are
formed starting with the formation of the immature virion form. The viral DNA and other core
components are packed in a single membrane vesicle, the origin of which is still unknown (Roberts et
al. 2008). Aggresomes, which normally retain misfolded proteins, have been suggested to resemble
the vaccinia replication factory (Wileman 2006). Proteolytic cleavage of the core proteins
subsequently leads to the genertion of the brickshaped intracellular mature virus (IVM) also called
MV. At this point the majority of virions halt their assembly process and the IVMs are released upon
cell lysis. The IVMs which do continue morphogenesis are again transported by microtubule towards
the site where they obtain their second membrane forming an intracellular enveloped virus (IEV).
The formation of this second membrane has not been elucidated but it is thought that it has either
an endosomal or a trans golgi origin. Subsequently they are transported to the periphery on
microtubules. To reach the plasma membrane they must cross a barrier, the cortical actin. This is
achieved by inhibiting RhoA signaling. The outer membrane of the IEV can thus fuse with the plasma
31 | P a g e
membrane and an actin polymerization cascade is induced underneath the IEV, which propels the
cell associated enveloped virus (CEV) away from the cell to infect neighboring cells. IEV can also leave
the cell via exocytosis creating an extracellular enveloped virus (EEV) (Figure 7 B). EEV can spread to
either adjacent cells but can also spread through the blood and infect distal cells. IVM and CEV/EEV
have different viral proteins on their membranes and are hence structurally, antigenically and
functionally diverse. CEV/EEV, also called EV, contain a host-derived lipid membrane and can
therefore subvert the complement allowing it to travel to distant sites transported by the blood
(Roberts et al. 2008). They mediate spread of the infection within the host while IVM are thought to
mediate host-host infection. Most research on vaccinia has been done in immortalized epithelial cells
however it is known that vaccinia prefers infection of polarized cells (Schmidt et al.2012).
3.1.3 Oncolytic virus
Vaccinia is an ideal virus to use for oncolytic purposes and it has been shown that it can be used to
treat skin tumors in xeroderma pigmentosum mouse models (Brun et al. 2012). It replicates and lyses
the host cell quickly, and infected cells are already destroyed 48-72 hours post infection.
Furthermore they have a broad tumor tropism that allows them to be used in the combat of various
tumors. The virus can be made specific to tumor cells by deleting certain genes such as the thymidine
kinase and/or VGF, which are both overexpressed in tumor cells and these will therefore be good
hosts for the handicapped virus to retain its full ability. Additionally viral replication and spread are
both linked to the activation of the EGFR-Ras pathway which is a pathway frequently activated in
human cancer cells. A great advantage of vaccinia virus is that they are able to travel systemically
through the blood. They can do this as EEV virions because this form is able to reach distant sites in
the body without being targeted by the immune system (Kirn et al. 2009). Normally more IMV are
produced but a mutation in the vaccinia genome can shift this balance towards a higher production
of EEV increasing the beneficiary systemic traveling characteristic. Individuals pre-vaccinated with
vaccinia, however, remain problematic as they contain neutralizing antibodies (nAbs) targeted
against the virus. To overcome this problem in these individuals alternative delivery routes are
explored which could evade these nAbs. For example different virion carriers are looked at which
could aid in the evasion of these nAb. Temporarily suppressing the immune system has also been
suggested as a method to deal with the virus being targeted for destruction. (Reinboth et al. 2012).
3.2 Vaccinia and autophagy
The link between vaccinia infection and autophagy has not been well characterized. Considering that
this virus replicates in the cytoplasm, one would imagine that autophagy will be induced as part of
the cell immune response. This is, however, not dominantly visible as vaccinia virus potently infects
32 | P a g e
its host cells. There are several viral proteins that could potentially be involved in the inhibition of
autophagy. Some of these have not been investigated for their direct interaction with autophagy,
such as N1, and others such as E3 have been implicated to be involved in blocking autophagy
(Orvedahl et al. 2008; Cooray et al. 2007; Grahan et al. 2008).
3.2.1 Replication
IVs are enclosed within a membrane of which the origin has not been fully elucidated. Not only is the
origin under debate, there is also contradicting literature concerning whether these virions have a
double or a single layer membrane though the single membrane theory takes the overhand. At first
autophagy was proposed as a potential origin for the replication factory due to the double
membrane characteristic. This was, however, disproved as Atg5-/- cells were shown to contain the
replication factories and fully sustained vaccinia replication. Conformation was obtained by knocking
down Beclin 1 proving that autophagy is dispensable for vaccinia replication (Zhang et al. 2006).
3.2.2 PKR pathway
Vaccinia leads to rapid cell death and therefore it would be unlikely that vaccinia would induce
autophagy upon infection as it has no need to prolong the host’s life span. Inhibiting the autophagy
process would be more important for the virus as it would aid in evading destruction of the virions as
well as it would reduce the host’s immune response. Autophagy has been shown to be induced by
the PKR/eLF2α pathway, which also mediates host shut off. This pathway, which is universally
blocked by viruses to evade autophagy, is actively inhibited by two different vaccinia viral proteins:
E3 and K3 (Orvedahl et al. 2008). The former consists of an amino terminal Z DNA binding domain
and a carboxy terminal dsRNA binding domain. E3 is essential for the pathogenicity contributing to
vaccinia neurovirulence. With the dsRNA binding domain it interferes with the dsRNA mediated
activation of PKR as well as with its dimerisation (Myskiw et al. 2011). The Z DNA binding domain has
been suggested to block the recognition of viral nucleic acids by endosomal TLR sensing through the
inhibition of autophagy. The exact mechanism how this domain would interfere with autophagy is,
however, unknown. It would be interesting to see whether any proteins involved in autophagy can
interact with this domain. Nonetheless the dsRNA binding domain and its ability to interact with PKR
as well as the fact that E3, in primary DCs, is an antagonist of the innate immune response suggest
that this protein could be involved in the inhibition of autophagy activation (Cao et al. 2012).
Furthermore K3, which mimics the 88 amino acids of the N-terminus of eLF2α, blocks the PKR kinase
catalytic site and consequently the intraction with its substrate, eLF2α (Bahar et al. 2011). These two
proteins give the vaccinia virus two distinct ways via which the PKR pathway could be blocked
indicating that this inhibition of autophagy and host shut down is of high importance for the
virulence of the virus.
33 | P a g e
3.2.3 Bcl-2 like viral proteins
Vaccinia also encodes several proteins such as N1, K7, A52 and B14 which are Bcl-2-like proteins
because they all contain a Bcl-2 like fold. None of these have been studied in the context of
autophagy. It is, however, known that viral Bcl-2-like proteins from other viruses can have the ability
to bind to Beclin 1 and subsequently inhibit formation of an autophagosome. A52 and B14 have been
shown to inhibit NFκB rather than apoptosis, which is mainly inhibited by N1 (Cooray et al. 2007;
Grahan et al. 2008). N1 is conserved in most vaccinia strains with the exception of the modified
Ankara virus, which contains a truncated version of the protein. The viral Bcl-2 like protein is able to
regulate inflammation favoring the virus. Additionally it modulates both NK response as well as the
activation of lymphocytes. Knocking down N1 did not affect the replication of the virus but the virus
was cleared more efficiently by the immune system. It also resulted in an increase of NK cells and a
decrease of T cells infiltrating the area of infection (Jacobs et al. 2008). Hypothetically if N1 is indeed
involved in the suppression of autophagy inhibiting N1 would result in an increase in autophagy. The
extent of the increase might not be very large as there are probably additional, both experimentally
proven as well as less known, pathways suppressing autophagy. The hypothetical increase in
autophagy and hence increased virus destruction are in line with the fact that without N1 the virus is
more easily cleared. However, the decrease in T cells involved upon infection with the N1 lacking
vaccinia contradict the involvement of N1 with autophagy. This is due to the fact that less T cells
would indicate less viral epitopes being presented at the cell surface in MHC. An increase in
autophagy would, conversely, result in increased degradation of the virus and hence a higher
availability of viral epitopes for MHC loading. With a higher concentration of these epitopes one
would expect an increase in T cells infilitrating the infected area. Considering this it could very much
be the case that N1 does not have anything to do with autophagy. The inhibition of viral clearance by
N1 might thus be due to a completely different mechanism. Nonetheless, during influenza virus
infection autophagy is not involved in MHC II antigen presentation (see 5.2.1), hence perhaps the
same holds true for vaccinia.
