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Effect of hepatitis C virus infection on signal transduction adaptor protein: TRAF6

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Effect of hepatitis C virus infection on signal transduction adaptor protein: TRAF6

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EFFECT OF HEPATITIS C VIRUS INFECTION ON
SIGNAL TRANSDUCTION
ADAPTOR PROTEIN: TRAF6

by

Mansi Narula

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)

AUGUST 2013

ii

ACKNOWLEDGEMENTS

First and foremost, I would like to thank my parents and friends for their continuous
support and love. Without their encouragement, I wouldn’t have been able to pursue my
dreams and be what I am today.

I would like to acknowledge my PI Dr. J.-H. James Ou. Thank you for allowing me to be
a part of this lab to work on my MS thesis project and for being a supportive mentor all
these years. I would also like to acknowledge my colleague researcher, Jiyoung Lee, for
the training and guidance she provided me throughout the duration of my time in the lab.
I feel I have grown both as an individual and as a scientist and feel prepared for my future
endeavour. I would like to thank all other lab members for their kind support and
encouragement.

Also, I want to thank all my committee members- Dr. Keigo Machida, Dr. William
Depaolo and Dr. J.-H. James Ou; for their time and consideration. I am glad to have the
honoured faculty members on my committee and for making this endeavour a success.

iii

TABLE OF CONTENTS

1.2 Tumor Necrosis Factor Receptor-Associated Factor 6 (TRAF6) 6
1.2.1 Important Features of TRAF6 6
1.2.2 TRAF6 and Viruses 7
1.2.3 TRAF6 Autoubiquitination 8

Acknowledgements ii
List of Figures iv

Abstract

v

Chapter One: Introduction

1

1.1 Hepatitis C Virus

1
1.1.1 HCV Replication Cycle 2
1.1.2 HCV Genome Organization & Polyprotein Processing 3
1.1.3 HCV Transmission and Epidemiology 4
1.3 Intracellular Protein Degradation Pathways 10
1.3.1 Autophagic Marker Proteins 11
1.3.2 Colocalization of TRAF6 and p62 12
1.3.3 HCV Induced Autophagy 13
1.4 HCV and Apoptosis 14
1.4.1 Programmed Cell Death 14
1.4.2 Apoptosis: Caspase Pathways 14
1.4.3 Acute HCV Infection Induces Apoptosis of Cultured
Hepatoma Cells

15
Chapter Two: Materials and Methods 17

Chapter Three: Results

22
3.1 Reduction in TRAF6 protein expression upon HCV Infection 22
3.2 TRAF6 protein degradation pathways 24
3.3 Effect of HCV Infection on TRAF6 transcription 28
3.4 Apoptosis –mediated depletion of TRAF6 during HCV Infection 29
3.5 Anti-Apoptotic Effect of TRAF6 31
Chapter Four: Discussion

33
References 37

iv

LIST OF FIGURES

Figure 1: Structure of HCV Particle
Figure 2: HCV Replication Cycle
Figure 3: HCV Genome Structure and Functions of Viral Proteins
Figure 4: Signaling mechanism by single-stranded RNA viruses
Figure 5: Interaction of p62 and LC3 in the process of Autophagy
Figure 6: p62/SQSTM1 Protein Structure
Figure 7: HCV Induced Autophagy
Figure 8: HCV Infection leads to reduction in TRAF6 protein expression

Figure 9: Induction of Autophagy by HCV Infection
Figure 10: Effect of HCV Induced Autophagy on TRAF6 degradation
Figure 11: Effect of Proteosomal activity on TRAF6 degradation
Figure 12: Effect of HCV Infection on TRAF6 transcription
Figure 13: Caspase-8 mediated TRAF6 depletion during HCV Infection
Figure 14: Effect of TRAF6 knockdown on HCV Induced Apoptosis and Pathogenesis

v

ABSTRACT

Hepatitis C virus (HCV) is a major cause of chronic liver disease and evasion of the host
immune system by HCV plays a key role in its pathogenesis. However, the interaction
between HCV and hepatocyte innate antiviral defense system is not well
understood. Tumor Necrosis Factor Receptor-Associated Factor 6 (TRAF6) is known to
be an important adaptor protein that mediates signaling not only from members of TNF
receptor superfamily, but also from the members of the Toll/IL-1 family. Here we report
the reduction in TRAF6 protein upon infection of a Human hepatoma cell line, Huh7.5,
by an adaptive mutant of JFH-1 strain (genotype 2a) of HCV. Inhibition of the two major
protein degradation systems, autophagy-lysosome and the proteosome pathway, did not
contribute in the recovery of total TRAF6 while inhibition of the HCV-induced apoptosis
led to an increased TRAF6 protein level. Further, we observed siRNA silencing of
TRAF6 resulted in enhanced cleavage of procaspase-8, indicating increase in apoptotic
activity upon TRAF6 knockdown. Taken together, our studies indicated that TRAF6
reduction due to HCV infection contributes in the induction of apoptosis by HCV in
hepatocytes. These results provide important information for understanding HCV
pathogenesis and raised the possibility of targeting this signaling protein to treat HCV
patients.

1

Chapter One: INTRODUCTION
1.1 Hepatitis C Virus
Hepatitis C virus (HCV) is a major cause of severe liver disease, with more than 170
million people infected worldwide (Sy and Jamal 2006). HCV, first identified in 1989, is
a member of the Flaviviridae family and belongs to the genus of Hepacivirus. It is a
small, enveloped virus containing a positive single-stranded RNA genome which is 9.6
kb in length (Houghton 2009). The complete genome of HCV itself acts as the transcript
encoding a polyprotein, which is slightly more than 3000 amino acids in length, plus a
small protein named F protein that uses an alternative reading frame. Hence, the HCV
virion is composed of the positive RNA genome, the core capsid, a lipid envelope, and
the two major viral envelope proteins, E1 and E2.

Figure 1: Structure of HCV Particle. The simplified model of a hepatitis C virus
particle consists of a core of genetic material (RNA), surrounded by
an icosahedral protective capsid protein, encased in a lipid envelope of cellular origin.
Two viral envelope glycoproteins, E1 and E2, are embedded in the lipid envelope.

2

1.1.1 HCV Replication Cycle
The life cycle of HCV begins by binding of the virus particle to the host cell surface
receptor followed by receptor-mediated endocytosis to internalize the virus into the cell.
In the host cell cytoplasm, the viral genome is uncoated and released from its capsid shell
in order to begin replication. Both cellular proteins and viral proteins facilitate the
progression of HCV through its replication cycle. The single stranded positive viral RNA
genome itself acts as the template to translate into HCV polyprotein by utilizing host
cellular components. This polyprotein is cleaved to produce four structural proteins
(C, E1, E2 and p7 ) and six nonstructural proteins (NS2, NS3, NS4A, NS4B,
NS5A and NS5B). The non-structural proteins are required for the replication of HCV
RNA, which then forms the genome of the new HCV virion particle. The positive-sense
HCV RNA is encapsidated within the structural protein shell and is known as the
nucleocapsid. The nucleocapsid gets its envelope by budding into the lumen of the ER.
Finally, infectious virions are transported through the Golgi to the plasma membrane and
released to infect new cells. The HCV life cycle is entirely cytoplasmic and thus, the viral
RNA does not need to enter the host cell’s nucleus at anytime during its replication cycle
(Tan, Pause et al. 2002).

3

Figure 2: HCV Replication Cycle. The HCV life cycle is divided into several steps in
the host cell, namely a. Cell Attachment b. Entry by Fusion c. Uncoating d. Translation
and Polyprotein Processing e. RNA Replication f. Viral Assembly and Envelope
Formation g. Transport and Virion Release. (Source: Asselah and Marcellin 2011)

1.1.2 HCV Genome Organization & Polyprotein Processing
The 5′ UTR region of HCV is uncapped and together with a portion of the core-coding
domain, folds into a complex secondary RNA structure, known as the Internal Ribosome
Entry Site (IRES) that directly binds to the host’s ribosomal subunits and cellular factors
to mediate subsequent translation into HCV polyprotein. The polyprotein is initially
encoded as a single Open Reading Frame (ORF) but later on, cleaved by cellular and
viral proteases into 10 different proteins namely, Core, E1, E2, p7, NS2, NS3, NS4A,
NS4B, NS5A and NS5B. The structural proteins are encoded by the N-terminal part of

4

the ORF, whereas the remaining portion of the ORF codes for the nonstructural proteins
(Chevaliez and Pawlotsky 2006).

Figure 3: HCV Genome Structure and Functions of Viral Proteins. HCV genome is
marked by IRES at the 5’end and the HCV polyprotein cleaved by cellular and viral
proteases into 10 different protein subunits. (Source: Bartenschlager, Penin et al. 2011).

1.1.3 HCV Transmission and Epidemiology
HCV has a narrow host specificity and tissue tropism. Nevertheless, it has a high
chronicity rate in humans (about 85%, depending on the age at infection). HCV
transmission is exclusively through direct blood-to-blood contacts between humans. This
persistent infection can be treated with medication, the standard therapy being a
combination of pegylated interferon and ribavirin. Overall, 50–80% of people treated are
cured. Those who develop cirrhosis or liver cancer may require a liver transplant though
the virus usually recurs even after transplantation. No vaccine against hepatitis C is
available till date. It is estimated that 130–200 million people, or ~3% of the world's
population, are living with chronic hepatitis C. About 3–4 million people are infected per
year, and more than 350,000 people die yearly from hepatitis C related diseases. Rates
have increased substantially in the twentieth century due to a combination of intravenous

5

drug use and intravenous medication or poorly sterilized medical equipment and thus
require strong actions to be taken in near future.

The existence of quasispecies population of HCV is the main reason behind its
chronicity. It is due to the fact that the HCV RNA dependent RNA polymerase (RdRp)
has no proofreading mechanism to correct errors during genomic replication, so mistakes
made by RdRp get permanently incorporated into new HCV RNA as mutations and result
in closely related but genetically distinct variants of HCV (Sumpter, Loo et al. 2005).

Though HCV infection induces a number of immune responses in the host, it fails to
prevent chronicity in most cases and does not confer protection against reinfection neither
with the same strain nor its variant as demonstrated in the chimpanzee model (Farci,
Bukh et al. 1997). Nevertheless, production of type I IFNs (IFN-β and distinct IFN-α
subtypes) and proinflammatory cytokines by virus-infected cells is the central event in
their antiviral immune response. HCV induced IFN and cytokine gene transcription is
mediated through distinct signaling pathways.
In the current study, we aim to determine such viral-host interactions by targeting the
adaptor proteins that mediate signaling pathways. TRAF6 is one such signal transduction
adaptor protein.

6

1.2 Tumor Necrosis Factor Receptor-Associated Factor 6 (TRAF6)
TRAF6 is one of the 7 members of the family of TRAF proteins associated with
mediating the signal transduction from members of the TNF receptor superfamily. All
TRAF proteins share a C-terminal homology region termed as the TRAF domain that is
capable of binding to the cytoplasmic domain of receptors and to other TRAF proteins. In
addition, TRAFs, except TRAF1, have RING and zinc finger motifs that are important for
signaling downstream events.

1.2.1 Important Features of TRAF6
TRAF6 protein, consisting of 522 amino acids, is a unique intra-cellular signal transducer
that mediates signaling not only from the members of the TNF receptor superfamily, but
also from the members of the Toll/IL-1 family. TRAF6 acts as an E3 ubiquitin ligase that
functions by ligating Lysine-63 linked poly-ubiquitin chains to itself and other signal
transducers. In the cell, it is located in the cytoplasm, cell cortex, nucleus and lipid
droplet of the specific tissue types including heart, brain, placenta, lung, liver, skeletal
muscle, kidney and pancreas. Stimulation of the cell with IL-1-beta or TGF-beta induces
oligomerization of TRAF6 molecules, followed by auto-ubiquitination which involves
the enzymes, UBE2N and UBE2V1, and leads to TRAF6 activation. Signal amplification
by TRAF6 involves the activation of multiple kinase cascades including the IkB kinase
(IKK), MAP kinase and Src-family tyrosine kinases (Wu and Arron 2003).

7

1.2.2 TRAF6 and Viruses
TRAF6 plays a well-documented role in the cell survival and inflammation by mediating
induction of pro-inflammatory cytokines such as IL-17 and type I IFNs. It mediates
signal transduction of several ssRNA, dsRNA and dsDNA viruses by activating the NF-
κB, AP-1 and IRF pathways. One of the important functions of TRAF6 is its ability to
activate IkB kinase (IKK) in response to proinflammatory cytokines thereby acting as a
signal tranducer in the NF-kB pathway. Upon viral infection, the innate immune system
recognizes viral nucleic acids and induces the production of proinflammatory cytokines
and Type I interferons (IFNs). Toll-like receptor 7 (TLR7) and TLR9 detect viral RNA
and DNA, respectively, in endosomal compartments and recruit myleiod differentiation
primary response gene 88 (MyD88) onto the cytoplasmic TIR domains of the receptor
complex. As Interleukin-1 receptor associated kinase (IRAK) is recruited to the receptor
complex in the cell membrane through the death domain interaction between MyD88 and
IRAK, TRAF6 transiently moves to the membrane via its interaction with IRAK, which
simultaneously induces binding of Transforming Growth Factor- Beta (TGFβ) activated
kinase 1 (TAK1) binding protein 1 and 2 (TAB1, TAB2) as well as TAK1 to TRAF6.
The IRAK-TRAF6-TAB1-TAB2-TAK1 complex then immediately detaches from the
membrane and translocates into the cytosol where the activated TRAF6 induces
ubiquitination of multiple downstream molecules such as TAB2 and IkB kinase γ (IKKγ).
This leads to the activation of a ubiquitous transcription factor, NF-kB (p50/p65), due to
phosphorylation and proteasomal degradation of IkB subunits followed by translocation
of activated NF-kB in the nucleus. Thus, TRAF6 mediates antiviral responses triggered

8

by viral DNA and RNA thereby acting as a key molecule in innate and adaptive immune
responses against viral infection (Konno, Yamamoto et al. 2009).