3.2.4 Atg12-Atg3 conjugate
A strong link between vaccinia infection and autophagy was made when Maloughney and colleagues
discovered that upon infection with this virus no autophagosomes are formed. Moreover, amino acid
deprivation-induced autophagosome formation is abolished during a vaccinia infection. This indicates
that vaccinia actively inhibits cellular autophagy. Infection did however cause high levels of the
lipidated form of LC3. These LC3-II puncta were different from the ones seen during starvation as
they appeared as aggregates. LC3 lipidation depends on Atg7 and in part on Atg 5. Lipidation of LC3
during vaccinia infection did not require these two factors as the corresponding knockout cells still
displayed the increased levels of LC3-II. This modification, however, depends on Atg3. Indeed
34 | P a g e
knocking down Atg3 resulted in loss of the increased LC3-II. Atg3 was shown to colocalise and to
form a conjugate with Atg12. This conjugate was found near the viral factories suggesting that the
virus is indeed responsible for the formation of this novel conjugate. The colocalisation of Atg12,
Atg3 and the replication factory could, however, not be visualized at once as there were no
commercial antibodies available which would make this possible (Moloughney et al. 2011). The exact
method via which this conjugate is formed and hence causes inhibition of autophagy has not been
elucidated yet. The conjugate was previously shown to be involved in mitochondrial homeostasis
highlighting the possibility that vaccinia hijacks an endogenous system/pathway (Radoshevich et al.
2010). The Atg12-Atg3 conjugate by itself is, however, unable to lipidate LC3 and therefore another
viral or perhaps cellular component is necessary. A candidate molecule would be an E1-like protein
because Atg7 is not required and consequently a replacement for this molecule might be encoded on
the vaccinia genome. A model showing the mechanism how vaccinia may inhibit autophagy via the
Atg12-Atg3 conjugate is depicted in figure 8. It has been proposed that this conjugate would
covalently link the E2-like and the E3-like enzymes involved in autophagy and thus short circuit the
LC3 lipidation system making use of a as of yet unknown viral protein. In parallel, the absence of
Atg12-Atg5, as Atg12 now binds to Atg3, will hamper the formation of the Atg12-Atg5-Atg16L
essential for the autophagosome biogenesis. These two events would act synergistically in blocking
autophagy (Moloughney et al. 2011).
Figure 8: Proposed model for Atg12Atg3
conjugation
and
subsequent
inhibition of autophagy. It has been
suggested that the novel Atg12-Atg3
conjugate,
generated
by
two
yet
unknown E2- and E3-like enzymes, shorts
circuits the LC3 lipidation process. This
along with the fact that possibly the
formation
of
this
novel
conjugate
abrogates the formation of the Atg12Atg5-Atg16L complex, inhibts autophagy.
Adapted from: Moloughney et al. 2011
35 | P a g e
3.3 Future directions
Autophagy is definitely inhibited by the vaccinia virus infection as was shown by Maloughney and
colleagues upon the discovery of the Atg12-Atg3 conjugate, which abolished formation of
autophagosomes. As mentioned in the previous section (2.2.4) LC3 lipidation could not be
accomplished by this conjugate itself and hence it was suggested that perhaps an virally encoded E1
like enzyme might be involved in this process (Moloughney et al. 2011). In addition, an E2- and an E3like enzyme are also necessary and might perhaps also be present in the viral proteome. If this is,
however, not the case perhaps host factors are needed which are recruited by other viral proteins.
To determine which viral proteins are essential for the formation of the Atg12-Atg3 conjugate as well
as for the subsequent lipidation of LC3, one could look at different mutant strains generated by
reverse genetics and identify whether there are specific mutations that lead to a phenotype where
the increased LC3-II production during infection in the absence of autophagy is lost. This is quite a
laborious task considering the fact that there are around 200 genes in the vaccinia genome. The
formation of the Atg12-Atg3 complex could be monitored by fluorescence microscopy but this is
currently not possible because there are no commercial antibodies available which allow the
detection of Atg3 and Atg12 (Moloughney et al. 2011). To this aim and for other possible
experimental approaches, it would be important to generate specific antibodies against these two
proteins as well as against the conjugate. If the knockout of a viral protein is found to alter this LC3
lipidation in the absence of an autophagy phenotype, its binding to the conjugate could be checked
by pull down assays. The Atg12-Atg3 conjugate has also been shown to be involved in maintaining
mitochondrial homeostasis. As a result, it cannot be excluded that a viral component hijacks a host
pathway involved in the function of the conjugate. Nonetheless, the inhibition of autophagy via the
formation of the Atg12-Atg3 conjugate appears to be very effective and consequently this is most
likely the main way how vaccinia blocks autophagy during infection. This observation would suggest
that other ways used by vaccinia to inhibit autophagy would be of minor importance.
Nonetheless these alternative mechanisms of inhibiting autophagy are still interesting to be
investigated more in depth. The Bcl-2 like proteins might not be the most interesting group of
potential autophagy inhibitors. It is in my opinion unlikely that N1 interacts with autophagy
mediators. This is due to the fact that if N1 is knocked down, apoptosis is activated upon infection
(Jacobs et al. 2008), since it is an anti apoptotic molecule. If N1 would additionally also be an
inhibitor of autophagy this knock down would counteract apoptosis. A high level of autophagy could
tilt the balance towards apoptosis, however, this seems unlikely since the Atg12-Atg3 conjugate
inhibiting autophagy would still be in place. Hence the idea that it blocks autophagy and the
knockout study showing increased apoptosis are not in line with one another. Additionally the
36 | P a g e
decrease of T cells in the infiltrate of immune cell post infection of a N1 knock out virus would
suggest that autophagy is not important in presenting viral antigens on the surface, which is possible
as it is also seen in influenza but more often autophagy is considered to play a role in this process.
Nonetheless if it does have a role in blocking autophagy knocking down N1 might not have any
significant effect on restoring autophagy since the formation of the Atg12-Atg3 conjugate will
overpower it in blocking autophagy. With these considerations in mind, it would be still interesting to
look whether Beclin 1 is a binding partner of one of the viral proteins containing the Bcl-2-like fold. If
an interaction with Beclin 1 is found, then these Bcl-2 like proteins would most likely play a minor
role in the inhibition of autophagy upon infection, but they could become important if the cells can
somehow inhibit or counteract the effects of the Atg12-Atg3 conjugate. It will be hard to test the
individual effect of the Bcl-2 like proteins on autophagy during vaccinia infection, but this could be
done if the Atg12-Atg3 conjugate formation is blocked. This is considering the fact that knocking out
either protein would have an effect on autophagy itself as well as the fact that no other proteins
which are involved are known very tricky. One could possibly make a form of Atg12 or Atg3 which is
not able to bind to the other but does maintain its function in autophagy. These mutants could
subsequently be transfected into a cell where the wt versions have been knocked down. To my
knowledge the exact interaction between these two proteins has however not been elucidated yet
hence this would provide the first step. Additionally if an essential viral protein is found associated
with the Atg12-Atg3 complex, it could be used to abolish its function by depleting it or mutating it
and the effect of N1 or other viral proteins could be analyzed in a infection context where the
principal mechanism used to inhibit autophagy is not present.
Furthermore the relevance of the PKR inhibition and thus the block of autophagy also requires
additional research. Again the absolute contribution to autophagy inhibition is most likely not very
large compared to the Atg12-Atg3 conjugation inhibitory pathway. Like the Bcl-2-like proteins, it will
be hard to determine the inhibitory effect of E3 and K3 by themselves, which will be even harder if
the Bcl-2-like proteins are also involved in blocking autophagy. The Z DNA binding domain of E3 has
been proposed to inhibit autophagy as it interferes with the recognition of viral nucleic acids; the
exact process, however, is not known (Cao et al. 2012). Perhaps this domain binds the viral nucleic
acids and thus shields them from being recognized by the autophagy machinery. This will allows
interfering with recognition without directly interfering with the autophagy machinery. Nonetheless
it could also possible that this domain interacts with Atg proteins and does indeed inhibit the
machinery. Whether any of the known Atg proteins interact with this domain could be tested with
the FISH technique or co-immunoprecipitation, or perhaps on an affinity column containing the
37 | P a g e
binding domain. Identification of a potential Atg binding partner will allow elucidating whether the
domain is directly involved in inhibition of autophagy.
In conclusion, the most promising pathway via which vaccinia inhibits autophagy preventing
destruction of the virus as well as presentation to the immune system is via the Atg12-Atg3 pathway.
The virus relatively quickly causes post infection cell death via necrosis, which might be aided by the
fact that autophagy is blocked. Nevertheless this principally serves the purpose of releasing the IVMs
inside the host for progressive infection. Although this is most likely the main mechanism of
autophagy inhibition, large parts of the pathway remain to be elucidated along with potential minor
inhibitory pathways.
38 | P a g e
4. Coronaviruses
4.1. Introduction
Coronaviruses (CoV) became public concern when the severe acute respiratory syndrome (SARS) CoV
surfaced in 2002 resulting in worldwide morbidity and mortality. Before the SARS emergence, CoV
were mostly a veterinary health issue. CoV
A
viral particle diameters range from 80 to 120
nm and are enveloped, pleimorophic or
spherical (Belouzard et al. 2012). The name
corona is derived from the appearance of
the virus under the EM as it has a crown
structure due to trimers of the spike (S)
protein on the surface of the virion. They are
single stranded positive sense RNA viruses
which containing a 5’ cap and have a length
B
between
26.2
characteristic
and
places
31.7
them
kb.