Figure 4: Signaling mechanism by single-stranded RNA viruses. The figure
represents the role of TRAF6 in mediating NF-kB signaling pathway activation in
response to the detection of ssRNA viral genome.

1.2.3 TRAF6 Autoubiquitination
TRAF6 is unique from other TRAFs in that it utilizes a distinct interaction motif, which
is found in its upstream activators. As such, TRAF6 has been implicated in directing the
signals from representative members of a diverse array of receptor families. TRAF6
activation leads to downstream activation of PI3K, the mitogen-activated protein kinase
(MAPK) cascade, and the transcription factor families NF-kB, NFAT, and IRF.
INFLAMMATION

9

Numerous biochemical and genetic studies have revealed mechanistic insights into
TRAF6 utilization of ubiquitination to propagate diverse signals. To date, the most
prominent model holds that upon activation via TRAF6 homo-oligimerization, the RING
finger ubiquitin E3 ligase domain complexes with a K63-specific E2 conjugating enzyme
(Ubc13/Uev1a) to mediate attachment of non-degradative K63-linked ubiquitin chains to
TRAF6 substrates, specifically TRAF6 itself (Deng, Wang et al. 2000). Furthermore, a
research group has identified a single critical Ub acceptor site residue (K124) in TRAF6
that is required for TRAF6 auto-ubiquitination. Mutation of this site abolishes TRAF6-
mediated NEMO ubiquitination, TAK1 and IKK activation and NF-κB activation
(Lamothe, Besse et al. 2007). Given that TRAF6 needs to be ubiquitinated in order to be
activated, it appears most likely that deubiquitination inactivates TRAF6.

There are seven different ways that a protein can be polyubiquitinated. Dependent on
which of its lysine (K) residues is linked to the ubiquitin monomer, the chains are called
K6, K11, K27, K29, K33, K48 or K63 ubiquitin chains. Classical K48 ubiquitin chains
were originally identified as the canonical signal to target proteins for proteasomal
degradation. In contrast, non-classical linkage types such as K63, K11, M1-linked chains
or single ubiquitin moieties (monoubiquitination) were thought to signal mainly for non-
proteolytical functions. These chain types are involved in controlling several processes
such as receptor transport, DNA repair and signaling. However, recent reports have
demonstrated that regulatory proteins of the cell cycle modified with K11 chains are also
targeted for the proteasomal pathway, whereas K63 chains can target substrates for
degradation via autophagy (Olzmann and Chin 2008).

10

1.3 Intracellular Protein Degradation Pathways
The Ubiquitin-Proteasome System (UPS) and the Autophagy-Lysosome Pathway are the
two main routes for eukaryotic intracellular protein clearance:
Macroautophagy (autophagy) involves the sequestration of cytoplasm by double-layered
membranes to form vesicles called autophagosomes, which ultimately fuse with
lysosomes to form autophagolysosomes in which their contents are degraded. The
formation of autophagosomes is regulated by the two interconnected conjugation
systems, Atg12-Atg5 and the conjugation of Atg8 to the lipid phosphatidylethanolamine
(Korolchuk, Menzies et al. 2009).

Proteasomes are barrel-shaped, multiprotein complexes that predominantly degrade
short-lived and misfolded endoplasmic reticulum proteins. Typically, proteins are
targeted for proteasomal degradation after being covalently modified with ubiquitin. The
conjugation of ubiquitin to the substrate protein employs three types of enzymes: E1
(ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin
ligase). Often, the ubiquitin itself forms a substrate for further rounds of ubiquitination,
resulting in the formation of a polyubiquitin chain. These polyubiquitin chains serve as a
recognition signal that allows substrates to be escorted to the proteasome via a set of
chaperone proteins (Korolchuk, Menzies et al. 2009).

11

1.3.1 Autophagic Marker Proteins
The formation of autophagosomes and the autophagic activity is marked by the existence
or absence of some important proteins:
Light Chain 3 (LC3), a mammalian homolog of yeast Atg8, is a reliable marker of
autophagosome formation, indicated by tracking the conversion of LC3-I to LC3-II.
During autophagy, the cytoplasmic form (LC3-I) is processed and recruited to the
autophagosomes, where phosphatidylethanolamine-conjugated form of LC3-I,
called LC3-II is generated by site - specific proteolysis and lapidated near to the C-
terminus. The hallmark of autophagic induction is thus the formation of cellular
autophagosome punctae containing LC3-II, while autophagic activity is measured
biochemically as the amount of LC3-II that accumulates in the absence or presence of
lysosomal activity.

Another marker protein, p62/sequestosome 1 (SQSTM1), is a ubiquitin-binding scaffold
protein that colocalizes with ubiquitinated protein aggregates in many neurodegenerative
diseases and proteinopathies of the liver. The protein is able to polymerize via an N-
terminal PB1 domain and can interact with ubiquitinated proteins via the C-terminal
UBA domain. Also, p62/SQSTM1 binds directly to LC3 and GABARAP family proteins
via a specific sequence motif. The protein is itself degraded by autophagy and may serve
to link ubiquitinated proteins to the autophagic machinery to enable their degradation in
the lysosome. Since p62 accumulates when autophagy is inhibited, and decreased levels
can be observed when autophagy is induced, p62 may be used as a marker to study
autophagic flux (Bjørkøy, Lamark et al. 2009).

12

Figure 5: Interaction of p62 and LC3 in the process of Autophagy. Autophagic
degradation of polyubiquitinated substrates is accomplished by binding of p62 to the
substrates followed by attachment of LC3 to the substrate-p62 complex through the LIR
domain on p62.

1.3.2 Colocalization of TRAF6 and p62
Interestingly, p62 contains several structural motifs which suggest that it might
participate in the formation of multimeric signaling complexes. These domains include an
N-terminal interaction domain (PB1) that binds to aPKC, a ZZ finger, a binding site for
TRAF6, two PEST sequences, and the ubiquitin binding domain.

Figure 6: p62/SQSTM1 Protein Structure (Source: Komatsu and Ichimura 2010)

The C-terminal UBA domain of p62 can bind both Lys 48-linked and Lys 63-linked Ub
chains but has a higher affinity for Lys 63 chains (Seibenhener, Babu et al. 2004). Also,

13

since p62 possesses a binding site for TRAF6, it was suspected that TRAF6 may become
trapped along with p62 and ubiquitin wherein, p62 serves as a scaffold to regulate K-63
polyubiquitination via interaction with TRAF6. Interestingly, loss of p62 interaction
affects TRAF6 K-63 activation as well. Cells devoid of p62 exhibit low basal TRAF6
polyubiquitination and expression of p62 enhances the autoubiquitination of TRAF6.
This has also been confirmed using p62
−/−
mice in which the absence of p62 reduced
activation of TRAF6. Thus, deletion constructs that remove different p62 domains reveal
that each is necessary for activation and regulation of TRAF6 or NF-kB (Wooten, Geetha
et al. 2005).