This
among the
longest RNA viruses. The genome, consisting
of 6 to 10 open reading frames (ORFs), is
enclosed in a helical nucleocapsid packed in
a host derived lipid bilayer (Perlman et al.
2009). The replicase proteins are encoded by
the first ORF, making up two-thirds of the
genome. The structural protein genes,
ultimately present in the virion envelope,
have a conserved order in the last third of
Figure 9: Coronavirus genome and structure. A. 3 different CoV
the ORF: the hemagglutinin esterase (HE),
genomes containing the in red colored ORF1 and the structural
which is not encoded for by all CoV, the S
proteins (HE), S, E, M, and N. B. The structure of CoV (not drawn
in scale). Adapted from Belouzard et al. 2012
protein , the envelope(E) protein, membrane
(M) protein and nucleocapsid (N) protein
(Figure 9). There are four different genera of
CoV, both based on serology as well as on
genetic studies: the alpha-, beta-, gamma-
39 | P a g e
and delta- CoV. Although the fundamental steps in the viral life cycle are conserved amongst the
different CoV genera, they use different receptors to initiate the infection life cycle (Belouzard et al.
2012).
4.1.1 Disease
CoV mainly causes intestinal and/or respiratory infections. Often CoV do not cause severe disease in
humans however they may manifest systematically (Kahn et al. 2005). 30% of all human colds can be
attributed to CoV. It is, however, difficult to culture CoV and hence to connect them with other
diseases (Engleberg et al. 4th edition). In humans the clinical effects from CoV can range from the
common cold, as mentioned before, to pneumonia and acute respiratory distress syndromes. The
latter was the case when SARS, a beta-CoV, emerged causing severe respiratory disease with a 10%
mortality rate (Gu et al. 2005). Furthermore the human CoV–NL63, an alpha- CoV, also leads to
severe respiratory tract disease (Wenhui et al. 2007). Similarly to most of the viruses,
immunocompromised individuals as well as newborns are more likely to develop severe pulmonary
diseases even from mild penetrant CoV (Kahn et al. 2005). The appearance of SARS also revealed the
ability of CoV to adapt from one species to another. This was discovered as SARS had characteristics
from both mammalian and avian CoV (Engleberg et al. 4th edition). This indicates that CoV can
undergo adaptations continuously during their life cycle and they can thus potentially cause severe
human diseases. It remains clear that CoV have a high economic impact because they affect a large
number of mammals, including dogs, cats, horses and other life stock and birds. In these animals they
cause respiratory and enteric diseases, and less commonly can also lead to hepatitis and neurologic
illnesses (Belouzard et al. 2012). Infection bronchitis virus (IBV) for example, is a gamma- CoV that
can lead to extreme economic losses in the poultry business (Cottam et al. 2011). Transmissible
gastroenteritis virus leads to diarrhea in pigs like bovine CoV does in cattle (Saif et al 2010; Kim et al.
2000) Infection with the mouse hepatitis virus 59A (MHV) strain, a beta-CoV, can result in
encephalomyelitis. This model is often used for research regarding multiple sclerosis (Tsuhako et al.
2009).
4.1.2 Infection mechanism
The infection cycle starts as the virion interacts with a specific protein/receptor on the surface of the
target cell triggering fusion. The S protein, a type 1 transmembrane protein, is crucial for these two
steps playing both a role in the tethering to the target membrane as well as the subsequent fusion of
the virion with it. The S protein is highly glycosylated and assembles in trimers. It consists of 2
domains: the S1 domain, at the N-terminus, is important for receptor binding, and the S2 domain at
the C terminus, which is highly conserved and is involved in membrane fusion (Bosch et al. 2003). The
40 | P a g e
different CoV make use of different
receptors to tether to the target
surface making the S protein the
determinant for cellular tropism.
MHV makes us of the adhesion
molecule CEACAM1, which is part of
the Ig superamily. Most alpha-CoV,
such as the feline and canine CoV,
bind to aminopeptidase N which is
also called CD13 and is present at
the apical domain of epithelial cells
of the enteric and respiratory tracts.
Figure 10: The life cycle of the CoV. CoV bind to their preferred receptor
While these different CoV recognize
and via a conformational change of the S protein fuse either directly with
the same receptor, they interact
the plasma membrane or after they are endocytosed. After fusion, the
with different amino acids. These
virions dissociate and release their genomic RNA into the cytoplasm. Here
binding domains are located in a
two large precursor polyproteins are made that are subsequently processed
non-homologous region. Sialic acid
by viral proteases. Replication-transcription complexes anchored into DMVs
are formed. Structural proteins are made from nested subgenomic RNAs
(SA),
the
known
receptor
for
and the genomic RNA is replicated. Subsequently the structural proteins
influenza, is also used as binding
and genome are assembled in the ERGIC. They form a particle by budding
interface by the IBV and the bovine
into the ERGIC and hence obtain an envelope. Lastly they are transported
CoV. The SARS and human CoV
through the Golgi and eventually are released via exocytosis into the
extracellular space. Haan et al. 2008
NL63
both
converting
bind
enzyme
angiotensin
2
(ACE2).
Furthermore SARS as well as IBV interact with DC-SIGN, which is a C-type lectin receptor on
macrophages and DCs known to be highjacked by HIV, because of the heavy glycosylation of the
spike protein.
Once the virion is attached to the membrane, it can enter the cell by either fusing directly with the
plasma membrane or by fusion after it is internalized via endocytosis. Both situations can take place
with MHV though fusion is principally triggered by binding to the receptor. IBV, on the other hand, is
endocytosed and fusion is induced by the lowering of the pH in the endosome. Fusion is the result of
a conformational change of the S protein and involves proteolytic cleavage, either by a viral or a host
protease. This change brings the fusion peptide, an apolar 15-25 amino acid long region predicted to
be in the S2 domain, in close contact with the transmembrane domain facilitating fusion. Once fused,
the virion dissociates and releases its RNA genome into the cytoplasm (Belouzard et al. 2012). The
41 | P a g e
entire processing and replication process takes place in the cytoplasm. Here two large polyproteins
are synthesized, which are cleaved by viral proteases into 16 non structural protein (nsps). These
nsps are essential for the formation of the RNA replication-transcription complex (Perlman et al.
2009). These replication complexes are anchored into DMVs, of which the origin has been the subject
of much debate as will be discussed later in this chapter (Reggiori et al 2008). DMVs are a very
elegant way via which the virus ensures that the viral RNA is protected from cellular defense.
Formation of both these DMVs and the replication translation complexes leads to the translation of
the structural proteins from subgenomic nested RNA. This organization permits the RNA to have a
poly-A tail and a cap to resemble cellular mRNA so that it can make use of the host translation
machinery. (Perlman et al. 2009). The genome is replicated by the viral RNA-dependent RNA
polymerase, which is very error prone. This tendency to make mistakes relates to the fact that CoV
relatively easily obtain mutations. At times, this can result in interspecies jumps. Indeed closely
related CoV were discovered in distantly related animals indicating that this was caused by recent
interspecies jumps (Belouzard et al. 2012). Additionally homologous recombination has been shown
to occur in CoV due to their large RNA size. Homologous recombination would result in a strain
adapting novel characteristic from another CoV upon co-infection (Thor et al. 2011). Newly made
genomic RNA is subsequently assembled with the structural proteins at the ER-Golgi intermediated
compartment (ERGIC) (Ruch et al. 2012). The newly formed virions bud inwards into the ERGIC
forming an enveloped virion that is extracellularly released via exocytosis (Boscarino et al. 2008)
(Figure 10).
4.2 Coronavirus and autophagy
DMVs are the hallmark of a CoV infection. The origin of these DMVs has been much debated. Due to
their appearance, one hypothesis was that perhaps CoV were derived from autophagosomes.
Experimental exploration of this hypothesis resulted in some contradictory results on whether
autophagy machinery is important for CoV-induced DMVs formation and hence replication. DMVs
seem to be derived from the ER as ER markers are found on DMVs as well as because they are found
close to the ER and occasionally even attached to it (Prentice et al. 2004; Zhao et al. 2007; Haan et al.
2008). The ER origin coincides with autophagosomes, which also appear to be of ER origin, though
they can also be derived from the mitochondrial outer membrane (Tooze et al. 2010). Furthermore
DMVs were shown to be decorated with LC3, an autophagy marker protein. Denison and collegues
additionally showed that Atg5, another important autophagy protein, was necessary for the
formation of DMVs in murine embryonic stem cells (Prentice et al. 2004;). This latter finding was
however contradicted by a paper in which Atg5 was shown to be dispensable for DMVs formation in
primary macrophages and low passage primary embryonic murine fibroblasts (Zhao et al. 2007). This
42 | P a g e
would indicate that autophagy is not at all required for CoV replication in these cells. Further
discrepancy arose as some labs were able to show LC3 colocalizing with the DMVs while others could
not (Prentice et al. 2004; Zhao et al. 2007; Haan et al. 2008).
4.2.1 Highjacking LC3-I
LC3 is present on the membrane of the DMVs in the LC3-I variant, which unlike the LC3 form present
on autophagosomes, is not lipidated (Reggiori et al. 2010). This explains why some groups were able
to find the colocalisation while others were not. They used different detection methods as some
utilized antibodies that recognized all LC3 while others made use of autophagosome specific GFPLC3, which is in the LC3-II form and consequently does not colocalize with DMVs (Prentice et al.
2004; Zhao et al. 2007; Haan et al. 2008). LC3-I knock down results in the severe impairment of viral
replication showing that LC3-I is indeed important for CoV life cycle. LC3-I is converted to LC3-II by
Atg7, amongst other Atg proteins. Hence since the DMVs have non lipidated LC3 in their membrane
there should be no need for Atg5 nor for autophagy. LC3-I is in fact not a functional autophagy
protein unless it is transformed into LC3-II. This discards the hypothesis that DMVs are
autophagosomes. Nonetheless this does not reveal the origin of these DMVs. There is, however,
another structure in the cell that also has LC3-I onto its membranes, namely the EDEMosomes
(Reggiori et al. 2010).
4.2.1.1 The ERAD tuning pathway
EDEMosomes are part of a mechanism of control of the ER-associated degradation (ERAD) pathway.
ERAD ensures quality control of proteins in the ER; misfolded proteins are dislocated into the cytosol
and degraded by the ubiquitin-proteosome pathway. Because proteins that are still in the process of
folding under normal conditions also attract the ERAD machinery, the ERAD factors need to be tightly
regulated in order to avoid that these intermediate proteins are degraded. One of the ways of
control is VIA ERAD tuning. During this process, the ERAD factors are selectively removed from the
ER. EDEM1 and OS-9 are a couple of these ERAD regulators and they are sorted out of the ER in
EDEMosomes. EDEMosomes are 200-800nm large single membrane vesicles, which are decorated
with LC3-I. Furthermore EDEMosomes are formed in the ER in a COPII protein coat-independent
mechanism. They subsequently deliver their content to endosomes for degradation. The exact
mechanism of this disposal, however, remains unknown (Bernasconi et al. 2011).
4.2.1.2 DMVs and EDEMosomes
DMVs have also been shown to contain EDEM1 and OS-9 suggesting that CoVs hijack the
EDEMosomes. These two ERAD proteins, however, are not essential for MHV replication (Reggiori et
al. 2010). Trapping ERAD proteins might be beneficial later during infection when the ER stress is
increased due to the high production of viral proteins. This would normally turn on the ERAD
43 | P a g e
pathway and thus degrade viral proteins, which can be minimized by trapping the ERAD proteins in a
different location (Reggiori et al. 2011). DMVs will also need to be modified EDEMosomes
considering the fact that EDEMosomes normally fuse with endosomes. As DMVs are the replication
site of CoV, they must somehow stop the vesicle from fusing with the endosomes. The exact
mechanism how EDEMosomes become DMVs has not been solved nor has the exact function of LC3-I
in the formation of these vesicles been unveiled.
4.2.1.3 Models
There are a few proposed models for the role of LC3-I in CoV infection as well as for the formation of
DMVs from EDEMosomes. LC3-I is part of a complex including SEL1L and either OS-9 or EDEM1
during steady-state delivering the ERAD proteins to the endosomes. It is however not clear whether
LC3-I binds directly or indirectly to Sel1L. This suggests that LC3-I would be part of an EDEMosome
cargo receptor (complex) (Bernasconi et al. 2012). A way for CoV to hence hijack the ERAD tuning
machinery and form DMVs would be to hijack this cargo receptor. This could perhaps be done by a
viral nsp. LC3-I could on the other hand participate in ERAD tuning/viral replication by attaching the
DMVs and EDEMosomes to the microtubule network. MHV replicative structures indeed have been
shown to move along microtubules to concentrate in the perinuclear region. Nonetheless
microtubules are not crucial for MHV replication and therefore LC3-I must have another key function
in CoV replication (Reggiori et al. 2011). The fact that LC3-I seems to associate with the cargo
receptor SEL1L, makes the hijacking theory by one or more viral proteins an attractive possibility.
How are the CoV-induced DMVs derived from the single membrane EDEMosomes? There are two
models that still need to be explored experimentally (Figure 11). The first involves the
reticulovesicular network (RVN) that is formed upon SARS infection. This modified ER has both
convuleted membranes as well as many interconnected DMVs. The mechanism of this model is
nonetheless unclear. In the second model DMVs could be the result of invaginations and subsequent
fusion of the extremities of a single membrane vesicle, i.e. an EDEMosome, regulated possibly by a
viral protein (de Haan et al. 2010). Further studies are needed to elucidate both the role of LC3-I as
well as the role of viral and host proteins in the formation of DMVs from the ER.
Though LC3-I once lipidated is a protein involved in autophagy it is now clear that autophagy itself is
not involved in the formation of DMVs and the replication of CoV. It does however show that
proteins involved in autophagy can have additional and different role within the cell.
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Figure 11: Hypothetical models for the
origin and formation of DMVs. DMVs
could originate from RVNs as seen in
SARS-infected cells where a large number
of interconnected DMVs are formed
(model 1). Another model is based on the
inward budding profile of DMVs and
these could form by invaginations and
subsequent fusion of the extremities of
this intermediate (model 2). This event
could be mediated by viral proteins.
Adapted from: de Haan et al. 2010
4.2.2 Induction of autophagy
Autophagy has been shown to be induced upon infection with CoV through nsp6, a membrane
spanning protein. Transfection of nsp6 introduction of IBV, SARS and MHV into cells resulted in the
induction of autophagy via an omegasome intermediate. Omegasomes are domains in the ER that
contain DFCP1, which is recruited to the ER upon activation of autophagy, and that recruit key
autophagy proteins such as LC3, Atg5 and Atg14. The mechanism by which nsp6 induces autophagy
does not rely on the classical inhibition of mTOR, induction of ER stress or activation of Sirun1. It is
currently unclear how this viral protein leads to the formation of autophagosomes. It might be due to
the enhanced production of Ptdlns(3)P which binds DFCP1, because higher levels of Ptdlns(3)P were
measured after cell transfection with nsp6. Nsp6 could also mimic Atg14 and directly recruit Beclin 1
to the ER. Furthermore it could inhibit Jumpy. Jumpy is a phosphatase that dephosphorylates
Ptdlns(3)P into PtdIns inhibiting autophagy. The ability to induce autophagy was however shown by
introducing solely nsp6 into the cells in absence of a CoV infection. This is not a physiological
situation hence it does not directly show that nsp6 induces autophagy during virus infection.
Autophagy could be triggered during virus infection by either RNA being present in the cytosol, which
could be sensed by the immune system, or perhaps viral protein complexes might induce autophagy
(Cottam et al. 2011). If nsp6 is part of a complex during infection this might also interfere with its
ability to induce autophagy. However if nsp6 indeed induces autophagy during viral infection this
would not seem beneficial for the virus. Considering the fact that autophagy could degrade viral
particles, induction of this pathway would not be advantageous. Conversely, perhaps nsp6 makes use
of autophagy to target certain immunomodulatory proteins, which are synthesizes in the ER towards
the autophagosome degrading them and consequently decreasing the host immune response.
Alternatively, as for the VZV, the virus needs to induce autophagy to ensure a longer life span of the
host cell considering the fact that they interact with the ERAD machinery decreasing the clearance of
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Figure 12: ERAD tuning and highjacking by coronaviruses. The ERAD pathway is important in the clearance of misfolded
proteins. Under normal conditions the ERAD factors need to be tightly regulated preventing premature intermediate
proteins from being degraded. This is also called ERAD tuning, where certain ERAD factors are selectively removed from
the ER. EDEM1, one of these ERAD regulators, along with OS-9, is sorted out of the ER in EDEMosomes. This is done by the
cargo receptor SEL1L, which simultaneously binds these ERAD factors and LC3-I. The exact binding of the latter has not
been determined yet. The EDEMosomes subsequently deliver their content to the endosome/acidic compartment for
degradation. CoV highjack these vesicles forming DMVs, where they replicate, through a mechanism that still remains
unknown. Additionally CoV-induced DMVs do not fuse with endosomes/lysosomes hence they must somehow inhibit this
step. Bernasconi et al. 2012
misfolded proteins which could result in cell death. These latter two possibilities would be beneficial
for the virus.
4.3 Future directions
There is not a clear link between autophagy and CoV. Autophagy was first thought to be hijacked by
CoV, as the virus replicates on DMVs, which are morphologically similar to autophagosomes.
However, the LC3 form decorating both membranes is different. While autophagosomes are
decorated with lipidated LC3, DMVs have LC3-I, the non lipidated version, on their membranes.