1.3.3 HCV Induced Autophagy
As previously mentioned, autophagy is a key metabolic process that can eliminate
unwanted, damaged and worn out constituents of the cells. However, some viruses use
this process to modulate their replication in host cells. Some of the best examples of such
viruses are HBV and HCV. Incomplete induction of autophagy by HCV via the UPR
pathway in Huh7.5 cells has been previously identified (Sir, Chen et al. 2008). In these
experimental studies, the authors showed that HCV induces the formation
autophagosomes but prevents the maturation of autophagosomes to autolysosomes so that
the contents of the autophagosome are not degraded and HCV can use them for its
replication.

14

Figure 7: HCV Induced Autophagy. HCV induces autophagosome formation but
blocks the fusion of autophagosome and lysosome during early stages of infection.

1.4 HCV and Apoptosis
1.4.1 Programmed Cell Death
Apoptosis, also known as programmed cell death, is a physiological process that occurs
in cells during development and normal cellular processes to maintain cellular
homeostasis. The useless, unwanted, or damaged cells die during apoptotic process. It has
been reported that cells altered beyond repair by normal mechanisms or cells that have
completed their programmed biological function begin the apoptosis process. Apoptosis
can be initiated by cell signals and induced via the stimulation of several different cell
surface receptors (e.g., Fas) resulting in caspase activation. The activation of caspases
(cysteinyl-aspartate-specific proteinases), a family of intracellular cysteine proteases,
play an important role in the initiation and execution of apoptosis and sequential
apoptotic processes. Poly-ADP-ribose-polymerase (PARP), a 116-kDa protein that binds
specifically at DNA strand breaks, acts as a substrate for certain caspases (e.g., caspases 3
and 7). The activated caspases cleave PARP into two fragments during early stages of
apoptosis. Thus, the cleavage of PARP serves as an early marker of apoptosis (Sarkar and
Li 2006).

15

1.4.2 Apoptosis: Caspase Pathways
Caspases, cysteine-aspartic proteases, are responsible for deliberate disassembly of the
cell into apoptotic bodies during apoptosis. They are present as inactive pro-enzymes that
are activated by proteolytic cleavage. Caspases 8, 9 and 3 play pivotal roles in apoptotic
pathways. Caspase 8 initiates disassembly in response to extracellular apoptosis-inducing
ligands and is activated in a complex associated with the cytoplasmic death domain of
many cell surface receptors for the ligands (Extrinsic Pathway). Caspase 9 activates
disassembly in response to agents that trigger the release of cytochrome c from
mitochondria and is activated when complexed with apoptotic protease activating factor 1
(APAF-1) and extra-mitochondrial cytochrome c (Intrinsic Pathway). Caspase 3 appears
to amplify caspase 8 and caspase 9 initiation signals into full-fledged commitment to
disassembly. Caspase 8 and caspase 9 activate caspase 3 by proteolytic cleavage and
caspase 3 then cleaves vital cellular proteins like PARP or other caspases.

1.4.3 Acute HCV Infection Induces Apoptosis of Cultured
Hepatoma Cells.
Cleaved caspase-3 previously demonstrated activation of a terminal pathway involved in
apoptosis that appears to cause the cytopathic effect in cultures of cells infected with
HCVcc. The initial presence of activated caspase-3 also coincided with peak levels of
intracellular viral RNA suggesting a causative link between the level of HCV replication
and cell death (Walters, Syder et al. 2009).
Data about the role of single HCV proteins, either in cultured cells or transgenic animal
models, however, are contradictory, as both pro- and anti-apoptotic effects have been

16

observed. Nevertheless, apoptosis induction upon HCV infection may critically
contribute to liver damage, while inhibition of apoptosis may result in HCV persistence
and development of hepatocellular carcinoma. Therefore, we do not actually understand
whether apoptosis is linked to the clearance or persistence of HCV infection.
Nevertheless, previous studies have also shown that caspase-dependent cleavage of
signaling proteins during apoptosis act as a turn-off mechanism for anti-apoptotic signals
(Widmann, Gibson et al. 1998). Therefore, the current study tries to explore the
relationship between HCV-induced apoptosis and TRAF6, an important signaling protein
that is known to activate antiviral immune responses against many viruses such as
Epstein-Barr Virus (Saito, Murata et al. 2013), encephalomyocarditis virus (Konno,
Yamamoto et al. 2009), etc.

17

Chapter Two: MATERIALS AND METHODS

2.1 Cell Lines and Cell Culture
Human Hepatoma (Huh7.5) cells, a cell line derivative of Huh7 that act as a stable
system for infection, were used for all experiments in this study. They were maintained in
Dulbecco's modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine
serum (FBS) and 1% nonessential amino acids (Invitrogen, CA).The cells were incubated
at optimum temperature 37°C with 5% CO
2.

2.2 Reagents and Antibodies
TRAF6, Ubiquitin, PARP and GAPDH antibodies were purchased from Santa Cruz
Biotechnology. The rabbit anti-core antibody was produced in rabbits using the HCV
core protein expressed in Escherichia coli (Lo, Masiarz et al. 1995). The rabbit anti-actin
antibody and a control rabbit IgG were purchased from Abcam. The mouse anti-caspase-
8 and rabbit anti-p62 antibodies were purchased Cell Signaling Technology; anti-LC3
antibody was from MBL International Corporation; and anti-rat tubulin antibody was
obtained from AbD Serotec. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit
and goat anti-mouse secondary antibodies were purchased from Abcam. Goat anti- rabbit
FITC was purchased from Thermo Scientific (Pierce). The mounting medium DAPI was
purchased from Vector Laboratories. The Autophagic inhibitor, Bafilomycin A1 and
specific caspase-8 inhibitor, Z-IETD-FMK, were purchased from Sigma, proteosome

18

inhibitor MG132 from Calbiochem; and goat anti-rabbit IgG supramagnetic particles
from Qiagen.

2.3 Production of HCVcc and Titration
JFH-1 (HCV genotype 2a), derived from a Japanese patient with fulminant hepatitis, was
used for infection purposes (Kato, Furusaka et al. 2001). The wild type virus was
produced in the laboratory by transfecting Huh-7.5 cells with in vitro transcribed full-
length HCV RNA. To do this, JFH-1 plasmids were linearized with XbaI, and 1µg of the
linearized DNA was used to generate in vitro-transcribed RNA using Megascript kit
(Ambion) according to the manufacturer's instructions. HCV RNA was transfected into
Huh-7.5 cells by electroporation. Supernatants from JFH-1 RNA-transfected cells were
harvested 3 days posttransfection, titrated, and pooled to generate laboratory stocks. HCV
genotype 2a thus derived was serially passaged by infection of naïve Huh-7.5 cells
multiple times to generate an adaptive mutant of JFH-1 which produces a high level of
infectious virus particles (Cai, Zhang et al. 2005). The virus was titrated using
Immunofluorescence staining of the JFH-1 positive cells. Huh7.5 cells were seeded in a
8-chamber plate at 40,000 cells/chamber. The next day, each well was infected with HCV
after serially diluting 10-fold in complete growth medium in duplicates. 24 hours post
infection; the cells were immunostained for core as follows: The cells were rinsed with
PBS and fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. After
washing with PBS, the cells were incubated with rabbit anti-core antibody at a dilution of
1:200 for 1 hour at 37°C in PBS containing 0.5% (w/v) saponin, 1% (w/v) bovine serum
albumin (BSA), 0.2% (w/v), and 0.02% (w/v) sodium azide. Primary antibody binding

19

was detected with fluorescein-conjugated goat anti-rabbit IgG. Nuclei were
counterstained with DAPI. The HCV core-positive cells were counted under the
microscope for titration. Non-infected cells used as negative controls were processed
identically. The viral titer is expressed as focus-forming units per milliliter of supernatant
(ffu/ml).

2.4 HCV Infection & Cell harvest
For protein and RNA detection, 70-80% confluent Huh7.5 cells in 10cm cell culture
plates were infected with HCV at 37°C for 3 h. An infection titre of 2-4 virion/cell was
usually used. Three hours post infection, the infected cells were incubated with fresh
high glucose-DMEM containing 10% FBS for 3 days. For protein lysate preparation,
cells were rinsed with PBS and lysed with the Radio Immune Precipitation Assay buffer
(50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1%
Triton X-100, 1X Protease Inhibitor Cocktail was freshly added before lysis). After
sonication, cell lysates were subjected to centrifugation to remove cell debris and protein
was estimated using Bradford Reagent (Bio-Rad). An aliquot of the supernatant
containing 30-100 μg of the protein (depending on the abundance of protein and the
strength of antibody detection) was used for gel electrophoresis. Samples were denatured
in Laemmeli buffer by boiling at 100°C for 5 minutes before loading onto the gel.

20

2.5 Imunoprecipitation, SDS-PAGE and Immunoblotting
For Immunoprecipitation, cell lysates were prepared as previously described. 500-750 µg
of total protein was incubated with 4-5 µg of primary antibody and incubated overnight
shaking at 4°C. Next day, 40-50 µl of supramagnetic particles were added to the
immunoprecipitated protein and incubated further for 1hr at 4°C.The protein-particle
complex was then washed thrice with RIPA (containing PIC) by magnetic separation
followed by one last wash with high-salt buffer (containing HEPES buffer, NaCl and
Triton X-100). The bead complex was denatured by adding Laemmeli buffer and boiling
the sample at 100°C for 5 minutes. Immunoprecipitated protein samples and total lysates
were then loaded onto a 10-15% SDS-PAGE gel (depending on the protein to be
detected). After the semi-wet protein transfer on polyvinylidene fluoride (PVDF)
membrane, the membrane was blocked with 5% non-fat milk in TBS containing 1%
Tween 20 (TBST) for 1hr and incubated with the primary antibody overnight at 4°C.
Next day, the membrane was washed three times with TBST buffer, incubated with the
HRP-conjugated secondary antibody for another 1 h. After further washes with TBST,
the membrane was exposed to chemiluminescent substrates (Pierce) and the image was
captured using the LAS-4000 imaging system (FujiFilm).

2.6 RNA Isolation, cDNA synthesis and semi quantitative RT- PCR
RNA was extracted from cultured cells using the Trizol reagent (Invitrogen) following
the manufacturer’s protocol. For semi-quantitative Reverse Transcription PCR (sqRT-
PCR), 2.5µg of total RNA was reverse transcribed with First Strand cDNA synthesis
using SuperscriptII RT reaction and Oligo d(T)
16
primers (Applied Biosystems), and 2µl

21

of the newly synthesised cDNA was amplified using AmpliTaq Gold DNA Polymerase
(Applied Biosystems). The primers designed for TRAF6
5'-GGATCCAGCCAGTCTGAAAG-3'(sense) and
5'-TGGGGACAATCCATAAGAGC-3' (antisense) and for β-actin control
5'-AGAGCTACGAGCTGCCTGAC-3'(sense) and
5'-AAAGCCATGCCAATCTCATC-3'(antisense) were used for the PCR reaction. PCR
cycling conditions were as follows: denaturation at 94°C for 1 min, annealing at 51.5°C
for 1 min and extension at 72°C for 30 secs. Thirty cycles were carried out for the
amplification of TRAF6 and β-actin. The PCR products were subjected to electrophoresis
in 1% agarose gel and visualized by ethidium bromide staining.

2.7 siRNA Transfection
Pooled siRNAs against TRAF6 (Santa Cruz Biotechnology) and Negative Control
(Ambion) were transfected with Lipofectamine 2000 (Invitrogen) into Huh7/Huh7.5 cells
following the manufacturer's instructions. Briefly, 5 × 10
5
cells seeded in a
60-mm dish were transfected with 8 μl of siRNAs (100 μM each) and then infected 4-6
hrs later with HCVcc for 48hrs followed by lysis for immunoblotting.

22

Chapter Three: RESULTS
3.1 Reduction in TRAF6 protein expression upon HCV Infection

To determine the effect of HCV Infection on TRAF6, Huh7.5 cells were infected with a
highly infectious variant of HCV JFH1. TRAF6 expression was determined at different
time points until day 3 post infection. As shown in Fig. 8A, HCV caused a gradual
reduction in the overall TRAF6 protein level with a significant decrease at day-3 post
infection. HCV core protein expression was visible as early as 24 hours after infection
and GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), an enzyme involved in
glucose metabolism, was used as the loading control.

It has previously been shown several times that TRAF6 is polyubiquitinated via a unique
chain formation at the Lysine residue 63 (K63) in the ubiquitin polypeptide sequence
(Lamothe, Besse et al. 2007). This kind of polyubiquitinated form of TRAF6 is the
activated form and functions to further activate all other downstream signaling events.
Given that TRAF6 needs to be ubiquitinated in order to be activated, it appears most
likely that deubiquitination inactivates TRAF6. Thus, in order to analyze the activity of
TRAF6 in HCV infected cells, the polyubiquitinated-TRAF6 protein expression was
determined. Briefly, Huh7.5 cells were infected for 3 days followed by
immunoprecipitation of TRAF6 protein and immunoblotting with anti-ubiquitin antibody.
As revealed in Fig. 8B, HCV infected cells expressed less polyubiquitinated-TRAF6
protein when compared to the basal level expression in non-infected cells. This indicated
reduced activation of TRAF6 in HCV infected cells.