These DMVs were instead shown to be derived from EDEMosomes. This was suggested as both
vesicles have the LC3-I on their membranes as well as both contain the ERAD regulators EDEM1 and
OS-9. However it could still be possible that CoV hijack LC3-I and that the two ERAD proteins are
actively recruited by CoV into the DMVs to shut down the ERAD (Reggiori et al. 2011). LC3-I has been
shown to associate with SEL1L, a cargo receptor, and EDEM1 and OS-9 providing evidence for a CoV
nsp hijacking of the cargo receptor and thus forming a vesicle with LC3-I on the surface (Bernasconi
et al. 2012) (Figure 12). Further research is necessary to determine how LC3-I binds this cargo
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receptor, directly or via an adaptor protein, and whether one of the nsp of CoV is able to bind either
SEL1L, another cargo receptor or perhaps binds the complete complex including LC3-I. The exact
mechanism via which the DMVs are actually formed is also subject for further research. Whether this
is via the RVN during SARS infection or whether RVN is only used for replication later in infection
once the cell is no longer able to make isolated DMVs. To determine the latter it is key to look at
whether CoV are also able to replicate within the convulated membranes. Another suggested model
involves the pinching off of invaginations of single membrane vesicles perhaps by a viral protein. It
would be good to start by investigating whether one of the viral proteins is in fact able to pinch off
or cleave something off the ER. Shedding more light on these processes would help in understanding
the exact mechanism via which CoVs manipulate the host cell to create their replication paradise
(Haan et al. 2010). Again this is not a direct link between virus and autophagy, which is the main
focus of this thesis. However it is important to note that proteins involved in autophagy seem to
additionally be able to function in different cellular processes, in this case LC3 in a non lipidated
form.
Moving on to the autophagy relevant part of this section it is fair to conclude that autophagy has not
been firmly linked to CoV. There is evidence that nsp6 induces autophagy, however, the experiments
were done by introducing solely nsp6 into the cells (Cottam et al. 2011). Thus the autophagyinducing characteristic of nsp6 might not be present once the cell is infected with the entire virus.
Nonetheless the paper brought to light some things that will be interesting to look at in future
experiments. Nsp6 being a transmembrane protein was predicted to be important in the replication
transcription complex as it would span the membrane of the DMVs. Additionally it was also shown to
be present on the ER membrane as some C-terminal residues were glycosylated. This would mean
that it would both induce autophagy and also be present on the DMVs independently from
autophagy. There are, however, two different nsp6’s found which could be either two different ones
or one could be post translationally modified or otherwise alternatively processed (Baliji et al. 2009).
Perhaps one of these can induce autophagy while the other is present at the DMVs playing part in
the replication of the virus. Either way it is important to explore whether nsp6 is the causative agent
for autophagy induction when cells are infected with the entire virus or whether autophagy is
induced via a completely different mechanism. If autophagy is indeed induced by nsp6, looking at
how it is induced, as it is not via inhibition of mTOR or induction of ER stess is an elegant next step.
Here one could look at Jumpy, Ptdnlns(3)P or whether it bind Beclin directly or perhaps induces the
PKR pathway (Cottam et al. 2011). On a side note, IBV nsp2, nsp7 and IBV-induced overexpression of
GADD34 result in antagonism of PKR. This is done by interacting with the phosphatase PPI resulting
in less phosphoryalted eIF2α and by participating in the dephosphorylation of eIF2α respectively
47 | P a g e
(Cruz et al. 2011). Though this has not been directly related to autophagy, it is known that the PKR
pathway can stimulate autophagy thus interacting with proteins involved in this pathway
counteracting the activity of PKR might also result in some inhibition of autophagy (Talloczy et al.
2006). Additionally the reason for autophagy induction, whether it is beneficial for the virus, would
be an intriguing next step. Perhaps as mentioned before, induction of autophagy by nsp6 is
somewhat specifically targeting immunomodulatory proteins to their degradation via autophagy.
This raises the question how this autophagy is then driven to be more specific to the level that the
virus benefits more than being destroyed. Again the virus could also simply need the autophagy to
elongate the life span of its host so that it can replicate more.
All together the relationship between autophagy and CoV has not been elucidated yet. These viruses
do show that autophagy proteins, such as LC3, can participate in processes other than autophagy.
Nonetheless one of the only links connecting autophagy and CoVs is nsp6 which seemingly induces it.
This, however, needs to be validated in the context of functional CoV infection where other factors
might influence nsp6 hence disturbing this potential role in inducing autophagy. Furthermore the
PKR pathway also seems to be inhibited by IBV. This has not been investigated in the light of
autophagy inhibition. Concluding only little has been revealed considering this virus and autophagy
and more research is required in order to gain substantial knowledge in this part of the field.
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5. Influenza
5.1 Introduction
Influenza viruses are double stranded negative sense segmented RNA viruses which contain a
genome with a size of approximately 10- 13.6 kilobases. There are three different types of influenza
viruses, A, B and C, which are all very similar in structure. They, however, are quite diverse and show
no cross reactivity with antisera, a characteristic that separates them from each other (Engleberg et
al. 4th edition). Influenza virus C contains the smallest genome as it has 7 segments compared to the
8 segments found in influenza virus A and B. Each segment of RNA is packed by the viral
nucleoprotein (NP) as well as by three viral polymerase proteins. The matrix protein surrounds the
encapsidated RNA segments and is in turn packed in a host cell-derived membrane. Two viral surface
glycoproteins, hemagglutinin (HA) and neuramidase (NA), are embedded in this membrane and play
an important part in viral entry and exit, respectively. HA forms a homotrimer on the surface of the
virion.
The focus of this chapter will be the influenza virus A. The 8 influenza A virus RNA segments encode
11 different viral proteins (Palese et al. 2007). The largest three segments contain three RNA
polymerase components as well as the polymerase basic protein (PBIF2), which promotes cell death.
The smallest segment produces the nonstructural proteins, ns1 and ns2, which compromise the IFNmediated response by preventing innate recognition and support the nuclear export of packed viral
RNA, respectively. The remaining segments encode HA, NA, NP and the two matrix proteins, M1 and
M2. M2 is important in the uncoating of the virion which will be discussed in more detail in the next
section (Gannagé et al. 2009). There are currently 16 different serotypes of HA and 9 serotypes of NA
known allowing various combinations. Antigenic shift and drift are two processes important for
influenza pathogenesis through the creation of novel HA/NA combinations (Engleberg et al. 4th
edition). Antigenic drift results in the accumulation of mutations in the antigenic sites of either HA
or/and NA. This happens in all influenza viruses but occurs more frequently in the influenza A virus,
which is more prone to mutations. In a time span of 3-5 years the drift can give rise to a novel strain
causing disease (Boni et al. 2006). Antigenic shift is what distinguishes influenza A from influenza B
and C viruses. It involves the exchange of HA and NA genes from one virus to another. This process is
also called reassortment and happens only in influenza A virus due to its ability to infect multiple
species. It can cause serious disease as the immune system will not be able to recognize the virus,
which has obtained novel surface markers, and may not be able to clear it successfully. Antigenic
shift is the cause of many flu pandemics that have occurred in the past (Smith et al. 2009).
49 | P a g e
5.1.2 Disease
The influenza A virus is the causative agent of the Spanish flu H1N1 pandemic in 1918 as well as for
the more recent pandemics such as the 1957 Asian H2N2 flu, the 1968 Hong Kong H3N2 flu and the
most recent swine H1N1 flu pandemic in 2009. This latter pandemic caused fewer deaths than the
common flu however was classified as a pandemic due to the quick spreading (Smith et al. 2009).
Nonetheless, influenza can causes severe respiratory diseases that are highly contagious through
airborne droplets. Influenza clinical manifestations include fever, malaise, headache, chills and
respiratory symptoms such as cough. It can lead to several complications ranging from pneumonia to
encephalitis. During mild cases of influenza, the pathology displays affected respiratory epithelial
cells with edema which show inflammation due to the infection. Additionally, mononuclear cell
infiltrates are observed. More severe disease manifestations are caused by influenza damaging the
tissues. In this case the lungs become hemorrhagic and airless as well as display necrotizing
tracheobronchiolitis and bronchitis (Engleberg et al. 4th edition). This tissue damage and the virus
itself cause lymphopenia as well as severe inflammation which can result in a cytokine storm and this
could be lethal. It is thought that a cytokine storm caused the high number of deaths during the
Spanish flu outbreak as well as during the H5N1 avian influenza A virus pandemic (Osterholm et al.
2005). This latter virus strain has been responsible for human deaths since 1997, having a mortality
rate of 50%. It is characterized by a rapid progression to acute respiratory distress syndrome (Ma et
al. 2011). Influenza causes thousands of deaths annually as well as enormous economic loss, tens of
billions of US dollars, due to the time needed to recover from debilitating infections. Thus it also
causes social disturbances (Gannagé et al. 2009). The influenza C virus is the least severe virus and is
nonseasonal. In contrast, the influenza A and B viruses can cause the severe respiratory diseases
described above and occur between October and April, peaking between December and March on
the northern hemisphere (Engleberg et al. 4th edition).
5.1.3 Infection mechanism
The influenza virus enters the lungs and infects cells of the respiratory tract such as nonciliated
epithelial cells, which are commonly the first to be infected (Engleberg et al. 4th edition). A terminal
SA linked to galactose is the preferred receptor for the influenza virus. Human viruses recognize a
α2,6 linkage while avian influenza binds to a α2,3 linkage. Swine influenza can recognize both of
them (Samji, 2009). The membrane protein HA binds to SA. HA0 is the precursor of HA. This
precursor consists of HA1, which directly binds to the SA, and HA2, which contains the fusion
peptide at its N-terminus. The fusion peptide is composed of an extremely conserved hydrophobic
50 | P a g e
sequence. HA is the protypical member
of the class I fusion protein family. When
HA binds to the SA, the virion is
internalized through endocytosis. In the
endosomes the low pH, between 5 and
6, triggers fusion of the viral membrane
and the endosomal membrane. The
fusion is induced by a conformational
change of HA. At the N-terminus, a long
helix is formed as an unstructured linker,
called a prehairpin, becomes helical. In
this conformation the fusion peptide is
launched into the target membrane and
is subsequently embedded, connecting
the viral and endosomal membranes.
Figure 13: The life cycle of Influenza A. Influenza tethers its target cell
through the binding of HA to SA. Subsequently the virus enters the
Parallel the c-helix packed into the Nterminal trimeric coiled-coil grooves is
cell via endocytosis. Inside the endosome, the low pH triggers a
inverted forming a six-helix bundle. Due
conformational change of HA that allows fusion of the endosomal and
to the fusion peptide and the six-helix
viral membranes releasing the RNA genome into the cytoplasm. The
bundle, the two membranes are brought
vRNPs are then translocated to the nucleus were transcription takes
place. The negative sense RNA needs to be transcribed into a positive
into close proximity greatly easing
sense RNA in order to replicate the genome. The genomic RNA is
merging (Belouzard et al. 2012). Apart
packed together with NP and exported out of the nucleus. The
from the conformational change, the
assembly of the virion takes place at the plasma membrane where
acidic environment also opens the M2
the viral membrane proteins concentrate. Finally the virion buds off.
During this event, NA plays a key part cleaving SA and ensuring that
the virus particle is released.
ion channel. M2 exists in tetramers and
forms a channel through which protons
can enter. Activation of this channel
Adapted from: http://nursingcrib.com/microbiology/influenza-viruslife-cycle/
results in an acidified viral core which
permits the release of viral RNA packed
in NP, called the viral ribonucleoproteins (vRNP). As mentioned before, vRNPs also contain the viral
RNA polymerase complex. The transcription and translation subsequently occurs in the nucleus. In
order to enter the nucleus, all the vRNP have a nuclear localization signal (NLS), although it is
unknown whether the NLS is the key signal for the nuclear entry. Entry could additionally take place
by binding importin α and β, which are also involved in nuclear import. As the influenza virus has a
negative stranded RNA genome, it must first be transcribed into positive sense so it can be used for
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viral RNA production (Samji, 2009). For replication of the viral RNA, there is no need for a primer due
to the fact that the viral RNA contains highly conserved untranslated regions (UTRs) on both the 3’
(12 nucleotides) as well as the 5’ site (13 nucelotides) (Dai et al. 2012). Furthermore, these UTRs are
also partially inverse complementary so that they can base pair with each other forming corkscrewlike configurations.
Influenza only encodes 11 proteins, as previously mentioned, hence has developed several ways to
make use of the host machinery. For example cap snatching is observed during the
replication/transcription process. Viral mRNAs contain a 5’ cap, however, there is no viral gene that
encodes for this cap. The virus is able to use the host 5’ caps to protect its mRNAs from degradation.
The cap is snatched by the viral protein PB2, which has an endonuclease activity, and cleaves the cap
of the host mRNAs. Next the RNA needs to exit the nucleus, only negative sense vRNPs are exported
from the nucleus. Once the vRNPs are exported out of the nucleus, the virions assemble and exit the
cell. Viral assembly takes place at the plasma membrane as influenza is an enveloped virus. The
virions exit the cell at the apical side through budding. The tail of M2 has been shown to be crucial
for the virion formation. M1 also plays an important part in the budding off from the plasma
membrane. Additionally, NA is needed to allow release of the novel virion from the plasma
membrane since it is attached to SA. In particular NA is able to cleave off the SA hence removing
them, which allows the release of the virus and the subsequent infection of adjacent cells (Figure 13)
(Samji, 2009).
5.2 Influenza and autophagy
The literature about the association between autophagy and influenza contains many discrepancies.
While some studies state that autophagy is involved in replication, others conclude the contrary
(Zhou et al. 2009; Gannagé et al. 2009). Not only the study of the virus replication and autophagy has
produced contradictory results, determination whether the influenza virus is able to mediate
autophagy maturation did so as well. While there is evidence for a maturation block by the virus,
there is also proof that this block does not occur and that autophagy is fully functional (Gannagé et
al. 2009; Law et al. 2010; Comber et al. 2011). Perhaps these differences are due to the fact that
different cell types were used to study the influenza infection or simply because the different aspects
of the interaction between autophagy and influenza infection were difficult to pinpoint. Since
technology has greatly improved and the progression of autophagy can be tested in several ways,
this latter point seems negligible. Taking into account that with HSV there is also discrepancy
between different cell types, this seems the more likely explanation (Gobell et al. 2011). All the
results and discrepancies will be reviewed in the upcoming sections.
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5.2.1 Induction of autophagy
Like most viruses influenza too induces autophagy upon entering the host cell, which has been
shown by EM as well as by increased levels of LC3-II. Infection with influenza produces reactive
oxygen species (ROS) via the nox2 NADPH oxidases. These ROS damage Atg4 leading to the
accumulation of LC3-II, which results in autophagosome formation and accumulation. The influenza
virus has moreover been shown to upregulate the autophagy proteins Atg7, Atg5 and Atg12 (Dumit
et al. 2012). If the ROS are eliminated for example using the antioxidant procyanidin, autophagosome
formation is decreased. This same component has been shown to decrease the expression of the
overexpressed Atg7, Atg5 and Atg12. This suggests that both the ROS and overexpression of the Atg
proteins are indeed partially responsible for the autophagosome formation (Dai et al. 2012). The
induction of autophagy in MDCK and A549 cells infected with H1N1 (A/WSN/33) is initiated at least
via the mTOR pathway as phosphorylation of p70S6K, a downstream effector of this kinase,
decreases upon infection indicating that mTOR is inhibited, something that induces autophagy (Zhou
et al. 2009). Surprisingly, however, A549 cells infected with the A/newcoledonia/20/1999 H1N1
strain do not seem to inhibit mTOR (Sun et al. 2012). This suggests that autophagy might also be
regulated by another pathway and that this might be strain-related. Although autophagy is induced
during the influenza virus infection, it does not contribute to antigen presentation as seen during
other viral infections. CD4+ T cell responses were analyzed in Atg7 knockout cells and no difference
was found in the responding CD4+ T cells compared to wt cells. Antigen presentation on MHCII was
instead mediated by proteasome-dependent pathways (Comber et al. 2011).
5.2.2 Replication
There is much debate on whether influenza needs autophagy for its replication. Inhibition of
autophagy in MDCK and human lung cancer A59 cell lines infected with H1N1 (A/WSN/33) and H9N2
(A/chicken/Beijing/04), both using a chemical approach as well as RNA interference, resulted in a
decrease in viral yield. This was however not due to inability to replicate the genome, as this happens
in the nucleus, but was due to a decrease in the levels of M2, which is important in the assembly,
entry and uncoating of the virus. Thus autophagy inhibition deregulates the viral protein turnover
decreasing the yield. This suggests that autophagy, which should clear intracellular pathogens, is
manipulated by influenza to deliver the right proteins for viral assembly (Zhou et al. 2009). Activation
of autophagy is hence biphasic meaning that a part of an antiviral response is misused to support the
viral replication (Ehrhardt et al. 2006). Additionally infecting autophagy deficient human
mucoepidermoid pulmonary carcinoma cells with H1N1 (A/Puerto Rico/8/34) decreased the viral
load. In this same paper cathepsin D was suggested to potentially be used by the influenza virus to
corrupt the autophagic machinery during infection. Cathepsin D, which is involved in autophagy and
apoptosis, is a cysteinyl-, aspartyl-, and serine-protease which is released from lysosomes upon
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activation. This was indicative as Pepstatin A, an inhibitor of cathepsin D, had a negtative effect on
viral reproduction (Matarrese et al. 2011). Gannagé and colleagues contrastingly showed that
infection of autophagy deficient mouse embryonic fibroblasts with H3N2 (A/Aichi/68) did not have
an effect on the viral yield. In particular, they showed higher concentrations of RNA and viral proteins
when autophagy was functional, however, this did not seem to affect the release of infectious virus
(Gannagé et al. 2009).
4.2.3 To block or not to block
A second main discrepancy in the field is whether autophagosomal maturation is blocked or not
during an influenza infection. It has been shown that the influenza virus can prevent fusion of
autophagosomes with lysosomes leading to an accumulation of autophagosomes. This phenomenon,
however, does not occur until later during infection. This suggests that viral or perhaps even cellular
factors need to be produced first before the fusion with the lysosome can be impaired. Several
different strains, H3N2 (A/Aichi/68), H1N1 (A/WSN/33) and H1N1 (A/PR8/34), displayed this
accumulation of autophagosomes. Transfection of M2 into the lung A59 cells is sufficient to cause
this autophagosome accumulation phenotype (Gannagé et al. 2009). Indeed when excluding M2, by
silencing M2 expression post infection, the autophagosomal accumulation phenotype was no longer
observed. Unexpectedly, M2’s proton channel pump function did not contribute to the block of
fusion with the lysosomes. Instead M2 co-immunoprecitpitated with Beclin 1 (Gannagé et al. 2010).
Beclin 1 is a protein frequently targeted by viral proteins. In complex with UVRAG, it is important in
the maturation of autophagosomes. Interaction with Beclin 1 at this time point could thus indeed
result in a block of the late steps of autophagy (Munz, 2011). The exact interaction between Beclin 1
and M2 is not yet unraveled. Protein-protein interactions and dynamics will have to be further
elucidated to determine the exact mechanism via which M2 interacts with Beclin 1 and if this is done
in the context of the UVRAG-containing complex.
Cells deficient for autophagy have increased susceptibility to apoptosis. The fact that the survival
mechanism, autophagy, is blocked late during infection could indicate that the block of autophagy
might be set in motion by the virus after it has successfully replicated. The exact benefit for the
influenza virus to induce cell death by inhibiting autophagy remains unclear. This benefit might lie in
depleting immune cells thus reducing the immune response. Macrophages are very sensitive to
influenza virus-induced cell death (Bender et al. 1998). Lung epithelial cells are as well and
consequently this could serve to lift the lung barrier allowing influenza to infect deeper into the lung
tissue (Baskin et al. 2009). Apoptosis additionally limits the pro-inflammatory cytokine response.
Furthermore the antigens are trapped in the autophagosomes and as a result they cannot be
presented on the MHC complexes on the cell surface. All together, these observation support a
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model where the virus induces cell death via apoptosis and it combines it with a block of autophagy
to possibly accelerate apoptosis of the infected cells with minimal immunogenicity (Gannagé et al.
2009). Nonetheless there are other reports where this block of autophagy was not detected. By both
assessing LC3 as well as p62 degradation, two separate studies revealed induction of functional
autophagy post infection. This was done in several cell lines including fibroblasts and primary DCs
(Law et al. 2010; Comber et al. 2011). This, hence, again raises the possibility that perhaps the
influenza virus life cycle is different depending on which strain as well as which cell type is used for
the experiments.
5.2.4 H5N1-induced autophagy-mediated cell death
The avian influenza virus H5N1 is, as mentioned above, highly pathogenic having a mortality rate of
50%. In embryonic mouse fibroblasts cell death was caused by influenza infection via autophagy.
Autophagy is mostly a survival mechanism but when there is too much autophagy in the cell, this can
contribute to the induction of cell death. H5N1 triggers this type of autophagy by inhibiting mTOR.
Accordingly, cells with hyperactive mTOR were not susceptible to H5N1. The exact mechanism of
how H5N1 inhibits mTOR is currently unknown, but there are indications that it is through the
activation of the TSC1/2 protein complex. Furthermore the poor cell viability caused by H5N1
infection could be rescued by treating the cells with an autophagy inhibitor. These results were
obtained using a virus that was inactivated suggesting that autophagy contributes to the
pathogenicity independently from viral replication (Ma et al. 2011). The HA protein of H5N1 was
shown to be potentially involved in inducing cell death via autophagy (Sun et al. 2012). If these
results stand, autophagy inhibitors could be useful to diminish the tissue damage caused by H5N1.
5.3 Future directions
Clarity about the exact role of autophagy during influenza virus infection has as not been provided
yet. A substantial amount of contradicting literature has been published over the years suggesting
that the difference in influenza virus strain as well as the difference in infected cells might have a
large influence on the way autophagy is manipulated by the virus (Figure 14) The only result that is
common in all the works is that influenza virus infection induces autophagy. One of the mechanisms
that was proposed was the inhibition of mTOR. Nonetheless H1N1, which inhibits mTOR during the
24 hours post infection, has also been shown to induce autophagy independent of mTOR 36 hours
post infection (Zhou et al. 2009; Sun et al. 2012). Two different strains (A/WSN/33 and
A/newcoledonia/20/1999 respectively) were used during these experiments. This difference along
with the different time post infection might have influenced the preferred pathway via which
autophagy was induced. It would be interesting to elucidate the pathway used by the second H1N1
strain to trigger autophagy and see whether mTOR is crucial at an earlier time point or not. It seems
55 | P a g e
peculiar that the main mechanism to induce autophagy is not important 36 hours post infection.
Perhaps the virus somehow induces excessive ER stress and hence causes autophagy via this way.
Further investigation should shed more light on the different pathways via which autophagy can be
induced by influenza.
The next step in the viral life cycle, replication, and the role of autophagy during this process is under
debate. Autophagy has been indicated to be necessary for replication as well as be dispensable for
this step of the virus infection. As mentioned in the replication section, the strains that have led to
these varying conclusions were different. Two different H1N1 strains and a H9N2 strain were shown
to decrease replication when autophagy was blocked while a H3N2 strain showed the opposite (Zhou
et al. 2009; Gannagé et al. 2009; Matarrese et al. 2011). This difference might partially account for
the different outcome. Furthermore the cells infected were distinct as well. MDCK, human lung
cancer A59 and mucoepidermoid pulmanry carcinoma cells infected with the influenza virus showed
an autophagy-dependent viral replication while mouse embryonic fibroblasts displayed an
autophagy-independent replication. Thus, autophagy dependent replication was seen in epithelial
cells which are the primary cells infected with the influenza virus (Zhou et al. 2009; Gannagé et al.
2009; Mataresse et al. 2011). Yet another difference was the methods via which viral yield was
measured. Gannagé and colleagues made use of an MDCK plaque assay while Zhou and Matarrese
resolved the viral yield using an HA assay. These different assays might also contribute to the
contradicting results as the two papers using the same assay both showed an autophagy-dependent
replication. It is key to approach the issue of autophagy and influenza virus replication by using the
same tools and techniques in order to establish firm conclusions.
At a later time point of the influenza virus infection, there is yet another discrepancy; whether
autophagy is blocked or remains functional throughout the whole infection. M2 was shown to be the
viral protein responsible for blocking autophagosome fusion with lysosomes in A59 cells. This block in
autophagy was observed with two different H1N1 strains as well as the H3N2 strain. M2 is able to
bind the subpopulation of Beclin 1 possibly in complex with UVRAG, which blocks autophagosome
maturation. The exact interaction between M2 and Beclin 1 has not been elucidated. It is know that
the binding site on M2 lies somewhere in the first 60 amino acids either at the ectodomain (1-24) or
at the cytoplasmic amphipatic helix (46-62) (Gannagé et al. 2009; Munz. 2011; Dumit et al. 2012).
Looking into the molecular details of this protein-protein interaction, perhaps using crystallography,
is an elegant next step, which would shed more light on the exact involvement of M2 in autophagy.
Using the same techniques, p62 accumulation and GFP-LC3, autophagy was shown to progress
normally in fibroblast infected with H1N1 (PR8), the same strain used in by Gannagé and coworkers
(Comber et al. 2011). Primary DCs also showed normal autophagy in cells infected with two different
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Figure 14: Autophagy and Influenza infection. The interaction of the influenza virus with autophagy is surrounded by many
discrepancies. Autophagy is induced upon entry of influenza virus into the cell most likely via the mTOR pathway. This has
been shown for both H5N1 and H1N1 strains but was also disproved for H1N1. Two different H1N1 strains were used so
perhaps this might explain the observed difference. Subsequently different reports have suggested that autophagy is
necessary for the replication while others have shown that autophagy is indispensible for viral replication. It is thought that
autophagy induction during early infection leads to cell survival allowing the virus to replicate. Nonetheless during late
infection, when the virus has successfully replicated, it causes apoptosis of the host cell. This is done via pro-apoptotic
proteins (that have not been discussed within this chapter) but also through autophagy. During the H5N1 avian influenza,
the increased in autophagy has been shown to be the cause for the induction of apoptosis. There are also some reports,
however, which indicate that autophagy is blocked through the inhibition of the fusion between autophagosomes and
lysosomes by the viral protein M2. M2 interacts with Beclin 1 and UVRAG thus blocking autophagosome maturation. This
accumulation of autophagosomes eventually leads to cell death. All question marks indicate processes with contradicting
results described in the literature
H1N1 strains and H9N2 (Law et al. 2010). In these three papers, autophagy was hence assessed by
the same techniques consequently the technique employed for the experiments is most likely not
the reason of the observed differences. Perhaps the sole difference of cell type used for the
experiment could explain the different, something that is not unprecedented because this was also
shown for the herpesviruses. To my best knowledge, there are no reports which use different strains
to infect the same cell type and consequently it is difficult to determine whether the strain could also
have an effect on the manipulation of autophagy in a specific cell type, though it seems a likely
scenario.
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Influenza virus infection eventually leads to a high susceptibility of the host cell to death as is seen
both in H5N1 infection, through the induction of autophagy, and in A59 cells infected with H1N1 or
H3N2, via a block of autophagy (Gannagé et al. 2009; Ma et al. 2011). Autophagy is a double-edged
sword, that is a survival mechanism in normal conditions but when it is excessively induced or
manipulated by the virus, it can contribute to the induction of cell death. Influenza virus induces
autophagy upon infection, which is most likely extending the life of the host cell so that the virus can
efficiently replicate (Gannagé et al. 2009). Additionally autophagy has been shown not to contribute
to antigen presentation during influenza virus infection, which reduces the need for the virus to
inhibit autophagy at an early time point (Comber et al. 2011). Inhibition of autophagy during the
H5N1 infections was suggested to limit the pathology of this strain (Ma et al. 2009; Sun et al. 2012).
The use of this approach as a therapy, however, needs a lot more research considering the fact that
inhibiting autophagy in all tissues might lead to many toxic side effects. Additionally, as was shown
by Gannagé and colleagues, inhibition of autophagy raises sensitivity to apoptosis so it might not be
so beneficial for fighting this avian influenza virus. Nonetheless as this infection has a mortality rate
of 50%, perhaps the side effects will not be as severe as the potential outcome of the disease and
hence might be useful anyway.
In conclusion, there is a substantial amount of discrepancies concerning the interaction between
autophagy and the influenza virus infection. The exact role of autophagy will need to be elucidated
both in the process of viral replication as well as in the maturation of autophagosomes occurs during
the infection (Figure 14). These investigations will most likely need to be done in a strain and/or cell
type specific setting as there is clear indication that divergence of strain and cell type might be the
cause of certain discrepancies which exist in the field. Furthermore it is advised to make use of the
same tools and techniques when analyzing certain aspects of the infection.
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6. Conclusions
In this thesis herpes, vaccinia, corona and influenza viruses have been discussed with regard to their
interaction with autophagy. Between these four viruses, the ability of herpesviruses to manipulate
autophagy has been most extensively studied. In contrast, there is no solid evidence linking CoV
infection with autophagy. This latter virus, however, exploits LC3 for its replication thus indicating
that autophagy proteins may also be involved in other processes (Reggiori et al. 2010). A recurring
issue in the previous chapters was contradictory results. Viruses such as herpes and influenza
displayed different effects on autophagy when different strains were used or when different types of
cells were infected (Gannagé et al. 2009; Law et al. 2010; Comber et al. 2011). For example HSV
blocked autophagy in fibroblasts but was unable to do this in DCs (Ramussen et al. 2011; Alexander
et al. 2007). Furthermore, although it seems beneficial for a virus to inhibit autophagy and interfere
with its potential destruction as well as with the presentation of epitopes on the cell surface
recruiting the immune system, some viruses appear to induce autophagy. Even viruses from the
same class such as the alphaherpesviruses behave differently. For example while HSV blocks
autophagy, VZV induces it to expand the host cell’s life span (Cavignac et al. 2010). The four viruses
are different both in biology as well as when it comes to their interaction with autophagy.
Nonetheless, some do share similar characteristics regarding this interaction.
The autophagy protein Beclin 1 is a very popular target when it comes to viruses. Inhibiting this
protein abolishes the formation of autophagosomes. Many of the members of the different classes of
herpes viruses contain a viral protein that targets Beclin 1. For HSV and CMV, ICP34.5 and TSR1,
respectively, are responsible for binding Beclin 1 (Cavignac et al. 2010; Hakki et al. 2006). The gamma
herpes viruses do not contain a homolog or ortholog of these proteins but encode v-Bcl-2 proteins.
These proteins, which contain a BH3 domain, can bind Beclin 1 inhibiting autophagy (Cavignac et al.
2010; Sinha et al. 2008). Although the EBV v-Bcl-2 has not been experimentally proven to bind Beclin
1 and thus to be important in subverting autophagy, it seems likely that it could be functionally
involved in this mechanism (Taylor et al. 2011). The gammaherpesviruses are not the only discussed
viruses that code for v-Bcl-2 proteins; they are also found in vaccinia for example. Like EBV, however,
these proteins have not been studied for their involvement in autophagy (Cooray et al. 2007; Grahan
et al. 2008). Influenza on the other hand targets Beclin 1 via the proton channel protein M2. The
exact interaction between these two proteins is, however, unknown (Gannagé et al. 2009).
Altogether, three out of these four viruses analyzed in this report have developed different strategies
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as well as shared methods to target Beclin 1. This indicates that Beclin 1 is a very potent target for
the inhibition of autophagy.
Apart from Beclin 1, targeting the PKR system is another shared characteristic between these viruses.
They all have been shown to inhibit this pathway, however, this has not always been linked with a
negative regulation of autophagy (Orvedahl et al. 2008; Orvedahl et al. 2007; Cruz et al. 2011). Apart
from initiating autophagy, PKR stimulates host shut off. The main reason for the viruses to target PKR
might solely be to surpass the host shut off. This targeting could however also contribute to the
inhibition of autophagy as was revealed for both the herpes and the vaccinia viruses (Alexander et al.
2007; Myskiw et al. 2011). Nonetheless the presence of additional ways to block autophagy such as
the presence of ICP34.5’s BBD and the formation of the Atg12-Atg3 conjugate respectively suggest
that the contribution via the block of PKR would be minimal.
Autophagy is not only blocked by viruses, some viruses exploit the machinery for their own benefit. It
is a survival mechanism and viruses could use this system to lengthen the host’ life span and thus
allow replication to occur for a longer time. This was shown for VZV (Cavignac et al. 2010).
Additionally in the latent stage of EBV, autophagy is induced by LMP1. This protein uses the
mechanism to regulate its own levels. This process needs to be regulated tightly and a so far
unidentified autophagy inhibitor, such as perhaps the v-Bcl-2 proteins, might play a role in this
regulation (Lee et a., 2008). KSHV, another gammaherpesvirus, encodes the protein RTA that likewise
induces autophagy via an unknown mechanism. This induction also needs to be tightly controlled and
might be balanced by viral proteins such as v-Bcl-2 (Wen et al. 2010). This interplay with the
induction of autophagy for the regulation of the latent stage is not seen in any of the herpes virus
even though they all share the latent characteristic. This might be due to the fact that latency is its
default stage for EBV and KSHV. Although there is no solid evidence linking CoVs to autophagy,
transfection of cells with viral nsp6 did show induction of autophagy. Whether this induction is in fact
also present during a CoV infection, is not known (Cottam et al. 2011). If this would indeed be the
case the reasons behind inducing autophagy are speculative. Perhaps the virus, like VZV, benefits
from lengthening the life span of the host. Another interesting speculation was suggested where
immunomodulatory proteins would be targeted to the autophagosomes by the virus to decrease the
immune response. This would suggest a somewhat chaperone-mediated system and would require
the virus to avoid the autophagy system to prevent its own degradation. Nonetheless if the PKR
antagonist encoded by CoV is able to overcome this induction, CoVs would fall in the category of the
autophagy-blocking viruses.
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The mechanism interacting with autophagy, mainly the inhibitory ones, seem to have developed
different molecules to target the same cellular protein. Nonetheless this does not hold true for all the
viruses covered in this thesis. The vaccinia virus, although also containing PKR inhibitors and v-Bcl-2
proteins, displays a novel way to block autophagy, namely the formation of an Atg12-Atg3 conjugate
(Moloughney et al. 2011). This has, to my knowledge, not been shown in any other virus but it
remains to be seen whether it is typical only for vaccinia. The viral proteins involved in the formation
of this conjugate have not been discovered yet and therefore it has not yet been possible to
determine whether other viruses encode proteins with a similar structure and/or function. This could
be the case since it is a very efficient system to inhibit autophagy hence perhaps other viruses have
either dependently or independently developed the same/a similar system.
In conclusion, the interaction with autophagy seems to be very important for numerous viruses. CoV
is the only virus discussed in this thesis that has not been definitely linked to the modulation of
autophagy. However there are some experimental indications that hint towards an involvement. This
interaction will need to be further studied in order to truly say whether CoVs manipulate the
autophagy system. The other three viruses all show clear links with autophagy, some stimulatory and
others inhibitory. A substantial amount of information has been gathered regarding the interaction
between these three viruses and autophagy. Nonetheless, in order to clarify the whole picture much
more will need to be unveiled. Autophagy and viruses have an undeniable connection nonetheless
many of the individual aspects are awaiting full discovery.
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