23

A. h.p.i 0 24 48 72 (hours)
HCV - + + +

B. h.p.i 0 72 (hours)
HCV - +

Figure 8: HCV Infection leads to reduction in TRAF6 protein expression.
A. Huh7.5 cells were infected with HCV for 0 (no infection), 24, 48 and 72 hours and
analyzed for total TRAF6 protein expression (Mol. Weight: 60kDa) by immunoblotting.
Also shown is the HCV core protein expression. B. Cells were infected with HCV for 0
and 72 hours followed by immunoprecipitation of TRAF6 with anti-TRAF6 antibody and
immunoblot with anti-ubiquitin to analyze the polyubiquitinated-TRAF6 protein. (IgG
produces a heavy chain band at around 55kDa). GAPDH served as a control in both cases
A and B. Both experiments were repeated atleast three times to confirm the results.

IP: TRAF6
IB: Ubiquitin
TRAF6
GAPDH
HCV core
HCV core
GAPDH
IgG
heavy chain
TRAF6-Ub

24

3.2 TRAF6 protein degradation pathways

As we know the two pathways for intracellular protein degradation are the
Autophagosome-Lysosome pathway and the Ubiquitin Proteosome System (UPS)
(Korolchuk, Mansilla et al. 2009), we tried to analyze the ability of each of these systems
to degrade the TRAF6 protein amount post HCV infection. To test this possibility, the
previously established induction of autophagy by HCV infection in hepatocytes (Ait-
Goughoulte, Kanda et al. 2008), was also confirmed in Huh7 and Huh7.5 cells. This was
done by taking into account the change from LC3-I to LC3-II form and degradation of
p62 upon HCV infection, as shown in Fig. 9A and 9B, respectively. These two proteins
serve as useful markers to study autophagic responses.

A. h.p.i 0 24 48 72 (hrs.)
HCV - + + +

Figure 9: Induction of Autophagy by HCV Infection. A. Huh 7 cells were infected for
0, 24, 48 and 72 hours with HCV and analyzed for the conversion of LC3-I to LC3-II that
indicates formation of autophagosomes. At 72 hours, LC3-II is assumed to recycle back
to LC3-I. Tubulin served as the loading control. (Source: Jiyoung Lee’s data). B. Huh7.5
cells were similarly infected as in A and analyzed for the degradation of p62 protein by
HCV autophagic activity. GAPDH, in this case, was used as a loading control. Both
results have been confirmed by performing the experiments several times.

B. h.p.i 0 24 48 72 (hrs.)
HCV - + + +

LC3-I

LC3-II
p62
HCV
core
GAPDH
HCV core
Tubulin

25

Thus, to investigate whether degradation in total TRAF6 and its polyubiquitinated form is
due to HCV induced autophagy, Huh7.5 cells were infected for 3 days during which they
were treated with Bafilomycin A1, a late-phase inhibitor of autophagy. A concentration
of 100nM of Bafilomycin A1, added 16 hours before lysing the cells, was sufficient to
inhibit the autophagic activity as corroborated by the accumulation of p62 protein. A
similar treatment of infected and uninfected cells with dimethyl sulfoxide solvent
(DMSO) served as the control. Though the autophagic-lysosome system degrades
numerous long-lived proteins, it had no effect on TRAF6 degradation in this case, as
shown in Fig. 10A.To further find out the effect of autophagy on degradation of
polyubiquitinated TRAF6, TRAF6 protein was immunoprecipitated from cell lysates of
Bafilomycin A1 treated infected cells (drug treatment under similar conditions as in the
previous experiment), and immunoblotted with polyubiquitin antibody. Surprisingly, it
led to the recovery of polyubiquitinated TRAF6 in HCV infected cells, as observed in
Fig. 10B.

A. h.p.i 0 72 0 72 (hrs.)
HCV - + - +
BafA1 - - + +

TRAF6
Actin
p62

HCV core

26

B. h.p.i 0 72 0 72
HCV - + - +
BafA1 - - + +

Figure 10: Effect of HCV Induced Autophagy on TRAF6 Degradation. A. Huh7.5
cells were infected for 0 (no infection) and 72 hours with BafilomycinA1 (100nM conc.)
treatment to inhibit basal level and induced autophagic activity, given 56 hours post-
infection and the level of total TRAF6 protein analyzed. p62 served as an autophagic
marker while actin served as the loading control. B. The cells were infected and treated
similar to A. and analyzed for polyubiquitinated-TRAF6 protein by immunoprecipitating
the total cell lysate with anti-TRAF6 antibody followed by immunoblotting with anti-
ubiquitin antibody. Both A & B were each repeated thrice to confirm the trend of results.

Another well- known pathway of protein degradation is through the proteosomal system.
To test the possibility of TRAF6 degradation by this pathway, HCV infected and
uninfected cells were treated with MG132, a proteosome inhibitor, added at 10µM final
concentration 6 hours prior to lysis of 72 hour infected cell population. DMSO was still
used as a loading control. Interestingly, there was no change in the expression of total
TRAF6 protein in infected cells with or without the addition of MG132 as shown in Fig.
11A.This shows no effect of the proteosome activity on non-modified TRAF6. In
IP: TRAF6
IB: Ubiquitin
TRAF6-Ub
IgG
Heavy chain

27

contrast, polyubiquitinated TRAF6 was still affected by the UPS as inhibition with
MG132 was able to recover ubiquitinated TRAF6 when compared to the infected cells
with the loading control, as in Fig. 11B.

A. h.p.i 0 72 0 72 (hrs)
HCV - + - +
MG132 - - + +

Figure 11: Effect of Proteosomal activity on TRAF6 Degradation. A. Huh7.5 cells
were infected for 0 (no infection) and 72 hours during which they were treated with the
proteosome inhibitor, MG132 (10µM conc.), 66 hours post-infection to analyze the level
of total TRAF6 protein. Actin served as the loading control. B. The cells were infected
and treated similar to as in A. and analyzed for polyubiquitinated-TRAF6 protein by
immunoprecipitating the total cell lysate with anti-TRAF6 antibody followed by
immunoblotting with anti- ubiquitin antibody. The results in both cases were repeated
thrice to confirm the pattern.

TRAF6
HCV
core
Actin
B. h.p.i 0 72 0 72 (hrs.)

HCV - + - +
MG132 - - + +

TRAF6-Ub
IgG
heavy chain

IP: TRAF6
IB: Ubiquitin

28

3.3 Effect of HCV Infection on TRAF6 transcription
Since there was no effect of autophagic and proteosomal inhibition observed on non-
modified TRAF6 protein, we tried to determine the change in its mRNA transcription
upon HCV Infection. Semi-quantitative Reverse Transcription (RT) - PCR was used to
do this particular experiment. Briefly, total RNA was extracted from the uninfected and
3-day infected cells, which was then reverse transcribed using Oligo d(T)
16
primers and
Reverse Transcriptase enzyme, followed by RT- PCR using specific primer sequence for
TRAF6 under the conditions mentioned earlier. Actin served as the loading control.
Surprisingly, there was no significant difference in the mRNA transcription of TRAF6 in
uninfected vs. HCV infected Huh7.5 cells, as revealed in Fig. 12A. To confirm these
results, TRAF6 protein expression was analyzed simultaneously and under similar
experimental conditions as for TRAF6 mRNA experiment. The results show reduction in
non-modified TRAF6 protein in HCV infected cells than that in uninfected cells,
regardless of no change in TRAF6 mRNA expression. Therefore, we conclude that HCV
infection does not affect TRAF6 at transcriptional level but causes a reduction in TRAF6
protein, post-translationally, as shown in Fig. 12B.

29

A. h.p.i 0 72 (hrs.)
HCV - +

Figure 12: Effect of HCV Infection on TRAF6 transcription. A. Huh7.5 cells were
infected for 0 (non-infected) and 72 hours with HCV and analyzed for TRAF6
transcription by sqRT-PCR. Actin was used as the loading control and the PCR products
were analyzed on 1% Agarose Gel. B. Cells infected under exactly similar conditions as
in A, analyzed for TRAF6 protein by immunoblot to compare with A. Both, A & B were
repeated thrice simultaneously.

3.4 Apoptosis –mediated depletion of TRAF6 during HCV Infection
Previous studies have demonstrated activation of apoptosis upon HCV infection in
hepatocytes contributes to HCV pathogenesis (Kalkeri, Khalap et al. 2001). In the current
study, we have shown HCV induced apoptosis by activation of the extrinsic pathway
leading to procaspase-8 cleavage, as shown in Fig. 13A. Appearance of cleaved caspase-
8 was observed at 48 hours and further increased at around 72 hours post infection (data
not shown). Data shown in Figure 13A represents the apoptotic activity in cells infected
with HCV stock for 72 hours. During apoptosis, poly (ADP-ribose) polymerase (PARP)
nuclear protein that normally functions in DNA damage detection and repair is also
cleaved, thereby serving as an apoptotic marker protein. This cleavage effectively
neutralizes the ability of PARP to participate in DNA repair, and contributes to a cell’s
commitment to undergo apoptosis.
B. h.p.i 0 72 (hrs.)
HCV - +

TRAF6
Actin HCV core
GAPDH
TRAF6

30

Caspases are known to cleave many intracellular proteins including signaling proteins
such as Akt-1, RasGAP and Raf-1 (Widmann, Gibson et al. 1998). To test the possibility
of HCV induced apoptosis to cause depletion of TRAF6 in order to inhibit its signaling
function, we infected Huh7.5 cells with HCV followed by treatment with caspase-8
inhibitor, Z-IETD-FMK. The inhibitor is specific in inhibiting procaspase-8 cleavage to
its active caspase-8 form. Briefly, the experiment was performed by infecting the cells
with HCV and adding the inhibitor 3 hours post infection at a concentration of 20µM.
The cells were then grown for 3 days in the presence of the inhibitor at the end of which
they were analyzed on a 10% SDS-PAGE protein gel for the change in total TRAF6
protein. Addition of DMSO to infected and uninfected cells served as a control drug.
Interestingly, the results revealed recovery of total TRAF6 in the presence of caspase-8
inhibitor in infected cells. Effective treatment of the cells with the inhibitor is explained
by the blockage of procaspase-8 cleavage as shown in Fig. 13B. Hence, we concluded
that the depletion of TRAF6 observed post HCV infection was mediated by the action of
active caspase-8.
A. h.p.i 0 72 (hrs.)
HCV - +

PARP

Cleaved
PARP fragment
Procaspase-8

Cleaved
Caspase-8
HCV core
GAPDH
B. h.p.i 0 72 72 (hrs.)
HCV - + +
Z-IETD-FMK - - +

TRAF6
Procaspase-8
Cleaved
Caspase-8
HCV Core
GAPDH

31

Figure 13: Caspase-8 mediated TRAF6 depletion during HCV Infection. A. HCV infection
induced apoptosis in Huh7.5 cells on day-3 post infection marked by cleavage of full-length
PARP into PARP fragment and cleavage of procaspase-8 into active caspase-8 fragments. B.
Cells were infected in the same way as in A. during which they were treated with caspase-8
inhibitor (Z-IETD-FMK). The total lysate was analyzed for total TRAF6 protein expression.
GAPDH was used as the loading control in case A and B. Both A & B have been repeated more
than three times.

3.5 Anti-Apoptotic Effect of TRAF6

The observation of the role of active form caspase-8, induced by HCV, in depletion of
TRAF6 gave rise to some of the obvious questions as to what effect, if any, does TRAF6
have on HCV pathogenesis and HCV-induced apoptotic activity. Previous articles on the
relationship between TRAF6 and apoptosis show positive regulation of caspase-8
dependent apoptosis by TRAF6 (He, Wu et al. 2006). To investigate this possibility in
case of HCV infection, siRNA knockdown of TRAF6 was done in HCV infected cells
and the protein expression of cleaved caspase-8 fragments was analyzed. Knockdown of
TRAF6 expression was done by a lab colleague, Jiyoung Lee, as follows: Huh7 cells
(from which Huh7.5 cells are derived) were infected with HCV 24 hours prior to siRNA
transfection and the cells were harvested 3 days post infection, when the knockdown
efficiency is assumed to be maximum. We then used the samples to analyze the change in
cleavage of procaspase-8. Surprisingly, TRAF6 knockdown in infected cells showed
enhanced caspase-8 activity as compared to the negative control siRNA transfected cells.
It reveals the role of TRAF6 as an anti-apoptotic signaling protein suppressing the
apoptotic effect of HCV as shown in Fig. 14. Also, increase in the expression of HCV
core protein upon TRAF6 knockdown is noteworthy in this experiment. It implies the
role of TRAF6 in partially blocking HCV pathogenesis and replication, though there is

32

need for further experimentation to verify this particular observation. To conclude, there
seems a strong possibility of caspase-mediated degradation of TRAF6 induced by HCV,
in order to establish efficient pathogenesis in hepatocytes.

h.p.i 0 72 72 (hrs.)
HCV - + +
siRNA NC NC TRAF6

Figure 14: Effect of TRAF6 knockdown on HCV Induced Apoptosis and
Pathogenesis. Huh7 cells were infected with HCV and treated with Negative Control
(NC) and TRAF6 siRNA 1-day post infection and analyzed for the cleavage in
procaspase-8 and HCV core protein expression 3 days post infection. Immunoblot of
TRAF6 shows successful siRNA knockdown and GAPDH was used as the loading
control. This experiment was repeated twice to confirm the result pattern.

Cleaved caspase-8
fragment
HCV core
GAPDH
TRAF6

33

Chapter Four: DISCUSSION

TRAF6 is known to play a well-documented role in cell survival and inflammation by
mediating induction of pro-inflammatory cytokines such as IL-17 and type I IFNs. It
mediates signal transduction of several ssRNA, dsRNA and dsDNA viruses by activating
the NF-κB, AP-1 and IRF pathways. Therefore, in the present study, we demonstrated the
effect of HCV, a ssRNA virus, on TRAF6 by analyzing its transcription and protein
expression in presence of HCV infection. Surprisingly, we observed gradual decrease in
total TRAF6 protein over time upon HCV infection with significant reduction after 3
days (Fig.8A).To make sure this observation was not due to the conventional effect of
conversion of total protein to its polyubiquitinated, active form, we demonstrated the
change in polyubiquitinated TRAF6 post infection. Unlike other viruses that activate
TRAF6 signaling, HCV led to reduction in the active form of TRAF6 in a similar fashion
as the reduction in total TRAF6 suggesting the role of HCV in negatively regulating the
protein expression (Fig.8B).
To further identify the biological pathway of TRAF6 degradation, we analyzed the effect
of HCV induced autophagy. Autophagy is one of the important mechanisms that degrade
long-lived as well as polyubiquitinated proteins that cannot be degraded by the
proteosome. Hence, blocking of autophagic activity results in accumulation of such
proteins. Interestingly, as we observed, HCV induced autophagy had no effect on total
TRAF6 depletion (Fig. 10A) but resulted in the recovery of its ubiquitinated form (Fig
10B). A probable explanation for the degradation of polyubiquitinated TRAF6 by
autophagy comes from the fact that p62 contains a TRAF6 binding domain as well as

34

ubiquitin binding domain (Wooten, Geetha et al. 2005) and hence, degradation of
ubiquitinated TRAF6 could be the result of colocalization with p62 followed by HCV
induced autophagic action on p62- ubiquitinated TRAF6 complex.
UPS is the other most common pathway for degradation of short-lived and ubiquitinated
proteins. Analyzing the contribution of this system in degradation of TRAF6, similar
results as for autophagic degradation were obtained since inhibition of the proteosome
had no effect on total TRAF6 expression (Fig. 11A) but lead to recovery in
polyubiquitinated TRAF6 (Fig. 11B). This can be explained by the inability of the system
to degrade non-ubiquitinated form of this protein while the depletion of its ubiquitinated
form may be affected by the presence of a deubiquitinase called A20 in the cytoplasm
that is known to deubiquitinate K-63 polyubiquitinated substrates and at the same time
contains a C-terminal domain that acts as a ubiquitin ligase attaching K-48 polyubiquitin
chains to the same substrate, targeting them for proteosomal degradation (Lin, Chung et
al. 2008). According to the previous research, polyubiquitinated TRAF6 could also serve
as one of the substrates for A20 and thereby, undergo proteosomal degradation. We have
initiated the research on HCV induced ubiquitinated TRAF6 degradation; it remains to be
seen, however, the type of Lysine polyubiquitinated residues on TRAF6 and the exact
mechanism of autophagy and UPS in mediating this degradation.
The inability to restore total TRAF6 protein expression by inhibiting protein degradation
systems led to the study of transcriptional changes in TRAF6 mRNA upon HCV
infection. Surprisingly, there was no significant difference in the transcription of TRAF6
in uninfected and infected cell culture (Fig.12A) which indicated that the changes are due
to some posttranslational mechanism instead of transcriptional change.

35

We then concentrated on yet another pathway for certain protein degradation as a normal
mechanism called apoptosis, accompanied by the activation of cleaving caspase proteins.
Since HCV is known to induce apoptosis in hepatocytes (Fig. 13A), there was a
possibility that the action of caspases may result in cleavage of total TRAF6 protein.
Interestingly, in HCV infected cells, inhibition of caspase-8 activity by blocking the
cleavage of procaspase-8 resulted in recovery of TRAF6 protein as compared to when no
inhibitor was present (Fig. 13B). This further opened more avenues in finding out the role
of TRAF6 in HCV induced apoptosis. Because TRAF6 knockdown resulted in enhanced
caspase-8 cleavage (Fig. 14), it is highly likely that TRAF6 plays an important role in
negatively regulating HCV induced apoptosis. This is further supported by the
observation that there was increased expression of HCV core protein after TRAF6
knockdown in infected cells. This and all other findings strongly support the importance
of TRAF6 in controlling HCV pathogenesis. Further, it will be interesting to determine
the binding sites of TRAF6 on caspase-8 and produce mutants unable to bind to these
sites, to confirm the direct colocalization of both proteins during HCV induced apoptosis.
Also, it is important to determine the role of TRAF6 in targeting HCV replication and
production of proinflammatory cytokines such as IRFs and type I IFNs in response to
HCV infection. Moreover, it would be relevant to analyze the NF-κB activity and
production of IAPs (Inhibitors of Apoptotic Proteins) in response to TRAF6 knockdown
in HCV-infected cells in order to find out the indirect effect of TRAF6 on HCV-induced
apoptosis. Lastly, finding out the type of lysine polyubiquitinated residues on TRAF6 and
the exact mechanism of autophagy and UPS in mediating this degradation would further
add credibility to the story of TRAF6 and HCV Infection. Another important aspect of

36

consideration would be the statistical analysis of the data found to further verify our
results.
In conclusion, our results demonstrated that HCV suppresses the protein expression
levels of TRAF6 in order to enhance the apoptotic activity of the infected host cells. This
in turn, plays an important role in establishing HCV pathogenesis. The current study
raises an opportunity to target this relationship to treat HCV infected patients.

37

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Abstract (if available)

Abstract Hepatitis C virus (HCV) is a major cause of chronic liver disease and evasion of the host immune system by HCV plays a key role in its pathogenesis. However, the interaction between HCV and hepatocyte innate antiviral defense system is not well understood. Tumor Necrosis Factor Receptor-Associated Factor 6 (TRAF6) is known to be an important adaptor protein that mediates signaling not only from members of TNF receptor superfamily, but also from the members of the Toll/IL-1 family. Here we report the reduction in TRAF6 protein upon infection of a Human hepatoma cell line, Huh7.5, by an adaptive mutant of JFH-1 strain (genotype 2a) of HCV. Inhibition of the two major protein degradation systems, autophagy-lysosome and the proteosome pathway, did not contribute in the recovery of total TRAF6 while inhibition of the HCV-induced apoptosis led to an increased TRAF6 protein level. Further, we observed siRNA silencing of TRAF6 resulted in enhanced cleavage of procaspase-8, indicating increase in apoptotic activity upon TRAF6 knockdown. Taken together, our studies indicated that TRAF6 reduction due to HCV infection contributes in the induction of apoptosis by HCV in hepatocytes. These results provide important information for understanding HCV pathogenesis and raised the possibility of targeting this signaling protein to treat HCV patients.

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Asset Metadata

Creator Narula, Mansi (author)

Core Title Effect of hepatitis C virus infection on signal transduction adaptor protein: TRAF6

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Publication Date 06/27/2013

Defense Date 04/22/2013

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag apoptosis,autophagy,HCV,NF-kB,OAI-PMH Harvest,polyubiquitination,TRAF6

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Ou, J.-H. James (committee chair), DePaolo, R. William (committee member), Machida, Keigo (committee member)

Creator Email mansinar@usc.edu,narula.mansi5@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-280450

Unique identifier UC11293772

Identifier etd-NarulaMans-1710.pdf (filename),usctheses-c3-280450 (legacy record id)

Legacy Identifier etd-NarulaMans-1710-1.pdf

Dmrecord 280450

Document Type Thesis

Format application/pdf (imt)

Rights Narula, Mansi

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA