The molecular studies of HCV RNA replication and translation

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Content
THE MOLECULAR STUDIES
OF
HCV RNA REPLICATION AND
TRANSLATION

by

Helene Minyi Liu

__________________________________________________________________

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)

May 2009

Copyright 2009 Helene Minyi Liu

ii
DEDICATION

This dissertation is dedicated to

my dearly loved family and friends.

iii

ACKNOWLEDGMENTS

To become a scientist in the life science field has been my dream since I
was 12 years old. I once forgot this dream, and had thought that it would never be
possible. Without these people’s favors, I could never finish my dissertation, let
alone the dream of the little girl.
I would like to first thank my mentor, Dr. Michael Lai, for his endless
support during my Ph.D study. I thank him for training me for logical thoughts,
for inspiring me great ideas as a virologist, and for guiding me the way to become
a great scientist.
Thanks to Dr. James Ou, who has been just as my second mentor. His
encouragement has walked me through the most difficult days in my Ph.D study.
Thanks to my closest colleague, Dr. Keigo Machida, who has been a great
teacher in the laboratory. It was my great pleasure to work with such a brilliant
scientist.
I appreciate Dr. Hideki Aizaki and Dr. Jessie Yi-ja Li, who motivated me to
study viral replication and taught me all the basic techniques in a virology lab. I
also thank to Dr. Keum S. Choi, who initiated the SYNCRIP project, and Dr.
Grace Guann-yi Yu, who has always been a great senior person to me.
I especially appreciate Michelle MacVeigh and Ernesto Barron, two experts
who also trained me well for confocal and electron microscopy studies.
Donna Sir and Wenling Chen, I thank them for their friendship and support.

iv
I truly appreciate my Father, who first told me not to give up my dream and to
be great. Thank him for encouraging me to study abroad.
My dearest husband, Peter Shih, I thank you for your eternal support, in every
way you can. There is so much more than what I can express in words.
Thank you, my Lord, for giving me such a great chance to make my dream
come true and for sending me my beloved family and friends.

v
TABLE OF CONTENTS
DEDICATION........................................................................................................ ii

ACKNOWLEDGMENTS ..................................................................................... iii

LIST OF FIGURES ............................................................................................... vi

ABSTRACT......................................................................................................... viii

CHAPTER 1 – Introduction to Hepatitis C virus ....................................................1
1.1 Classification of HCV .......................................................2
1.2 Genome Structure ..............................................................2
1.3 Viral proteins .....................................................................5
1.4 HCV life cycle .................................................................12
1.5 Host factors involved in HCV replication .......................15
1.6 Systems for study HCV life cycle and pathogenesis .......18

Table 1-1. Possible receptors for HCV.................................13

CHAPTER 2 – SYNCRIP (Synaptotagmin-Binding, Cytoplasmic
RNA-Interacting Protein) is a host factor involved in
Hepatitis C virus RNA replication...............................................20
2.1 Abstract ...........................................................................20
2.2 Background and Rationale...............................................21
2.3 Material and Method........................................................22
2.4 Results..............................................................................27
2.5 Discussion........................................................................41

CHAPTER 3 – The translation of HCV RNA is colocalized with and
dependent on RNA replication.....................................................45
3.1 Abstract............................................................................45
3.2 Background and Rationale...............................................46
3.3 Material and Method........................................................47
3.4 Results..............................................................................53
3.5 Discussion........................................................................76

CHAPTER 4 – Conclusion and Future Directions ................................................81

BIBLIOGRAPHY..................................................................................................86

vi
LIST OF FIGURES

CHAPTER 1

Figure 1-1. The organization of HCV Genome ..........................................4
Figure 1-2. A simplified illustration of HCV life cycle............................12
Figure 1-3. Immunofluorescence staining of PTB and BrUTP-labeled
newly-synthesized HCV replicon RNA.................................17
Figure 1-4. Illustrations of HCV full-length and subgenomic replicon
RNA. ......................................................................................18

CHAPTER 2

Figure 2-1. Membrane flotation assay showed relocalization of
SYNCRIP to DRM in HVC replicon cells ............................28
Figure 2-2. SYNCRIP collocalization with de novo-synthesized HCV
RNA in HCV replicon cell.....................................................31
Figure 2-3. SYNCRIP knockdown by siRNA in HuhHyg HCV
replicon cells ..........................................................................33
Figure 2-4. SYNCRIP knockdown by siRNA in HuhN1b replicon cells 35
Figure 2-5. Deficiency of full-length HCV replication in SYNCRIP
knockdown Huh7 cells...........................................................36
Figure 2-6. siRNA-knockdown of SYNCRIP inhibited HCV
replication activity in vitro replication assay.........................39
Figure 2-7. Immunodepletion of SYNCRIP inhibited HCV replication
activity in in vitro replication assay.......................................41

CHAPTER 3

Figure 3-1. The translocation of newly-synthesized HCV RNA..............54
Figure 3-2. The translocation of newly-synthesized HCV RNA from
ER fraction to Golgi fractions................................................55
Figure 3-3. Nocodazole treatment blocks the translocation of newly-
synthesized replicon RNA .....................................................56
Figure 3-4. Increase in replicon RNA translation in nocodazole-
treated HCV replicon cells.....................................................57
Figure 3-5. Double-staining of newly-synthesized replicon RNA and
peptide....................................................................................59
Figure 3-6. Live images of double-staining of newly-synthesized
replicon RNA and peptide .....................................................61
Figure 3-7. Immunogold staining and electronmicroscopy ......................64

vii
Figure 3-8. DRM supports in vitro translation of replicon RNA..............66
Figure 3-9. The distribution of eIF3 .........................................................67
Figure 3-10. .Benzothiadiazine treatment in Huh-N1b replicon cells .......70
Figure 3-11. .HCV RNA translation is dependent on the RNA
transcription .........................................................................71
Figure 3-12. .Deficiency of translation in HCV replication-deficient
replicon and full-length RNA .............................................74
Figure 3-13. .Translation of the replicating and non-replication
replicon RNAs .....................................................................75

CHAPTER 4

Figure 4-1. The hypothetical model of coupled replication/translation
of HCV RNA .........................................................................85

viii
ABSTRACT

Hepatitis C virus RNA replication requires not only the viral replicase but
also the host cytoskeletons, membrane structures, and cellular factors. Several
hnRNPs, such as polypyrimidin-tract-binding protein (PTB) and La autoantigen
were found to regulate both HCV RNA replication and translation. Another
hnRNP, SYNCRIP (synaptotamin-Binding, Cytoplasmic RNA-Interacting Protein,
or NSAP-1), was found to regulate HCV-IRES dependent translation. In my first
part of dissertation, we identified SYNCRIP as a positive regulator of HCV RNA
replication.
With a growing number of “dual-function” proteins which are able to
regulate HCV RNA translation and replication, it is interesting to investigate how
the two important steps in HCV life cycle are temporally and spatially regulated.
Therefore, we initiated a series of experiments to examine if HCV RNA
translation is dependent on its replication. The colocalization of newly-
synthesized viral RNA and peptide were detected in live cell and under
electronmicroscopy. It is also found that the replication-deficient replicon or full-
length HCV RNA has deficiency in translation as well. Our findings together
suggest that the replication and translation of HCV RNA are coupled and occur in
the same cellular compartments termed “replicasome”.

1

Chapter 1
Introduction to Hepatitis C virus

Hepatitis C virus (HCV) was first discovered twenty years ago (Choo et al.,
1989). Besides Hepatitis B virus, HCV is the most important etiological agents of
blood-borne viral hepatitis in the world. It has been estimated that 3% of the
worldwide population, 170 million people, are infected with HCV (Lauer and
Walker, 2001). HCV transient infection induces acute hepatitis, which may cause
no symptoms or may result in hepatitis accompanied by jaundice, but fulminant
liver failure is rare. However, 70-80% of patients develop the persistent infection
of HCV causes chronic liver disease leading to chronic hepatitis, liver cirrhosis,
and hepatocellular carcinoma. The high frequency of established persistent HCV
infection in immunocompetent adults indicates that HCV interferes with many
aspects of both innate and adaptive immune responses of the host. The currently
available treatments for HCV cause unpleasant side effects, and there is no
existing valid vaccine for HCV. Therefore, the understanding of the viral life
cycle and the mechanism of pathogenesis of HCV will be very helpful for the
development of effective therapeutic approach to stop disease progress.
1.1 Classification
The genome of HCV was identified and classified as the sole member of the
Hepacivirus genus of Flaviviridae family, which includes the classical

2
flaviviruses (for example, yellow fever, dengue and tick-borne encephalitis
viruses), the animal pestiviruses (for example, bovine viral diarrhoea virus) and
GB viruses A (GBV-A), GBV-B and GBV-C.
HCV isolates are classified into 6 genotypes and several subtypes. The
nucleotide sequences differ by 30–35% between genotypes (Simmonds et al.,
2005). The response to interferon-alpha treatment is batter in patients infected
with genotype 2 or 3 than those infected with genotype 1. Within an HCV
genotype, several subtypes (designated a, b, c and so on) can be defined that differ
in their nucleotide sequence by 20–25%. Without a proof-reading ability of HCV
viral polymerase, NS5B, mutations arising from RNA replication are easily
detected. The term “quasispecies” refers to the genetic heterogeneity of the
population of HCV genomes that coexist in an infected individual.

1.2 Genome structure
HCV is an enveloped RNA virus with a single, positive-stranded RNA
around 9.6 kb in length (Reed and Rice, 2000). As a member of Flaviviridae,
HCV has a positive-strand RNA genome that is composed of a 5'-non-translated
region (NTR), which includes an internal ribosome entry site (IRES), one open
reading frame (ORF), and a 3'-NTR. With an IRES structure at the 5’ of HCV
RNA, the RNA molecule can bind to 40S complex directly, minimizing the
requirement of the initiation factors (Mottola et al., 2002). The 5’- and 3’-NTR
are highly-structured RNA sequence with functions in regulation of HCV RNA
replication and translation (Back et al., 2002; Dutkiewicz and Ciesiolka, 2005;

3
Fang and Moyer, 2000; Mizutani et al., 2000; Reigadas et al., 2001; Smith et al.,
1995; Varaklioti et al., 1998). Flanked by the 5’- and 3’- NTR, the single ORF of
HCV viral genome encodes a large viral polyprotein with 10 HCV proteins
organized in the order: NH
2
-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-
COOH (Grakoui et al., 1993; Lohmann et al., 1999).

4

5
1.3 Viral proteins
The single polyprotein translated from HCV RNA undergoes a complex series
of co- and post-translational cleavage events catalyzed by both host and viral
proteinases to yield the individual HCV structural (C, E1, E2 and p7) and
nonstructural (NS2, NS3, NS4A, NS4B, NS5A and NS5B) proteins. An
additional HCV protein, F (for frameshift protein) or ARFP (for alternate reading
frame protein), generated by an overlapping reading frame in the core protein
coding sequence, has been proposed.
1.3.1 Structure proteins
Core protein, the nucleocapsid protein of HCV, is located at the very N-
terminus of the polyprotein. The core protein has two distinct domains: an N-
terminal two-third domain of highly charged amino acids and a C-terminal
domain (aa. 120-191) of hydrophobic residues (Lai and Ware, 2000; Nolandt et
al., 1997). The N-terminal domain is responsible for the interaction with nucleic
acid and the formation of core homodimer or multimers. Therefore, this domain is
likely responsible for the constitution of the viral capsid.
Interestingly, this HCV core protein has been suggested not only to participate in
viral assembly but also to possess multiple regulatory functions inside the cells.
These functions include activation of transcription factors, suppression of
translation, and activation of signal transduction pathways. Moreover, HCV core
protein has been shown to regulate lipid metabolism, cellular proliferation, and
apoptosis. However, a significant controversy remains regarding various functions
of this protein. This review summarizes the synthesis and the multiple biological

6
functions of HCV core protein and discusses controversies surrounding its many
functions.
E1 and E2 are the viral envelope glycoproteins. It has been suggested that
E1 and E2 interact to form heterodimers, which have been proposed as a
functional subunit of HCV virions (Dubuisson et al., 1994; Ralston et al., 1993;
Yi et al., 1997). The heterodimer formation has been shown to occur within ER,
and it depends on a prolonged association with calnexin, a chaperone protein
(Dubuisson and Rice, 1996). HCV E1 protein interacts with the core protein
(Smith et al., 1995), and this interaction is important for virus assembly. It is
believed that E2 protein contains the receptor-binding domains (McCaffrey et al.,
2007). E2 protein consists of three hypervariable regions (HVR1, HVR2, and
HVR3) at the N-terminus (Troesch et al., 2006; Yagnik et al., 2000). The
variation was assumed to be caused by random mutation and selection of mutants
capable of escaping from neutralizing antibodies produced in the host (Kaplan et
al., 2003).
Recently, several genotypes of HCV are found to be lymphotropic (Kondo
et al., 2007; Sansonno et al., 2007). Such lymphotropic HCV strain specifically
infects lymphocyte but not hepatocyte, which is the common host for other HCV
strains. By chemira virus studies, it is suggested that swapping E1 and E2 region
between the lyphotropic and hepatotropic strains of HCV can change the tropism
(Machida et. al, unpublished data). These studies indicate that the envelope
proteins of HCV are not only critical for virion-receptor interaction but also
important for tropism determination.

7
p7 protein was first shown to form hexamers as a viroporin, which
mediates cation permeability across membranes (Griffin et al., 2003; Pavlovic et
al., 2003). Structure predictions suggest that p7 comprises two
transmembrane(TM) helices connected via a short cytoplasmic loop (Carrere-
Kremer et al., 2002). Subgenomic HCV replicons do not contain p7,
demonstrating that it is not necessary for RNA replication. It has been recently
reported that HCV relies on p7 function for efficient assembly and release of
infectious progeny virions from liver cells in a genotype specific manner (Jones et
al., 2007; Sakai et al., 2003; Steinmann et al., 2007), suggesting that p7 is an
important virulence factor that may modulate fitness and, in turn, virus
persistence and pathogenesis.
1.3.2 Nonstructural proteins
Nonstructural proteins of HCV are also termed as “replicase” (Lai et al., 2003).
The first nonstructural protein, which is closely associated with the structural
proteins, is NS2. NS2 is a transmembrane protein with at least four
transmembrane domains (Santolini et al., 1995; Yamaga and Ou, 2002). The C-
terminus region of NS2 and the N-terminus of NS3 have been shown to be a
NS2/NS3 metalloprotease (Kiiver et al., 2006; Welbourn and Pause, 2007). The
NS2/NS3 metalloprotease activity is stimulated by Zinc ions (Zn
2+
) and can be
inhibited by EDTA (Pieroni et al., 1997). The NS2/NS3 metalloprotease is
responsible for the autocatalytic cis-cleavage of NS2/NS3 junction for NS3
maturation, which is essential for viral replication (He et al., 2008; Reed et al.,
1995). It has been shown that the C3 junction of transmembrane domain (TMD) 1

8
and TMD2 of NS2 is the most susceptible region for chimera virus construction
(Pietschmann et al., 2006).
NS3 protein has been characterized as a serine protease at its N-terminus,
which is required for cis-cleavage of NS3/NS4A junction and the trans-cleavage
of NS4A/NS4B, NS4B/NS5A, and NS5A/NS5B junctions (Bartenschlager et al.,
1994; D'Souza et al., 1994; Lohmann et al., 1996). Besides cleaving polypeptide
for the nonstructural protein maturation, NS3 protease is also found to cleave
several host targets, such as Cardif, an adapter protein in the RIG-I pathway (Foy
et al., 2005; Meylan et al., 2005). Such cleavage may result in deficiency of IRF-3
and IRF-7 response, which are important innate immune responses downstream of
the RIG-I signaling pathway. The C-terminus of NS3 has nucleoside triphosphate
(NTPase)/helicase activity (Tai et al., 1996; Tai et al., 2001), which may be
important in regulating the HCV RNA translation and replication (Jennings et al.,
2008; Wardell et al., 1999). NS3 has also been shown to be associated with actin
filament and microtubule, and the association between NS3 and the filaments can
mediate the interaction between HCV replication complexes and the host
cytoskeleton (Lai et al., 2008b). Recently, several studies have suggested that
beside HCV core protein, NS3 also has oncogenic potential and inhibitory effects
on DNA repair (Budhu et al., 2007; He et al., 2007; Lai et al., 2008a; Zemel et al.,
2000). How NS3 affects and alters the cellular signal pathways is a current topic
that has been under investigation.
NS4A is a cofactor for NS3 (Failla et al., 1994; Hahm et al., 1995; Hong et al.,
1996; Tomei et al., 1996). NS4A is required for the cis-cleavage of NS3/NS4A

9
junction for NS3 maturation (Lin and Kim, 1999; Tai et al., 2001); this cleavage
can only occur in cis. X-ray crystallography revealed that the central domain of
NS4A forms a strand that contributes to the formation of an eight-stranded barrel
with the amino-terminal domain of NS3, stabilizing NS3 to the ER or and ER-like
modified compartment (Brass et al., 2002; Mottola et al., 2002).
NS4B is a very hydrophobic intergral membrane protein (Grakoui et al., 1993;
Lin et al., 1994). It has been shown that NS4B, expressed alone or from the full-
length HCV genome, can induce membranous web in the cytoplasm (Egger et al.,
2002; Konan et al., 2003). All of the HCV proteins, including core, E1, E2, NS3,
NS4A, NS4B, NS5A, and NS5B were found colocalized on the membranous web
structure, indicating that the membranous web structure may be the site for the
formation of and HCV replication complex (Egger et al., 2002). The N-terminal
amphipathic helix in NS4B was also reported to mediate membrane association
and subcellular localization of the replication complex (Elazar et al., 2004).
Palmitoylation of NS4B has been identified at the C-terminal Cysteines and is
critical for the polymerization of NS4B (Moradpour et al., 2004). Such lipid
modification may be important for the membranous web and replication complex
formation and therefore affects the replication of HCV RNA.
NS5A is a phosphoprotein which is preferentially modified at serine residues
(Neddermann, 2009; Tanji et al., 1995; Yi et al., 1997). The hypophosphorylated
form of NS5A is detected as a 56-kDa protein, and the hyperphosphorylated form
of NS5A migrates at 58-kDa. NS5A phosphoryaltion has been observed in the
absence of other viral proteins, suggesting autophosphorylation or

10
phosphorylation by cellular kinases of NS5A (Kwong et al., 1999; Vyas et al.,
2003). NS5A has vital roles in HCV RNA replication. A membrane-anchoring
region was mapped to the N-terminal 30 a.a. of NS5A which form a highly
conserved amphipathic α-helix (Brass et al., 2002). A later study demonstrated
that the N-terminal amphipathic helix is not only necessary and sufficient for
membrane localization, but also important for HCV replication since mutations
disrupting helix formation impaired HCV replication (Elazar et al., 2003). NS5A
can also bind to NS5B and mediate the anchorage of NS5B RNA-dependent RNA
polymerase (RdRp) to the membrane structures (Shirota et al., 2002). However,
NS5A is so far the only NS protein which can be supplied in trans for HCV RNA
replication (Appel et al., 2005). Besides the important functions in RNA
replication of NS5A, NS5A can directly interact with and inhibit PKR, a protein
kinase involved in the cellular antiviral and antiproliferative response (Gale et al.,
1998; Wu et al., 2008). The direct binding between NS5A and the interferon
sensitivity-determining region (ISDR) suggests that NS5A may conter resistance
to the antiviral effects of interferon (Gale et al., 1998). It is believed that both
NS5A and E2 proteins can inhibit PKR activity, which then allows HCV
replication in the presence of an IFN-induced antiviral response (Gale et al., 1998;
Wu et al., 2008). We have found that NS5A expression can induce the expression
of TLR4. LPS, which is a ligand of TLR4, can stimulate the downstream pathway,
leading to the expression of beta interferon (IFN- β) and interleukin-6 (IL-6)
(Machida et al., 2006), and further leading to Nanog expression and oncogenesis

11
(Machida et al., 2008b), suggesting a role of NS5A in HCV-induced
hepatocellular carcinoma.
NS5B is an RNA-dependent RNA polymerase (RdRp), which is
responsible for both positive and negative strand synthesis for HCV replication
(Ago et al., 1999; Kao et al., 2000; Lohmann et al., 1997). NS5B shares the
common characteristics with other RdRp in the presence of the polymerase GDD
motif and the Magnesium ion (Mg
2+
) binding motif (Poch et al., 1989; Yamashita
et al., 1998). The purified recombinant NS5B polymerase, however, does not have
template specificity for RNA-dependent RNA synthesis (Yamashita et al., 1998;
Zhong et al., 2000). Therefore, additional viral or cellular factors may be required
for the template specificity of RNA replication in vivo.

1.4 HCV life cycle
HCV particles present in clinical samples have been partly characterized.
Enveloped virions are sensitive to detergent and to chloroform, with a diameter of
about 50 nm. Robust in vitro HCV cell culture was not available until the year of
2006. Scientists had to use HCV replicon construct and pseudo particle system to
study the replication of HCV RNA and the viral attachment to the receptor
molecule. Many of the important steps of HCV life cycle were revealed by these
systems, such as the discovery of one of the most critical receptor of HCV, CD81
(Petracca et al., 2000; Pileri et al., 1998), and various of host factors which are
required for HCV RNA replication (Ali et al., 2002; Krieger et al., 2001;
Lohmann et al., 1999).

12

13
A robust replicating HCV 2a strain was cloned from a patient with fluminant
hepatitis C, named as JFH1 (Wakita et al., 2005). Since then, the studies of HCV
life cycle have been made a great progress. The first step of HCV infection is the
viron attachment to the receptor molecule. So far several hepatotropic and
lymphotropic receptors have been identified, including CD-81, Claudin-1, SR-B1,
LDL receptor, and CD-86 (Bartosch et al., 2003; Germi et al., 2002; Heo et al.,
2006; Masciopinto et al., 2001; Pileri et al., 1998; Tan et al., 2003)(Summarized
in Table 1-1).

Biological Function Expressing tissue Entery step Tropism
CD-81
Tetraspanins, regulating
the fusion of
mononuclear phagocytes
ubiquitous
early binding
steps
H/L
Claudin1 tight junction protein
epithelial cells,
liver cells
Later
internalization
H
SR-B1
Class B
Scavenger Receptor
liver and adrenal
tissues
N/A H
LDL
receptor
endocytosis of
cholesterol-rich LDL
in all nucleated
cells
N/A H
CD-86
regulation of
lymphocyte
development
activated
lymphocytes
N/A L
Table 1-1. Possible receptors for HCV. For tropism: L, lymphotropic; H, hepatotropic;
N/A, not studied.

After viral particle is internalized, the viral RNP is released into the
cytoplasm. The viral RNA first undergoes translation to synthesize the
polypeptide, from which the RdRp (NS5B) is released by the NS3-mediated
cleavage. NS5B then replicates the positive-stranded HCV RNA into a negative-

14
stranded template by its RNA-dependent RNA polymerase activity. The negative-
stranded template can be used for the amplification of positive strand. The
positive-stranded RNA molecules can be used for either RNA translation directly
or for the next round of RNA replication. It has been shown that both steps are
dependent on membrane structures (El-Hage and Luo, 2003; Gosert et al., 2003;
Honda et al., 1996; Moradpour et al., 2004; Shi et al., 2003; Svitkin et al., 2005).
In my dissertation, we will study the regulation of HCV RNA translation by its
replication.
HCV core protein has been shown to be highly associated with lipid
droplet (LD) budding from the ER (Hope et al., 2002; Roingeard et al., 2008).
With such a high concentration of core protein, LD is likely to be the site for
HCV RNA encapsidation. Indeed, LD has been shown to be required for HCV
virus production (Boulant et al., 2007; Miyanari et al., 2007). How the HCV RNA
is targeted to the core-associated LD is not clear. However, besides core protein,
NS5A is also shown to partially localized at LD (Targett-Adams et al., 2008), or
colocalized with LD-associated core protein. NS5A is therefore a possible RNA-
binding protein with LD localization signal to target HCV RNA to LD for
encapsidation.
HCV core has been shown to bind to stem-loop II of the HCV 5’NTR to
initiate the viral assembly (Kunkel and Watowich, 2002). It is possible that the
presence of the abundant amount of HCV core proteins, which are synthesized
during viral replication, inhibits the RNA translation and initiates the viral RNA
packaging by binding to the 5’UTR of HCV genomic RNA. E1 and E2 may be

15
glycosylated at Golgi, and then transported back to ER membrane for envelope
synthesis. Efficient assembly and release of infectious HCV progeny virions from
host cells rely on p7 function (Jones et al., 2007; Sakai et al., 2003; Steinmann et
al., 2007).

1.5 Host factors involved in HCV replication
HCV has a very limited tissue tropism. In addition to the envelope protein and
receptor interaction, the host cellular factors may play important roles in HCV
replication.
1.5.1 membranous structures
Most of positive-strand RNA viruses induce a distinct membrane alteration
that provides the necessary structural scaffold for RNA replication. Distinct
patterns of intracellular membrane changes were induced by expression of all
structural and nonstructural proteins in the context of the entire HCV polyprotein
(Egger et al., 2002). As mentioned above, all HCV proteins are associated with
membrane structures, either through direct binding to the membrane or by
protein-protein interactions. In vitro studies also confirmed that membrane
structure is required for HCV viral protein maturation (Svitkin et al., 2005). The
determinants for membrane association and the protein-protein interactions
involved in formation of the HCV replication complex are so far poorly
understood.
1.5.2 hnRNP and vesicle-associated proteins

16
Besides the viral NS proteins, several host factors, including the human
homologue of the 33-kDa vesicle-associated membrane protein-associated protein
(hVAP-33) (Gao et al., 2004), polypyrimidine-tract-binding protein (PTB) (Fig.
1-3) (Aizaki et al., 2006; Chang and Luo, 2006; Domitrovich et al., 2005), La
antigen (Domitrovich et al., 2005) and host geranylgeranylated proteins and fatty
acids (Kapadia and Chisari, 2005) have recently been shown to be involved in
HCV replication. Some of these host factors, such as PTB and La autoantigen,
was initially found to regulate HCV translation (Ito and Lai, 1999) by binding to
the 5’ and 3’-untranslated regions (UTR) of HCV RNA. Later studies showed that
some of these host factors also directly regulate HCV RNA replication either by
participating in the formation of the RNA replication complex (e.g., VAP-33)
(Gao et al., 2004) or by binding to the viral RNA (e.g., PTB) (Chang and Luo,
2006; Mottola et al., 2002). A recent study showed that another host protein,
synaptotagmin-binding, cytoplasmic RNA-interacting protein (SYNCRIP), also
named NS-1-associated protein (NSAP1), binds to the N-terminal of the core
protein-coding region of HCV RNA and enhances HCV Internal Ribosomal Entry
Site (IRES)-dependent translation (Back et al., 2002).

17

Figure 1-3. Immunofluorescence staining of PTB and BrUTP-
labeled newly-synthesized HCV replicon RNA. Huh7 and HCV
replicon cells are both treated with actinomycin D and then labeled
with BrUTP. PTB is mainly distributed in the nucleus in the Huh7
cells. In the Huh7 cells harboring a HCV replicon RNA, PTB was
partially recruited to the site of newly-synthesized replicon RNA.
1.5.3 cytoskeleton
In addition to the membrane structure and cellular proteins that are required
for HCV RNA replication, cytoskeleton also plays an important role in HCV
RNA replication. As discussed in Chapter 1.3.2, HCV NS3 protein can mediate
the binding between HCV replication complex and actin filament or microtubule
(Lai et al., 2008b), indicating that HCV replication is dependent on not only
special membrane structure but also the cytoskeleton. In the replicon studies,
long-term treatment of nocodazole, a chemical which can depolymerize
microtubule, has been found to inhibit HCV replication (Bost et al., 2003).
Cytoskeletons such as microtubules are also important in regulating cellular
membrane trafficking pathways and organelle localizations. Therefore,
cytoskeleton requirements may greatly affect the replication of HCV.

18

1.6 Systems for study HCV life cycle and pathogenesis
As briefly described in Chapter 1.4, HCV cell culture system was not
available until recent years. So far the only in vivo HCV infection relies on
chimpanzee model. HCV genomic and subgenomic replicon has been used for
studies in HCV RNA translation and replication (Fig. 1-4). In this system, HCV
replication is very robust so that the HCV RNA and proteins can be detected by
Northern blot and Western blot.

Figure 1-4. Illustrations of HCV full-length and subgenomic replicon
RNA. A selection marker or a luciferase reporter is expressed under the
control of HCV IRES.

Recently, two cell culture systems of HCV are available. A HCV 2b strain
culture system for studying HCV infection was established in B-cell culture
(Machida et al., 2008a; Sung et al., 2003). This is a model of lymphotropic HCV

19
infection. The other culture system was established with genotype 2a, JFH1 strain,
in Huh7.5 cells (Wakita et al., 2005). Both systems are useful tools to study the
complete life cycle of HCV, and can be used for recombination studies.
In vitro labeling in both HCV replicon cells, HCV full-length transfected or
HCV-infected cells with BrUTP or fluorescence-tagged UTP provides a practical
means to visualize viral replication sites in the cell. In my dissertation, in vitro
labeling was widely applied with several fluorescence-tagged chemicals under the
treatment of cellular or viral transcription/translation inhibitors to observe the
subcellular localization of HCV RNA replication and translation.

20
Chapter 2
SYNCRIP (Synaptotagmin-Binding, Cytoplasmic
RNA-Interacting Protein) Is a Host Factor
Involved In Hepatitis C virus RNA Replication

2.1 Abstract
Hepatitis C virus (HCV) RNA replication requires viral nonstructural proteins as
well as cellular factors. Recently, a cellular protein, synaptotagmin-binding,
cytoplasmic RNA-interacting protein (SYNCRIP), also known as NSAP1, was
found to bind HCV RNA and enhance HCV IRES-dependent translation. We
investigate whether this protein is also involved in the HCV RNA replication. We
found that SYNCRIP was associated with detergent-resistant membrane fractions
and colocalized with newly-synthesized HCV RNA. Knock-down of SYNCRIP
by siRNA significantly decreased the amount of HCV RNA in the cells
containing a subgenomic replicon or a full-length viral RNA. Lastly, an in vitro
replication assay after immunodepletion of SYNCRIP showed that SYNCRIP was
directly involved in HCV RNA replication. These findings indicate that
SYNCRIP has dual functions, participating in both RNA replication and
translation in HCV life cycle.

21
2.2 Background and rationale
As described in Chapter 1.5.2, besides the viral NS proteins, several host
factors, including polypyrimidine-tract-binding protein (PTB) (Aizaki et al., 2006;
Chang and Luo, 2006; Domitrovich et al., 2005) and La antigen (Domitrovich et
al., 2005) have been shown to be involved in some steps of HCV replication cycle.
PTB and La autoantigen, were initially found to regulate HCV protein translations
(Ito and Lai, 1999; Mottola et al., 2002) by virtue of their binding to the 5’ and 3’-
untranslated regions (UTR) of HCV RNA. Later studies showed that some of
these host factors also directly regulate HCV RNA replication by binding to the
viral RNA (e.g., La, PTB) (Chang and Luo, 2006; Mottola et al., 2002). A recent
study showed that another host protein, synaptotagmin-binding, cytoplasmic
RNA-interacting protein (SYNCRIP), also named NS-1-associated protein
(NSAP1), binds to the N-terminal of the core protein-coding region of HCV RNA
and enhances HCV Internal Ribosomal Entry Site (IRES)-dependent translation
(Back et al., 2002).
A recent report showed that Synaptotagmin-binding, cytoplasmic RNA-
interacting protein (SYNCRIP), also named NS-1-associated protein (NSAP1),
binds to the N-terminal of the core protein-coding region of HCV RNA and
enhances HCV Internal Ribosomal Entry Site (IRES)-dependent translation (Back
et al., 2002).
SYNCRIP is a member of cellular heterogeneous nuclear
ribonucleoprotein (hnRNP) family, to which PTB also belongs. hnRNPs are well-
known for their abilities to bind to cellular proteins and RNAs to facilitate many

22
biological processes. Interestingly, SYNCRIP has previously been shown to be
involved in mouse hepatitis virus (MHV) RNA replication (Choi et al., 2004b).
Since SYNCRIP binds to HCV RNA at a site close to the 5’-end of the RNA, it is
likely that SYNCRIP may also affect the replication of HCV RNA. If this is the
case, SYNCRIP will have duel functions in both RNA replication and translation,
similar to other duel-purpose hnRNPs, such as PTB. Our goal of this study is to
investigate whether SYNCRIP may indeed be involved in HCV RNA replication
in addition to its role in translation.

2.3 Material and Methods
2.3.1 Cells
Huh7 cells were grown at 37 ºC in Dulbecco’s modified Eagle medium
(DMEM) supplemented with 10% fetal bovine serum (FBS) and nonessential
amino acids. Huh7N1b and HuhHyg replicon cells harboring an HCV
subgenomic replicon RNA derived from the HCV-N strain (Guo et al., 2001)
were grown in the same medium containing 0.5 mg/ml of G418 or 100µg/ml of
Hygromycin (Shi et al., 2003).
2.3.2 Antibodies and drugs
The primary antibodies used for the analyses in this study were sheep anti-
BrdU polyclonal antibody (BoiDesign, ME), mouse anti-BrdU monoclonal
antibody (Caltag, CA), anti-Calnexin monoclonal antibody (Abcam, MA), anti-
GS27 monoclonal antibody (Abcam, MA). Brefeldin A and Nocodazole were
purchased from Sigma, and Actinomycin D was from Fisher. The polyclonal anti-

23
SYNCRIP antibody was generated in rabbits by peptide (amino acid 140 to 152)
injection (Mizutani et al., 2000).
2.3.3 Labeling and immunofluorescene staining of de novo-synthesized viral
RNA
Labeling of de novo-synthesized viral RNA, immunofluorescence staining and
confocal microscopy were modified from the previously described procedures
(Kanestrom et al., 1998). Briefly, Huh7 or replicon cells were plated on 8-well
chamber slides at a density of 1 x 10
4
cells per well. Two days after seeding, cells
were incubated with actinomycin D (10 µg/ml) for 1 hour to inhibit cellular RNA
synthesis. Subsequently, 2 mM of bromouridine triphosphate (BrUTP) was
tranfected into cells at 4˚C for 15 minutes using FuGENE 6 transfection reagent
according to the manufacturer’s instructions (Roche Molecular Biochemicals, IN).
The cells were washed with phosphate-buffered saline (PBS) twice and cultured
at 37˚C for different incubation durations with DMEM supplemented with 10%
FBS. After incubation, cells were washed twice with PBS and subsequently fixed
by 4% formaldehyde for 1 hour at 4˚C. For permeabilization, the cells were
treated with 0.1% Triton X-100 (TX-100) (Sigma-Aldrich, St. Louis, MO) in PBS
supplemented with 1% FBS for 30 minutes at room temperature. Primary
antibodies were diluted in PBS containing 1% bovine serum albumin (BSA) and
incubated with cells for 1 hour at room temperature. After three washes in PBS,
the cells were incubated with fluorescein isothiocyanate (FITC)-conjugated or
Rhodamine-conjugated secondary antibodies diluted at a 1:100 with PBS
containing 5% BSA for 1 hour at room temperature. The cells were then washed

24
three times in PBS and mounted in Vectashield (Vector Laboratories, Burlingame,
CA).
2.3.4 Membrane flotation and detergent solubilization assay
The membrane flotation assay was performed as previously described (Shi et
al., 2003). Briefly, cells were first lysed in 1 ml of hypotonic buffer [10 mM Tris–
HCV (pH 7.5), 10 mM KCl, 5 mM MgCl2] and passed through a 25-gauge needle
20 times. Nuclei and unbroken cells were removed by centrifugation at 1000g for
5 minutes in microcentrifuge at 4 ºC. Cell lysates were then mixed with 3 ml of
72% sucrose in low-salt buffer [LSB, comprising 50 mM Tris–HCl (pH 7.5), 25
mM KCl, and 5 mM MgCl
2
] and overlaid with 4 ml of 55% sucrose in LSB,
followed by 1.5 ml of 10% sucrose in LSB. The sucrose gradient was centrifuged
at 38000 rpm in a Beckman SW41 Ti rotor for 14 hours for 4 ºC. After
centrifugation, 1-ml fractions were taken from the top of the gradient, and each
was added 1.7 ml of LSB to dilute sucrose and concentrated by being passed
through a Centricon YM-30 filter unit (Millipore, Bedford, MA). One half of each
sucrose gradient fraction was separated by 12% SDS-PAGE and transferred to
nitrocellulose membrane. After blocking, the membrane was incubated with the
primary antibody for 1 h at 37 , followed by the appropriate species ℃ -specific
horseradish peroxidase conjugate, for an additional 1 h at 37 . Bound antibody ℃
was detected by the ECL-plus system (Amersham, Piscataway, NJ).
2.3.5 Transfection of siRNAs and HCV full-length RNA
The siRNAs against SYCRIP are 19-nt sequences located at nt 189-107 and
nt140-1438, respectively, of SYNCRIP open reading frame (ORF) and were

25
synthesized by Integrated DNA Technologies, Inc. (Coralville, IW). siRNAs were
designed to target two different sites of the human SYNCRIP gene (5’-
CUAUCGUGGUGGAUAUGAAGATT-3’, and
(5’-AGACAGUGAUCUCUCUCAUGUTT -3’) chosen with the siRNA target
finder software from Ambion (Choi et al., 2004b) . Replicon or Huh7 cells were
grown in 10% FBS-DMEM without antibiotics. For transfection, cells were plated
to a density of 10
5
cells per well in a 24-well plate on day 1. Three µl of a 20-µM
stock of siRNA duplex was mixed with 47 µl of Opti-MEM (Invitrogen, CA) on
day 2. In a separate tube, 3 µl of Lipofectamine 2000 (Invitrogen, CA) was
resuspended in 12 µl of Opti-MEM, followed by incubation at RT for 7 min. The
two mixtures were combined and allowed to sit at RT for 25 min. After the
incubation, 35 µl of Opti-MEM was added and the 100 µl mixture was directly
added to the well containing 500 µl of growth medium. On day 3, cells were
trypsinized and split into a well of the 12-well plate. On day 4, cells were
retransfected using 6 µl of siRNA with 6 µl of Lipofectamine 2000. On day 5,
cells were harvested either for Western blot analysis or for RNA isolation.
pHCV-1bhyb, which contains a full-length HCV RNA hybrid sequence of
genotype 1a and 1b under the control of T7 polymerase promoter, has been
described previously (Choi et al., 2004a). Full-length HCV RNA was in vitro
transcribed through T7 promoter to obtain a positive-sense HCV RNA of about
9.6 kb. HCV full-length RNA was then transfected into cells one day after siRNA
re-transfection with Mirus Trans-IT mRNA transfection reagents (Mirus Bio, WI).
Briefly, 1 μl of Booster reagent and 1 μl of Trans-IT reagent were added into 100

26
μl OPTI-MEM sequentially, followed by 1.5µg of in vitro transcribed HCV RNA.
The mixture was incubated for 3 min at RT and added into each well of a 12-well
plate containing 1 ml of fresh DMEM supplemented with 10% FBS. Cellular
RNA was isolated from each well at 0 to 4 days after HCV RNA transfection.
2.3.6 Cell-Free replication assay and immunodepletion experiment
Cell lysate of replicon or control Huh7 cells were prepared by a modified
protocol (Mottola et al., 2002). The cells grown in 100-mm-diameter dishes were
washed with cold washing buffer (150 mM sucrose, 30 mM HEPES [pH 7.4], 33
mM ammonium chloride, 7 mM KCl, 4.5 mM magnesium acetate), followed by
treatment with lysolecithin buffer (250 µg/ml of washing buffer) for 2 min. Three
milliliters of washing buffer were added to each culture plate. The buffer was
removed by aspiration. The cells were collected by scraping in 120 µl of
incomplete replication buffer (100 mM HEPES [pH 7.4]; 50 mM ammonium
chloride; 7 mM potassium chloride; 1 mM spermidine; 1 mM [each] ATP, GTP,
and UTP; 10 µM CTP), transferred to a new tube, and lysed gently by pipetting
15 times. The cell suspension was centrifuged at 1,600 rpm in a microcentrifuge
for 5 min at 4˚C.
For immunodepletion experiment, 40 µl of cytoplasmic fraction
(supernatant) obtained as above was treated with 1% Nonidet P-40 (NP-40)
(Boehringer Mannheim, Quebec, Canada) at 4˚C for 1 hr and incubated with 0.1
µg or 1.0 µg of the indicated antibody with an adjusted amount of PBS at 4˚C for
4 hr with rotation. After incubation, sample was incubated with
32
P-CTP (30 µCi;
800 Ci/mmol), 10 µg of actinomycin D per ml, and 800 U of RNase inhibitor per

27
ml (Promega Corporation, Wis.) for 3 hr at 30°C. Extraction of RNA from the
total mixture was performed with the TRI Regent (Molecular Research Center,
Inc., Cincinnati, OH). The RNA were precipitated and eluted in 10 µl of RNase-
free water. The replication products were analyzed by gel electrophoresis on 1 %
formaldehyde agarose gel.Results

2.4 Result
2.4.1 SYNCRIP relocalized to detergent-resistant membrane fraction in
HCV replicon cells.
It has been shown that HCV RNA replication occurs in detergent-resistant
membrane (DRM) fractions (El-Hage and Luo, 2003; Mottola et al., 2002; Shi et
al., 2003). The nonstructural proteins of HCV are associated with the DRM
structures containing Caveolin-2, strongly suggesting that the viral replication
complex has properties of lipid rafts (Gao et al., 2004; Shi et al., 2003). To
determine whether SYNCRIP is in the RNA replication complex, we performed
membrane flotation analysis of HCV replicon cells, followed by immunoblotting
with anti-SYNCRIP antibody to examine the possible presence of SYNCRIP in
the detergent-resistant membrane fractions, where the HCV replication complexes
reside. We found that SYNCRIP was present mostly in the cytosolic fractions
(fractions 6-9, Fig. 2-1) in both Huh7 and HuhN1b cells, but a small fraction was
associated with the membrane (fractions 2-4, Fig. 2-1). After treatment with
Triton X-100 at 4 ºC, some SYNCRIP was still associated with the membrane in
HuhN1b cells; in contrast, almost all of SYNCRIP was solubilized in Huh7 cells.

28
Longer exposure of immunoblotting was performed, and SYNCRIP was still not
found in the DRM fractions of TX-100 treated Huh7 cells. As a control, Calnexin,
a marker protein of ER membrane, was concentrated exclusively in the membrane
fractions (fraction 2-3) in the absence of detergent treatment in both Huh7 and
HuhN1b cells (Fig. 2-1, panel 3). After the cells were treated with cold detergent,
Calnexin was redistributed entirely to the soluble fractions, indicating that
Calnexin was associated with detergent-soluble membrane, which is a known
characteristic of unmodified ER. These data suggested that SYNCRIP is
predominantly a cytoplasmic protein, but is relocalized to the DRM fractions
(fraction 2-3 after TX-100 treatment) in the HuhN1b replicon cells.

29
The relocalization of SYNCRIP protein, but not Calnexin, to the DRM
fraction in the HuhN1b replicon cell indicated that SYNCRIP may be specifically
recruited by HCV RNA to the replication complexes, since SYNCRIP binds to
HCV RNA (Back et al., 2002).

2.4.2 SYNCRIP colocalized with de-novo synthesized RNA in HCV replicon
cells.
Previous studies on HCV replicon cells have shown that the newly synthesized
HCV RNA and the viral nonstructural proteins colocalized with each other on the
distinct speckle-like structure in the cytoplasm of the replicon cells (Gosert et al.,
2003; Shi et al., 2003). To examine whether SYNCRIP is associated with HCV
RNA synthesis in the speckle structures, BrUTP labeling was performed in HCV
replicon cells transfected with peGFP-SYNCRIP (a gift from Dr. Mizutani, The
University of Tokyo). Briefly, two days after transfection of peGFP-SYNCRIP,
BrUTP was transfected into actinomycin D-pretreated cells (Kanestrom et al.,
1998). Immunofluorescence staining with sheep anti-BrdU polyclonal antibody
(Biodesign, ME) was then performed (Kanestrom et al., 1998). Under this
condition, all of the BrU-label represents HCV RNA since the cellular
transcription is inhibited by actinomycin D treatment. The BrU-labeled RNA was
present either in distinct speckle-like structures or in large spherical particles in
the cytoplasm of the replicon cell (Fig. 2-2), consistent with our previous report
(Shi et al., 2003). These two patterns probably represent two different states of
viral RNA synthesis. No BrU-labeled RNA was found in Huh7 cells without an

30
HCV replicon. SYNCRIP was also localized in the cytoplasm in Huh7 cells
without HCV replicon, but in a more diffuse pattern than that of BrU label in
HCV replicon cells. It was found that eGFP-SYNCRIP was partially colocalized
with BrU-labeled RNA in the replicon cells (Fig. 2-2), indicating that only a
portion of SYNCRIP was recruited to the HCV RNA replication site. This finding
is consistent with the fractionation profile, which showed that SYNCRIP is
primarily a cytosolic protein and that only a portion of SYNCRIP is relocalized to
the DRM fractions in the replicon cells (Fig. 2-1). This phenomenon was also
observed with PTB in the replicon cell (Fig. 1-3) (Aizaki et al., 2006), in which
only a small portion of PTB was relocalized to the cytoplasm, whereas the
majority remained in the nucleus. These results suggested that a portion of
SYNCRIP is localized to the HCV replication complex, implying that SYNCRIP
is involved in HCV RNA replication.

31

2.4.3 SYNCRIP is important in HCV replication.
To determine the biological role of SYNCRIP in HCV RNA replication, we
monitored HCV RNA levels in HCV replicon cells in which the endogenous
SYNCRIP was knocked down with the RNA interference method (Aizaki et al.,
2006; Wagner and Garcia-Blanco, 2002). HuhHyg replicon cells were transfected
with either SYNCRIP-specific ( siRNA 1 and 3) (Choi et al., 2004b) or
nonspecific (NS) siRNA. Protein analysis by immunoblotting was performed with
rabbit polyclonal anti-SYNCRIP antibody, and HCV RNA level was monitored

32
by using Taqman quantitative realtime RT-PCR (Gao et al., 2004). The cells
transfected with SYNCRIP siRNA showed a significant reduction of the
endogenous SYNCRIP by day 2 post-transfection (Fig. 2-3, A). After re-
transfection with the same siRNAs respectively, almost no endogenous SYNCRIP
could be detected. Correspondingly, SYNCRIP siRNA-transfected HCV replicon
cells showed a 70-80% reduction of HCV RNA by day 2 after retransfection with
the siRNA as compared to that in the cells transfected with the nonspecific siRNA
(Fig. 2-3, B).

33

HuhN1b replicon cells were also used to confirm the result obtained using
HuhHyg replicon cells. SYNCRIP knock-down was achieved by siRNA
transfection, and the viral protein expression and RNA levels were examined in a
time-course study. While the non-specific siRNA transfection did not
significantly affect SYNCRIP expression, the expression level of SYNCRIP was
dramatically decreased after the transfection of specific siRNA against SYNCRIP,
and the re-transfection of siRNA maintained the knock-down effects (Fig. 2-4, A).
When SYNCRIP was knocked-down by 70%, a 50% decrease in intracellular

34
replicon RNA were detected from day 2 p.t. (Fig. 2-4, B). The decrease in NS3
expression was also detected in siRNA 3-transfected cells; however, the viral
protein detected in the total cell lystate was not decreased until 4 days after
siRNA transfection. It is indicated that SYNCRIP may regulate HCV RNA
translation and RNA replication through different mechanisms.
It has been reported that SYNCRIP interacts with HCV RNA fragment
spanning nt 342 to 374, corresponding to the N-terminus of the core protein-
coding region (Back et al., 2002). Since this region is immediately downstream to
the neomycin-phosphotransferase gene in the HCV replicon, it is possible that the
observed involvement of SYNCRIP in the HuhN1b replicon cells was due to the
possible effects of SYNCRIP on the expression of phosphotransferase. To rule out
this possibility, we further examined the role of SYNCRIP in the replication of
HCV full-length RNA (pHCV-1b-hyb) without the neomycin phosphotransferase
gene.

35

36

37
Huh7 cells were first transfected and re-transfected with SYNCRIP-specific
siRNA to knock-down the endogenous SYNCRIP protein significantly; one day
after retransfection of siRNA, the cells were transfected with the replication-
competent full-length HCV RNA (HCV-1b) (Choi et al., 2004a). The SYNCRIP
expressions and NS3 levels in siRNA transfected cells were examined by
immunoblotting. SYNCRIP expression was significantly decreased in cells
transfected with specific siRNA (siRNA 1 and 3) (Fig. 2-5, A). NS3 expression
was also affected by endogenous SYNCRIP level; in siRNA1- and siRNA 3-
transfected cells, NS3 was detected in the first 2 days post transfection of HCV
full-length RNA, but became undetectable after day 3 (Fig. 2-5, A). Total
intracellular HCV RNA was determined sequentially at various days by
quantitative real-time RT-PCR. In the cells transfected with the nonspecific
siRNA, HCV RNA titer gradually increased during the first 72 hr post-
transfection in agreement with the published report (Choi et al., 2004a) (Fig. 2-5,
B). In contrast, in the cells transfected with the SYNCRIP-specific siRNA, HCV
RNA titer decreased steadily over the same period of time (Fig. 2-5, B). Although
NS3 was detected in the first 2 days after HCV RNA transfection in SYNCRIP-
knocked down cells (Fig. 2-5, A), there was no sign of HCV replication (Fig. 2-5,
B). HCV RNA titer constantly decreased in SYNCRIP-knocked down cells. There
was a slight increase in intracellular HCV RNA level at day 5 post transfection of
HCV full-length RNA. It may be due to the increase in SYNCRIP protein level.
This result suggested that endogenous SYNCRIP is directly involved in HCV
replication, but not through the suppression of the expression of neomycin

38
phosphotransferase gene. This result indicates that SYNCRIP is involved in HCV
replication by affecting either HCV RNA replication or translation, or both.

2.4.4 SYNCRIP inhibited HCV RNA replication in vitro.
The siRNA knock-down approaches showed that once SYNCRIP protein
level was decreased, the HCV RNA titer would be correspondingly decreased.
Since SYNCRIP has been shown to be directly involved in HCV translation
(Back et al., 2002), the inhibition of HCV RNA replication in SYNCRIP-
knockdown cells may have resulted from the indirect effect of inhibition of
translation; namely, the viral NS protein synthesis was inhibited, and thereby viral
RNA synthesis was decreased.
To distinguish the effect of SYNCRIP on RNA replication from that on
translation, we designed experiments to separate viral RNA replication from viral
translation. We employed an in vitro replication assay using crude membrane
fractions of the HCV subgenomic replicon cells (Gao et al., 2004; Mottola et al.,
2002), after the endogenous SYNCRIP had been knocked down by the siRNA
approach. We also performed in vitro RNA replication assay after SYNCRIP was
depleted with the anti-SYNCRIP antibody from the cell lysates.

39

Immunoblotting showed that the amount of SYNCRIP in the HuhN1b
replicon cells was substantially reduced by the specific siRNA treatment for two
days (Fig. 2-6). At this time, the amount of NS5A was only partially reduced. Cell
lysates from siRNA-treated replicon cells were treated with TX-100 at 4 °C for 30
minutes and fractionated by sucrose gradient centrifugation to isolate DRM
fraction (Aizaki et al., 2006; Mottola et al., 2002). The DRM fractions from these
cell lysates were then used for in vitro replication assay. The HCV RNA synthesis
was detected as single band of
32
P-labeled RNA. The result showed that there was
no detectable RNA replication activity at all in the DRM fractions from the

40
SYNCRIP siRNA-transfected replicon cells when compared with those from the
non-transfected or nonspecific siRNA-transfected replicon cells (Fig. 2-6). Since
there was still a significant amount of NS5A remaining in the siRNA-transfected
cells, the total lack of the in vitro replication activity in SYNCRIP knocked-down
replicon cells suggested a direct role of SYNCRIP in HCV RNA replication.
We further performed an immunodepletion experiment to remove
SYNCRIP form the DRM fraction and assessed the effects on HCV RNA
replication in vitro. For immunodepletion, the DRM fractions from replicon cell
lysates were incubated with a rabbit anti-SYNCRIP polyclonal antibody to
deplete the endogenous SYNCRIP from the lysate. After incubation, samples
were used for cell-free synthesis of HCV RNA. The results showed that the
treatment with anti-SYNCRIP antibody inhibited the replication activity in an
antibody concentration-dependent manner, whereas a control anti-Ig antibody did
not inhibit any activity at the same or an even higher antibody concentration (Fig.
2-7 and data not shown). As a control, anti-VAP33 (VAP-A) antibody also
inhibited HCV RNA replication, similar to the previous result (Hamamoto et al.,
2005), whereas anti-Calnexin antibody did not.

41

These results combined suggested that SYNCRIP is directly involved in
HCV RNA replication, in addition to its role in regulating the translation of HCV
RNA. Since SYNCRIP is colocalized with the newly synthesized HCV RNA, it is
likely that SYNCRIP is a part of the HCV RNA replication complex and
participates in viral RNA synthesis.

2.5 Discussions
The results above show that SYNCRIP can modulate HCV RNA replication.
It has been previously reported that SYNCRIP can also enhance HCV IRES-
dependent translation (Back et al., 2002). Thus, similar to PTB and La
autoantigen, SYNCRIP has dual functions in HCV life cycle. This may be a
common characteristic of HCV RNA-binding proteins.

42
In our study, the relocalization of SYNCRIP to the DRM fractions in HCV
replicon cells indicates that SYNCRIP is associated with the RNA replication
complex, which is localized in this membrane fraction. It is interesting to note that
the distribution of Calnexin was slightly different between the control and the
replicon cells; there was some shift of Calnexin toward the lighter sucrose
gradient fractions, probably cased by the alteration of cellular membrane
structures and the associations of HCV NS proteins to ER membrane structure in
HCV replicon cells (Egger et al., 2000; El-Hage and Luo, 2003; Gosert et al.,
2003; Mottola et al., 2002). Nevertheless, SYNCRIP was clearly localized in the
DRM fraction, where as Calnexin was not.
The immunodepletion experiments in the current and previous studies have
shown that antibody against PTB, hVAP-A, hVAP-B, and SYNCRIP can inhibit
HCV RNA replication activities specifically. Previous studies have suggested that
the HCV RNA replication complexes are protein complexes with the newly-
synthesized RNA being contained within (Tai et al., 2001), and that subtilisin
protease treatment could disrupt the replication complexes. However, it was also
reported that the HCV replication complexes were resistant to proteinase K
treatment at room temperature (Aizaki et al., 2004; Quinkert et al., 2005). The
latter study suggested that the HCV replication complexes were very compact,
and therefore the accessibility of immunoglobulin to the specific protein target in
the replication complex may be limited. However, the ability of these antibodies
to inhibit HCV RNA replication suggested that those complexes may not be so
compact and are accessible by immunoglobulin molecules under these conditions.

43
The genome of positive-stranded RNA viruses, such as HCV, poliovirus,
and coronavirus, serve as a template for both translation and the synthesis of
negative-strand RNA, the latter of which is, in turn, the template for synthesizing
more positive-strand RNA. The positive-strand RNA can also be packaged to
form new viral particles. Since the same positive-strand RNA can participate in
different steps of the viral life cycle, the temporal or spatial regulation is very
important. It is likely that the regulation is through RNA-protein interactions.
When in complex with specific RNPs, the RNA can be utilized specifically in
different steps. With limited numbers of genes in the viral genome, the regulation
likely requires the participation of various host factors interacting with the viral
RNA or viral proteins.
There are many known host and viral RNA-binding proteins that can
facilitate positive-strand RNA replication, such as Tat protein binding to the TAR
structure in HIV1 RNA (Dingwall et al., 1989; Wagner and Garcia-Blanco, 2002)
and poly(rC)-binding protein binding to the cloverleaf structure of poliovirus
RNA (Blyn et al., 1996; Gamarnik and Andino, 2000). However, not so many
RNA-binding proteins have been reported to have dual functions in viral RNA
replication and translation. Recently, La and PTB, are found to regulate both
RNA replication and translation of HCV, probably as a result of their ability to
bind to HCV RNA (Aizaki et al., 2006; Domitrovich et al., 2005). Host factors
with dual-regulatory functions may play important roles in switching the RNA
from translation to replication or replication to translation. For example, PCBP
regulates translation-replication switch in poliovirus life cycle (Back et al., 2002;

44
Gamarnik and Andino, 1998). It was reported previously that stem-loop I and II
are critical for HCV RNA replication, and stem-loop II, III, and IV are important
for HCV RNA translation (El-Hage and Luo, 2003; Fukushi et al., 2001; Qi et al.,
2003). La autoantigen was shown to bind to loop IV of HCV 5’NTR (Mottola et
al., 2002). Although there is no evidence that La binds to stem-loop I or II, La can,
nevertheless, regulate HCV replication (Domitrovich et al., 2005). Similarly,
SYNCRIP was reported to bind at nt 342 to 374 (Back et al., 2002), a region
essential for HCV IRES-driven translation but not HCV replication. Yet we found
significant positive regulatory effect of SYNCRIP in HCV RNA replication. It is
possible that the binding of SYNCRIP to HCV RNA alters the secondary
structure of the RNA or recruits other required factors to facilitate the assembly of
the replication complex.
The mechanism of switching between translation and replication of HCV
RNA is still unclear; conceivably, it may be regulated by these dual-function
proteins which are involved in both replication and translation. It will be
interesting to determine in the future whether the relative ratio of these proteins
may trigger the switch.

45
Chapter 3
The translation of HCV RNA is colocalized with
and dependent on RNA replication

3.1 Abstract
Hepatitis C virus (HCV) RNA replication occurs on the distinct detergent-
resistant membrane (DRM) structures near the endoplasmic reticulum; however,
the site of viral protein translation is not yet clear. In this study, we showed that
the DRM structure that supported HCV RNA replication could also support HCV
protein translation, and that the nascent HCV peptides derived from cap-
independent translation colocalized with the newly-synthesized HCV RNA.
Furthermore, the HCV proteins encoded from the HCV replicon were translated
from the DRM fraction, whereas the control reporter protein from the expression
plasmid was translated from the soluble and non-DRM fraction. The Cy5-UTP
and BIDOPY-FL-lysine-tRNA double labeling in live cells showed that all the
HCV protein synthesis occurred at the sites of HCV RNA synthesis. Electron
microscopy further showed that HCV RNA and protein syntheses took place on a
multilayered vesicle structure. The translation of HCV proteins is dependent on
active RNA synthesis: inhibition of RNA synthesis resulted in decreased HCV
viral protein synthesis before there was significant decrease in the total amount of
HCV RNA, and the replication-defective HCV RNA could not translate any

46
protein. When the movement of HCV RNA from the site of RNA synthesis to the
Golgi complex was blocked by nocodazole, HCV protein translation was
enhanced, suggesting that the translation of viral proteins occurred near the site of
RNA synthesis. Our findings together suggest that the replication and translation
of HCV RNA are coupled and occur in the same subcellular compartments termed
“replicasome”.

3.2 Background and rationale
Membrane-association of the viral proteins is essential for HCV replication,
at both steps of RNA transcription and translation(Lee et al., 2004; Moradpour et
al., 2004; Simmonds et al., 2005). HCV viral proteins, except NS3, are associated
with membranes though some of the protein topologies are not well
understood(Gosert et al., 2003).
HCV subgenomic replicon cells were widely used as an in vivo system of
HCV replication. Although the replicon system cannot support production of
infectious virus, it is believed that the replicons recapitulated faithfully the viral
intracellular life cycle, including HCV RNA replication(Pietschmann et al.,
2006) . Using the replicon system, many host factors were identified to be
involved in HCV RNA replication, including hVAP-33 (Gao et al., 2004), PTB
(Chang and Luo, 2006; Domitrovich et al., 2005), La autoantigen (Domitrovich et
al., 2005), and host geranylgeranylated proteins and fatty acids (Kapadia and
Chisari, 2005). Some of these host factors, such as PTB and La autoantigen, were
also found to regulate HCV viral translation as well(Ito and Lai, 1999) through

47
their binding to the 3’ UTR of HCV RNA. The identification of host proteins with
“dual-functions” in regulating both translation and transcription implies the
possibility of coupled transcription/translation of HCV RNA.
The balance between viral RNA transcription and translation is critical for
the replication of positive-stranded viruses, since the same RNA is used both for
translation and as the template for negative-strand RNA synthesis. Transcription
of poliovirus has been reported to be dependent on the translational activity of the
viral RNA(Novak and Kirkegaard, 1994). On the other hand, the translation of
Sindbis virus and vesicular stomatitis virus has been reported to be
transcriptionally dependent (Sanz et al., 2007). Coupling of transcription-
translation has been well documented to have advantage in maintaining the
stability of the RNA molecule in bacteria (Iost and Dreyfus, 1994; Iost and
Dreyfus, 1995) and also to respond to regulatory signals coordinately.
Considering the fact that the RNA transcription machinery and translation
machinery are well separated in the cell since they require different structural and
nonstructural components and involve different molecular mechanisms, the
reported coupling between viral RNA transcription and translation is puzzling.
We therefore set out to examine the sites of HCV RNA replication and protein
translation in the cells.

3.3 Material and Method
3.3.1 Cell lines, HCV full-length and subgenomic constructs.
Huh7 or Huh7.5 cells were grown at 37°C in Dulbecco’s modified Eagle medium

48
(DMEM) supplemented with 10% fetal bovine serum (FBS) and nonessential
amino acids. Bicistronic HCV-N1b replicon was derived from the HCV-N
strain(Guo et al., 2001) with a neomycin-phosphotransferase (NPT) gene for
selection. Huh-N1b replicon cell line and Huh-Neo cells containing an NPT
gene(Gao et al., 2004) were grown under the same conditions as Huh7 cells using
the same media containing a supplement of 0.5mg/ml G418.

3.3.2 In vitro transcription and electroporation of HCV full-length and
subgenomic RNA.

HCV JFH1 and JFH-GND constructs were obtained from Dr. Wakita’s lab
(Wakita et al., 2005). Bicistronic replicon with either firefly luciferase (FFLuc) or
Renilla luciferase (RLuc) gene was derived from HCV1bneo(Guo et al., 2001) by
replacing NPT with either FFLuc or RLuc reporter gene. To prepare the template
for in vitro transcription, the plasmids were digested by Xba I and Mugbean
nuclease and Gel-purified. For electroporation, Huh7 or Huh7.5 cells were
trypsinized, washed and resuspended in serum-free DMEM. HCV replicon RNA
or JFH full-length RNA were transcribed in vitro by T7 MegaScript (Ambion). A
total of 6 to 10 μg of RNA and 10
7
Huh7 cells were mixed and incubated on ice
for 5 minutes and subjected to an electric pulse at 975 µF and 220 V. Cells were
immediately transferred to 8 ml of DMEM containing 10% FBS for incubation.

3.3.3 Labeling and immunofluorescene staining of de novo-synthesized

49
viral RNA and newly-translated peptides.
Labeling of de novo-synthesized viral RNA, immunofluorescence staining
and confocal microscopy were modified from the previously described
methods(Aizaki et al., 2006). Briefly, Huh7 or replicon cells were plated on 8-
well chamber slides at density of 1 x 10
4
cells per well. Two days after seeding,
cells were incubated with actinomycin D (10 g/ml) for 1 hour to inhibit cellular
RNA synthesis, and in some experiments, with 50 M cap-dependent translation
inhibitor NSC119893(Novac et al., 2004)

or 20 nM of hippuristanol(Bordeleau et
al., 2006) for 1 hour to inhibit most of cellular protein synthesis. Subsequently, 2
mM of bromouridine triphosphate (BrUTP) was transfected into cells at 4°C for
15 min using Fugene 6 transfection reagent according to the manufacturer’s
instructions (Roche). Transcend biotinyl-Lys-RNA (Promega) (Iborra et al., 2001)
was added along with BrUTP into the Fugene 6 solution for double-labeling of
nascent peptide and newly-synthesized HCV RNA. The cells were washed with
phosphate-buffered saline (PBS) twice and incubated at 37°C with DMEM
supplemented with 10% FBS for different periods of time. After incubation, cells
were washed twice with PBS and subsequently fixed by 4% formaldehyde for 1
hr at 4°C. For permeabilization, the cells were treated with 0.1% Triton X-100 in
PBS supplemented with 1% FBS for 30 min at RT. Primary antibodies were
diluted in PBS containing 1% bovine serum albumin (BSA) and incubated with
cells for 1 hr at RT. After three washes in PBS, the cells were incubated with
fluorescein-isothiocyanate(FITC)-conjugated or Rhodamine-conjugated
secondary antibodies, or Texas-red-conjugated streptoavidin diluted at a 1:100

50
with PBS containing 5% BSA for 1 hr at RT. Then the cells were washed three
times in PBS and mounted in Vectashield (Vector Laboratories).

3.3.4 Immunoprecipitation of the metabolically-labeled viral proteins.
Huh7 cells, Huh-Neo cells, and Huh-N1b replicon cells were used in this
experiment. Briefly, the cells were pretreated with or without nocodazole (or
Benzothiadiazine) for 4 hours to 16 hours respectively. Cells were then incubated
in methionine-free DMEM containing the various chemicals described in the text
for 1hr.
35
S-methionine and fresh medium were added to and incubated with the
cells for 4 hours. The cell lysates were immunoprecipitated with either HCV
patient sera or monospecific antibodies against HCV viral proteins. Neomycin-
phosphotransferase antibody was also used to monitor the NPT translation. The
immunoprecipitates were separated on SDS-PAGE gels and followed by
autoradiography.

3.3.5 Analysis of intracellular viral RNA by Northern blotting and real-
time RT -PCR.
To determine the quantity of RNA by real-time PCR, a single-tube reaction
was performed by using the TaqMan EZ RT-PCR Core Reagents (Applied
Biosystems). Duplicate reactions for RNA standards and the samples were
performed in 20- μl volume using 1.0 μl of HCV RNA, primers from HCV 5’ non-
coding region (5’ GAG TGT CGT GCA GCC TCC A 3’ and 5’ CAC TCG CAA

51
GCA CCC TAT CA 3’) of the HCV 1b sequence(Simmonds et al., 2005), and a
fluorescent probe [5’ (FAM) CCC GCA AGA CTG CTA GCC GAG TAG TGT
TGG (TAMRA) 3’] spanning these two regions. The RT step was performed at 60
°C for 50 min, followed by 1 min at 50 °C. The amplification condition was as
follows: 95 °C for 5 min and 50 cycles of denaturation at 94 °C for 15 sec,
annealing at 55 °C for 10 sec, and extension at 69 °C for 1 min. Using the ABI
Prism 7900 program, standard curves of the assays were obtained automatically
by plotting the three hold values against each standard dilution of known
concentration (10
1
–10
6
copies per reaction) of HCV genotype 1b transcript. The
same software was used to calculate the coefficients of regression. Values were
normalized to that of GAPDH (Applied Biosystems). Each test was done in
triplicate and averages were obtained.

3.3.6 Fractionation of ER and Golgi membrane
The procedure was based on the published method(Choi et al., 2004a). Cell
lysates were applied to a discontinuous sucrose gradient composed of layers of
2M, 1.3M, 1.0M and 0.6M sucrose. The ER fraction was concentrated at the
interface between 0.6M and 1.0M sucrose, and the Golgi fraction was
concentrated at the interface between 2M and 1.3M. To determine the signal of
3
H-Uridine-labeled RNA, the fractions were then passed through DE81
membranes to concentrate the labeled RNA. The membranes were then counted
by scintillation counter, and the ratio of signals from the ER and Golgi fractions
were calculated.

52
3.3.7 Membrane preparation and in vitro translation assay.
The membrane preparation was performed as previously described(Aizaki et
al., 2004). The cells were starved in methionine-free culture medium for 30
minutes and then lysed in 0.5 ml of hypotonic buffer and passed through a 25-
gauge needle 20 times at 4°C. Unbroken cells and cell debris were removed by
centrifugation at 1,000 × g for 5 min at 4°C. To identify detergent-resistant
membrane, the supernatant was treated with 1% Triton X-100 on ice for 30
minutes. The cell lysates were then mixed with 3 ml of 72% sucrose in low-salt
buffer (LSB) and overlaid with 4 ml of 55% sucrose in LSB, followed by 1.5 ml
of 10% sucrose in LSB. The sucrose gradient was centrifuged at 38,000 rpm in a
Beckman SW41Ti rotor for 14 h at 4°C. One-milliliter fractions were taken from
the top of the gradient, and each was diluted by PBS and then concentrated by
passage through a Centricon YM-30 filter unit (Millipore). Membrane fractions
(fraction 2-3) and soluble fractions (fraction 6-7) were then pooled separately and
mixed with equal volume of 2X Extraction buffer supplied with
35
S-Methionine
(Favre and Trepo, 2001). After 30 minutes of incubation at 30°C, each reactions
were diluted with RIPA buffer and immunoprecipitated with NS3 and NPT
antibodies respectively.
3.3.8 Immunogold staining and electronmicroscopy
The method was modified from a previous study(Iborra et al., 2001). Briefly,
the 2mM BrUTP and the biotinyl-lys-tRNA were co-transfected into Huh7 and
Huh-N1b cells by Fugene 6 at 4°C for 15 min. The cells were pelleted down and
fixed with half-strength Karnofsky’s fix (2% paraformaldehyde, 2%

53
Glutaraldehyde, 0.1m Cacodylate buffer [pH 7.4]) for examining the details of
ultra-structure, or with Psi fix (2% paraformaldehyde, 0.2% Glutaraldehyde, 0.1M
phosphate buffer) for detecting immunogold staining. For immunogold staining,
monoclonal anti-BrdU antibody, 10nm or 30nm Gold-conjugated anti-mouse IgG,
and 20nm Gold-conjugated streptoavidin were used.

3.4 Results
3.4.1 The newly-synthesized HCV RNA is colocalized with ER marker
and shifts along with anterograde membrane trafficking.
To visualize the replication of HCV RNA in the replicon cells, we
performed BrUTP labeling in the actinomycin D-treated Huh-N1b replicon cells.
After 15 minute labeling, the RNA was chased for 30 minutes to 3 hours, and then
stained with anti-BrdU antibody. By co-immunofluorescence staining with
individual organelle markers, including calnexin for ER, ERGIC53 for the ERGIC
and GS27 for Golgi apparatus, the
subcellular localization of BrU-labeled newly-synthesized HCV RNA was
determined. The results showed that HCV RNA was initially colocalized only
with the ER; after 3 hours of chase, the majority of the labeled HCV RNA exited
from the ER and colocalized with Golgi apparatus instead (Fig.3-1, A and B). We
did not observe any significant colocalization between BrU-labeled HCV RNA
and ERGIC53. As a control, the BrUTP signals could not be detected in the
actinomycin D-treated Huh7 cells, while the immunofluorescence-staining

54
patterns of the ER, ERGIC, and Golgi apparatus appeared similar in Huh7 and
Huh-N1b cells (data not shown).

55

The viral RNA movement was further confirmed by biochemical analysis.
The actinomycin D-treated Huh-N1b cells were labeled with
3
H-uridine for 30
minutes and chased for 30 minutes to 3 hours. The labeled cell lysates were
separated into ER and Golgi fractions. Immunoblotting studies showed that the
ER and the Golgi apparatus were efficiently separated by this procedure. The
relative ratio of
3
H-uridine-labeled RNA in the Golgi and the ER significantly
increased over time (Fig. 3-2). The result is consistent with the interpretation that
the newly-synthesized HCV RNA was transported from the ER to the Golgi-
derived membrane. When the cells were treated with nocodazole, which
depolymerizes microtubules and therefore inhibits the anterograde membrane
trafficking(Lippincott-Schwartz et al., 1995; Watson et al., 2005), BrU-labeled
RNA remained colocalized with calnexin even after 3 hours (Fig. 3-3). Under
these conditions, the morphology of cells was not altered by the treatment. These
results suggest that the anterograde membrane trafficking may be involved in the

56
transport of HCV RNA after its synthesis. This transportation presumably is
required for certain steps of the HCV life cycle, such as translation of RNA or
assembly of viral particles.

3.4.2 HCV RNA translation is increased when anterograde membrane
trafficking is blocked.
We next tested if the transportation of the newly-synthesized HCV RNA
from the ER to the Golgi-derived membrane is required for HCV RNA translation.
Huh-N1b cells were pre-treated with nocodazole, and then subjected to
35
S-

57
Methionine metabolic labeling. We determined the amounts of
35
S-Methionine-
labeled Neomycin-phospho-transferase (NPT) and HCV NS3 protein, both of
which are encoded from the HCV replicon RNA under the control of separate
IRES elements. We found that after a 4-hour nocodazole treatment, the total
amounts of these two proteins were notably increased (Fig. 3-4, A and B).
Quantitation of the proteins showed that there was a 40%-50% increase in both
NPT and NS3 protein synthesis after the nocodazole treatment. By comparison,
NPT synthesis in a neomycin-resistant Huh-Neo cells, in which NPT was
expressed from an integrated plasmid, was not affected by the nocodazole
treatment (Fig. 3-4, B), indicating that the increase in protein synthesis by
nocodazole treatment was not a global effect but an HCV RNA-specific event.

58
These results are opposite to our prediction and raise a possibility that the
newly-synthesized HCV RNA may be used for RNA translation immediately after
RNA synthesis before it is transported away from the ER. This result raised an
intriguing possibility that HCV RNA replication and translation are colocalized
around the ER.

3.4.3 HCV RNA transcription and translation are colocalized at the same
subcellular site.
To visualize possible colocalization of the replication and the translation
machinery in the cells, double-labeling of the HCV replicon cells with BrUTP for
RNA replication and with biotinyl-Lysine-tRNA for protein translation was
performed. The replicon cells were pre-treated with both actinomycin D and
NSC119893 to minimize the background of cellular transcription and translation.
NSC119893 inhibits cap-dependent translation(Novac et al., 2004), which is the
major mechanism for cellular protein translation but not HCV translation.
Therefore, the metabolic labeling with biotinyl-Lys-tRNA in the presence of these
inhibitors represents mostly the newly-synthesized peptides from the HCV
replicon RNA, which is under the control of the HCV IRES or the EMCV IRES.
The immunofluorescence staining with BrUTP and biotinyl-Lys-tRNA showed
that the newly-synthesized HCV RNA (BrUTP) and viral peptides (biotinyl-
Lysine) were mostly colocalized at the peri-nuclear region (Fig. 3-5). These
results suggest that these two processes are closely associated.

59

To further investigate the relationship between HCV RNA replication and
translation, Huh-N1b replicon cells were co-transfected with Cyanine-5-UTP
(Cy5-UTP), and BODIPY-FL-lysine-tRNA (FL-lys-tRNA), followed by time
series confocal microscopy of live cells. Cellular transcription and translation
were inhibited by pre-treatment with actinomycin D and hippuristanol, the latter
of which is an eIF4A inhibitor(Bordeleau et al., 2006). It has been reported the
HCV IRES-mediated translation does not require the activity of eIF4A (Mottola
et al., 2002); therefore, the HCV-IRES translation will not be affected by
hippuristanol, whereas the cap-dependent translation and many other IRES-
mediated translations, such as CrPV-IRES translation, will be inhibited. The Huh-
N1b cells were labeled with Cyanine-5-UTP (Cy5-UTP) and BODIPY-FL-lysine-

60
tRNA (FL-lys-tRNA) for 15 min on ice to decelerate the intracellular trafficking
and the enzyme activities. In the absence of actinomycin D and hippuristanol,
Huh7 Cells without HCV replicon showed nuclear labeling of Cy5-UTP and
cytoplasmic labeling of FL-lys-tRNA (Fig. 3-5, B), whereas Huh7 cells treated
with both actinomycin D and hippuristanol could not be labeled by either Cy5-
UTP or FL-lys-tRNA (data not shown). These results indicate that these labels
specifically represent HCV RNA and protein syntheses respectively. Under these
conditions, the Huh-N1b cells were first labeled by Cyanine-5-UTP (red) during
the first 12 minutes, while no FL-lys-tRNA label (green) was seen (Fig. 3-6).

61

61

62
Interestingly, in many red spots (RNA), there is a yellow spot at their
center, which represents colocalization of HCV RNA and protein. This is
particularly evident after 12 minutes. After 15 minutes, some green label (protein)
began to appear around the yellow center, as if the protein radiates away from the
center. At this time, all the red labels were completely taken over by the yellow
and green labels. These results suggest that all of the HCV protein synthesis
occurred at the site of the newly synthesized HCV RNA and that all of the newly
synthesized RNA was used for translation immediately after they were
synthesized. At later time points, some red labels began to reappear around the
green spots (after 33 minutes, Fig. 3C, lower panel), indicating the dissociation of
the RNA from the site of translation. These findings suggest that HCV protein
translation is coupled with new RNA synthesis.
We next examined the BrU-labeled and Biotinyl-lys-tRNA-labeled HCV
replicon cells by immunogold staining under electron microscopy. The cells were
treated with actinomycin D and/or hippuristanol and were labeled with BrUTP
alone or with Biotinyl-lys-tRNA. A preliminary study showed that significantly
greater number of vesicles in the perinuclear region of the Huh-N1b replicon cells
than that in the Huh7 cells (Fig. 4A), consistent with the previous report(Gosert et
al., 2003). The vesicle structures probably represent sites of RNA replication. At
early time points (15 minutes), only the BrUTP but not biotinyl-lys-tRNA labels
were detected (Fig. 3-7, B); after 60 min chase, both BrUTP signals and Biotinyl-
lys-tRNA signals were detectable (Fig. 3-7, C). Both the BrUTP and Biotinyl-lys-
tRNA signals were often in the same vesicles and appeared to be located next to

63
each other. Very often, the both particles were detected on the same membrane.
The details of the membrane could not be further elucidated because the cell-
fixing condition used to allow maximum immunogold detection did not yield
good histological resolution. We have further used a different cell-fixing
condition that allows better histological resolution but poorer immunogold
accessibility. Under this condition (single BrUTP labeling), the BrU signals were
detected mainly in the vesicles of the replicon cells (Fig. 3-7, D) but not in the
naïve Huh7 cells (data not shown). The labeled RNAs were often found on multi-
layered membrane vesicle, both at the inner surface and outer surface.
Interestingly, some of the vesicles contained ribosomes on their outer surface.
These results indicate that the replication and translation of HCV RNA are
coupled in the same subcellular compartments.

64

65
3.4.4 The detergent-resistant membrane fraction supports HCV RNA
translation in vitro.
It has been shown that the HCV RNA replication complex resides in
detergent-resistant membrane (DRM) fractions, which can support HCV RNA
replication in vitro (Aizaki et al., 2004; Mottola et al., 2002; Shi et al., 2003). To
further demonstrate that the replication and translation of HCV RNA are in the
same subcellular compartment, the detergent (0.1% Triton X-100 at 4ºC)-resistant
membrane fraction and the soluble fraction from the HCV replicon cells were
isolated as previously described(Aizaki et al., 2004). We used the DRM fraction
to carry out in vitro translation according to the modified procedure (Favre and
Trepo, 2001), and the
35
S-Methionine-labeled NPT and NS protein products from
the in vitro translation reaction were detected by immunoprecipitation. The data
showed that the DRM fractions from the Huh-N1b cells could translate both NS3
and NPT from the bicistronic replicon RNA (lane 5, Fig. 3-8); the soluble
fractions could not synthesize NS3 (lane 6, Fig. 3-8), but did synthesize some
NPT, probably due to excessively harsh treatment of DRM. The same results were
obtained for the crude membrane preparation (prepared in the absence of
detergent treatment) (lanes 7 and 8, Fig. 3-8), in which NS3 and NPT were
translated from the membrane fraction, but not the soluble fraction. For
comparison, NPT was translated mainly from the soluble fractions of Huh-Neo
cells, in which NPT was expressed from a separate cellular mRNA (lanes 2 and 4,
Fig. 3-8). The DRM fraction of Huh-Neo cells could not support NPT translation
(lane 1, Fig. 3-8). It is notable that NPT from the Huh-N1b cells was slightly

66
larger than the NPT from Huh-Neo cells because the former was fused with the
N-terminus of HCV core protein. These results indicate that the DRM fraction
that supports HCV RNA replication also supports HCV protein translation.

To confirm this result, we next determined whether eIF3, the only required
translation initiation factor for HCV IRES (Mottola et al., 2002; Robert et al.,
2006), was present in the DRM fractions of the Huh-N1b cells (Fig. 3-9). The
eIF3 in Huh7 cells was distributed in both the membrane (fractions 2-3) and the
soluble (fractions 5-9) fractions. After the detergent treatment, eIF3 could only be
detected in the soluble fraction. In contrast, in Huh-N1b cells, some eIF3 could be
found in the DRM fractions, indicating that the required factor for HCV
translation was present in the DRM, supporting the findings that the DRM

67
fraction could support in vitro translation of the HCV replicon RNA. The results
suggested that both replication and translation of HCV RNA took place in the
DRM fractions.

3.4.5 HCV RNA translation is dependent on the transcriptional activity of
the RNA.
The above findings suggested that HCV RNA was used for translation
immediately after transcription, and that translation may take place in the vicinity
of the RNA transcription factory. Thus, these two events may be coupled. In
order to analyze if HCV RNA translation is closely linked to RNA transcription,
we used a specific NS5B polymerase inhibitor, Benzothiadiazine(Hirashima et al.,
2006; Ishida et al., 2006; Tedesco et al., 2006), to inhibit HCV RNA synthesis.
We first determined the efficiency and specificity of the inhibitor on
3
H-
uridine incorporation (Fig. 3-10). Huh7-N1b replicon cells were pretreated with or

68
without Benzothiadiazine for 16 hours and then with actinomycin D for an
additional 1 hour prior to
3
H-uridine labeling. Under this condition,
3
H-uridine is
incorporated only into HCV RNA(Choi et al., 2004a). The data showed that, in
Huh7 cells, actinomycin D almost completely inhibited uridine incorporation.
However, in Huh-N1b cells, actinomycin D did not completely inhibit
3
H-uridine
incorporation; this residual incorporation likely represents HCV RNA synthesis,
as confirmed by autoradiography of the RNA products. The residual RNA
synthesis was inhibited by Benzothiadiazine. This conclusion was also supported
by Br-UTP labeling followed by immunostaining of Huh-N1b cells, which
showed that all of the labeling accumulated as speckles around the perinuclear
region; these speckles were not visible in the presence of Benzothiadiazine (Fig.
3-10, B). The results indicate that Benzothiadiazine inhibits viral RNA synthesis
specific
by Benzothiadiazine, the existing HCV RNA molecules were relatively stable in
ally.
We also studied the effects of Benzothiadiazine on the steady-state level
of replicon RNA by realtime RT-PCR analysis. The data showed that even after
16 hours of treatment, the total amounts of replicon RNA in the cells was not
significantly affected (Fig. 3-10, C). The quality of HCV RNA was not affected
by the treatment even after 16 hours. After 2 days of treatment, the replicon RNA
level was decreased by about 50%. After 5 days, the RNA level dropped to 10%
(Fig. 3-10, D). As a comparison, nocodazole, which was reported to inhibit HCV
RNA replication(Bost et al., 2003), had a smaller effect on the accumulation of
HCV RNA. The result indicated that, although HCV RNA synthesis was blocked

69
the cell. Thus, the total amount of HCV RNA will not change significantly at least
within the first 16 hours after the addition of Benzothiadiazine.

70

71

Having established the specificity of Benzothiadiazine on HCV RNA
synthesis, we then preformed metabolic
35
S-Met labeling in replicon cells treated
with Benzothiadiazine and assessed HCV protein translation (Fig. 3-11, A). At 4
hour post-treatment, the total HCV RNA titer and the quality of RNA were not
significantly affected, as determined by realtime RT-PCR and Northern blot (Fig.
3-11, B). However, the amounts of HCV NS3 and NPT translated in the replicon
cells were significantly decreased after the Benzothiadiazine treatment. In contrast,
in Huh-Neo cells, the neomycin-resistant stable cell line, Benzothiadiazine
treatment did not affect the translation of NPT, indicating that Benzothiadiazine
specifically inhibited HCV RNA translation.
We further used a replication-defective replicon RNA (GND mutation in
the NS5B region) to assess the requirements of RNA replication for HCV protein

72
translation. After electroporation of the wild-type HCV replicon and its GND
mutant RNAs into Huh7 cells, viral protein syntheses were determined by
35
S-Met
labeling overnight followed by immunoprecipitation. Both of the replicon RNA
and the GND mutant could be translated in vitro, producing equivalent amounts
of NS2 to NS5B proteins (data not shown). However, after electroporation, the
GND mutant yielded very little NS3 as compared with the wildtype replicon RNA
(Fig. 3-12, A). It is notable that the total amounts of wildtype and mutant RNAs
were equivalent overnight after RNA electroporation (Fig. 3-12, B).
We further used the infectious HCV clone JFH1 and its replication-
defective mutant, JFH1-GND, to perform a time-course study of HCV protein
translation. Huh7 cells transfected with JFH1 or JFH1/GND were labeled with
35
S-Met for 0-8, 8-16, or 16-24 hours post-transfection, followed by
immunoprecipitation. The remaining amounts of these two RNAs at 24 hours
post-transfection were almost the same (Fig. 3-12, C). However, only JFH RNA
yielded detectable NS3 and NS5A at 16-24 hours p.t. (Fig. 3-12, D). The kinetics
of HCV protein synthesis corresponded well with the kinetic of HCV RNA
synthesis following JFH1 RNA transfection as previously reported(Pietschmann
et al., 2006). In contrast, the JFH/GND mutant did not yield detectable amount of
NS3 or NS5A, despite the presence of the GND mutant RNA comparable to JFH1
RNA. The result further supported the conclusion that the replication activity of
HCV RNA is required for the HCV RNA translation.
Further, we compared the translation activity of a bicistronic Firefly
luciferase replicon RNA (Luc-Rep) and the comparable but replication-defective

73
Renilla luciferase replicon GND mutant (RLuc-RepGND) (Fig.7A). Both
constructs had been tested by in vitro translation assay to ensure that the both
reporters were functional (data not shown). Initially, the both luciferase activities
were equivalent; however, the FFLuc/RLuc ratio increased over time (Fig. 7B),
indicating that the replication-competent HCV RNA was preferentially translated.
The reverse paired control structures, RLuc-Rep and Luc-GND, also gave a
similar result (Fig. 7C). From all the results above, we conclude that HCV RNA
replication is required for the translation of HCV RNA.

74

75

75

76
3.5 Discussion
The mechanisms of replication and translation of HCV RNA have been
extensively studied in the past few years. Cellular membrane alterations, namely,
membrane webs or vesicle-like structures, have been observed in the perinuclear
region in the Huh7 cells harboring an HCV replicon RNA(Gosert et al., 2003).
These membrane structures are thought to be related to HCV replication.
However, the exact subcellular localization of HCV RNA replication and
translation is still unclear. Evidence has been presented that HCV RNA
replication occurs on the DRM possibly derived from the ER or Golgi(Aizaki et
al., 2004; Shi et al., 2003). Intuitively, the newly synthesized RNA is thought to
be transported subsequently to the site of the cellular translation machinery. In
this study, we showed that the subcellular site for HCV protein translation is very
close to that for HCV RNA replication and that the translation is dependent on the
replication activity of the RNA. In other words, the newly synthesized RNA is
used for translation immediately before it is transported away, presumably to the
site of viral assembly. Significantly, the movement of the HCV RNA replication
complex is not required for translation. Rather, RNA replication appears to take
place in the same subcellular complex, so that blocking the RNA movement
enhanced protein translation. This mechanism of coupled RNA replication and
translation may explain the previous findings that many cellular proteins are
involved in both the replication and translation steps in the HCV life cycle.
This scenario is different from that of poliovirus replication (Iborra et al.,
2001). The movement of poliovirus viral RNA mediated by microtubule has been

77
reported to be associated with the activity of the replication complex(Egger and
Bienz, 2005). While the inactive replication complexes reside at microtubule-
organizing center (MTOC), the replicating viral RNA is localized at the
perinuclear sites(Egger and Bienz, 2005). In contrast, we found that the blockage
of HCV RNA movement by nocodazole increased protein translation, at least
initially. It was observed that the newly-synthesized HCV RNA failed to exit
from ER after the nocodazole treatment; thus, the cytoskeleton-assisted movement
of the newly-synthesized HCV RNA is not required for RNA translation.
Furthermore, the increase of translation level of HCV RNA after the nocodazole
treatment means that HCV RNA normally is synthesized near the ER and directly
used for RNA translation, without being transported via microtubule or actin
filaments to another site for protein translation. Thus, in HCV, the observed
movement of the viral RNA from the ER to the Golgi appears to be required for
other steps of HCV replication, such as viral assembly, rather than protein
translation. This conclusion is consistent with the finding that HCV replicon,
which does not encode the core protein, behaved much like the JFH1 HCV RNA
(Pietschmann et al., 2006) in the characteristics of RNA replication and
translation.
It has been observed that the translation and transcription of poliovirus
RNA are coupled, in the sense that the transcription is dependent on the viral
translation in cis(Novak and Kirkegaard, 1994). Insertion of an early termination
codon resulted in lower efficiency of RNA replication. The translation and
replication are regulated by the binding of different cellular or viral proteins to the

78
5’ UTR(Gamarnik and Andino, 2000; Perera et al., 2007; Toyoda et al., 2007). In
our study, we found that the translation of HCV RNA was directly linked to the
transcription activity of the RNA molecule. Furthermore, the time course study in
live cells showed that HCV RNA is synthesized first, which is then followed by
the translation of viral proteins from the newly synthesized RNA. Importantly, all
of the newly synthesized proteins were found to correspond to the sites of newly
synthesized RNA. Thus, HCV protein translation appears to be tightly linked to
and depend on RNA replication. This concept is novel to the known mechanisms
of RNA virus translation and transcription.
Both the replication of HCV RNA and the maturation and assembly of the
HCV viral proteins require the support of membrane structures (El-Hage and Luo,
2003; Gosert et al., 2003; Honda et al., 1996; Moradpour et al., 2004; Shi et al.,
2003; Svitkin et al., 2005). The membrane structure provides a microenvironment
to protect the HCV RNA(Tai et al., 2001), which is vital for the RNA replication
and translation. Besides, the membrane structure can also concentrate the required
factors for carrying out RNA replication and translation. In addition, the shielding
of viral RNA, including the double-strand RNA intermediate, by the membrane
structure may prevent cellular innate immune response, such as the PKR
pathways, which may result in the shut-off of translation machinery by eIF2
phosphorylation. The idea of a replication factory containing elevated local
concentrations of viral and cellular factors for HCV RNA replication and HCV
RNA translation, rather than a replication mechanism that affects the entire cell,
may explain the persistency of HCV infection.

79
Many of the positive-strand RNA viruses induce host membrane alteration
during their replication (Ahlquist, 2006), such as poliovirus(Lyle et al., 2002) and
alphaviruses(Kujala et al., 2001). Whereas poliovirus replicates in an
autophagosome-like membrane structure by remodeling the ER(Suhy et al., 2000),
alphaviruses replicates on the cytoplasmic surface of lysosomes and
endosomes(Froshauer et al., 1988). Our electron microscopic studies of HCV
replicon cells showed that the HCV RNA replication and translation were
associated with multi-layered membranous vesicles. Although the origin and
composition of this membrane structure are not yet known, it is highly likely that
this membrane structure is associated with, or derived from, the ER, which is also
suggested by previous reports(Simmonds et al., 2005). It is also possibly
generated from the Golgi apparatus, considering that this membrane structure has
characteristics of detergent-resistance. Finally, it may also be derived from other
cellular membrane structures, such as autophagosomes or lysosomes.
Whether the HCV RNA translation and replication sites are located in the
exactly same membrane structure or at two very neighboring sites is not clear.
Judging from our electronmicroscopy results, it is more likely that both RNA
replication and translation sites are in the same membrane structure, and both are
membrane associated. The live cell images showed that these structures are
similar to those of vaccinia virus, in which viral replication occurs in a distinct
cytoplasmic “factory”. Our studies further showed that the immunogold particles
representing the newly synthesized HCV RNA and those representing the newly
synthesized proteins are in the same vesicles. Most interestingly, some of the

80
newly synthesized RNA and proteins appear to be on the opposite sides of the
multilayered membrane of the same vesicle. Furthermore, the RNA-immunogold
particles appear to move from the inside to the outside of the membrane
chronologically after RNA synthesis. This may explain why the initial movement
of HCV RNA after synthesis was not blocked by nocodazole. The outside
membrane is lined with ribosomes. We propose a term, “replicasome”, to describe
this machinery for HCV RNA replication and translation, which is a discrete
structure concentrating the required viral and cellular factors locally for viral
RNA and protein synthesis.
This “replicasome” consists of the DRM, which contains all the required
factors for HCV RNA, such as eIF
3
and 18S rRNA. In addition, many of the host
proteins which regulate both HCV RNA replication and translation are also
fractionated in the DRM. Therefore, the replicasome may contain the newly-
synthesized viral proteins as well as the host factors such as PTB and SYNCRIP
(Aizaki et al., 2006; Domitrovich et al., 2005). It is not known which protein is
critical for coordinating the replication and translation of HCV RNA; however,
the host RNA-binding proteins, such as PTB and SYNCRIP, together with HCV
NS proteins, may bind to the HCV RNA to induce a structural change that
facilitates translation or transcription.

81
Chapter 4
Conclusion and Future Directions

The replication of positive-strand RNA virus has been an interesting topic to
me. By the method of BrUTP labeling in the replicon cells or HCV RNA
transfected cells, the viral replication can be monitored. The subcellular
localizations of the newly-synthesized RNA can be shown by co-staining with
other cellular organelle markers. In the time-course study, the translocation of the
BrU-labeled HCV RNA from ER to the Golgi apparatus was observed. It is
known that the transportation from ER to the Golgi is the anterograde membrane
trafficking pathways, which can be an ERGIC-dependent or independent process.
From the co-immunofluorescence stating data, we did not observe the
colocalization of BrUTP labeled RNA and the ERGIC complex; however, there
was a close association between the staining signals. Whether the translocation of
HCV RNA is dependent on ERGIC is not determined at this point. It is possible
that the intermediate steps of the anterograde membrane trafficking are very
dynamic, and therefore the colocalization may not be detected. Nevertheless, the
membrane structures have been shown to be important in several steps in the
HCV viral life cycle, such as replication, translation and the virus envelope
assembly.

82
In previous studies, Nocodazole treatment was shown to depolymerize
microtubule and also to inhibit HCV RNA replication (Bost et al., 2003). We
compared the inhibition of NS5B inhibitor, Benzothiadiazine, treatment to
nocodazole treatment in Huh-N1b replicon cells. At 2 days after treatment, the
HCV RNA titer dropped 50% in the nocodazole treatment group compared to the
untreated cells, whereas NS5B inhibitor, Benzothiadiazine treated cells had 40%
lower HCV RNA titer. The nocodazole treatment result was consistent with
previous report. The results indicated that the HCV RNA had a high stability after
transfection; although the RNA was not replicating, the RNA was only slowly
degraded. At day five after treatments, the Benzothiadiazinethe treated cell has
80% decreases in the treated replicon cells, compared to 40% decrease in the
nocodazole-treated replicon cells. This result indicates that compared to the
indirect inhibition by treatment of nocodazole, the direct inhibition of replication
by Benzothiadiazinethe has a higher efficiency of inhibition of the HCV RNA
replication in a long-term treatment.
In previous HCV studies, due to the lack of the cell culture system of HCV,
the replicon system was introduced. It was reviewed that the replication of HCV
replicon RNA and the full-length RNA shared the same mechanism (Pietschmann
et al., 2006). Indeed, in our study, the same characteristics of coupled HCV RNA
replication and translation were both observed in the replicon system and the
JHF1 strain transfected Huh-7 cells. It was recently reported that HCV NS5A
protein, which is a critical co-factor for HCV RNA replication, was associated
with HCV core protein (Miyanari et al., 2007; Shi et al., 2002; Shirota et al.,

83
2002). In the replicon system, HCV core does not express, however, we did not
observe much difference in terms of the pattern of BrU-labeled newly synthesized
RNA.
Many studies reported that translation and transcription of poliovirus are
coupled. In the case of poliovirus, the transcription is linked to the viral
translation in cis (Novak and Kirkegaard, 1994). Insertion of an early termination
codon resulted in inefficiency of replication in poliovirus. It was also reported that
the translation and transcription was regulated by the binding of different cellular
or the viral proteins to the 5’ UTR (Gamarnik and Andino, 2000; Perera et al.,
2007; Toyoda et al., 2007). The higher the translation rate, the lower the
transcription rate is of the poliovirus RNA. In our study of HCV RNA, the
translation of the RNA was directly linked to the transcription activity of the RNA
molecule, which is novel to the known mechanisms of RNA virus translation and
transcription.
By electromicroscopy, it was clearly shown that the HCV RNA replication and
translation were associated with membrane compartment. Although the origin and
the components of the membrane structure are not known to date, it is highly like
to be associated with the ER, and also possibly with the Golgi apparatus, or other
cellular membrane structures, such as autophagosomes or lysosomes.
The membrane structure provides a micro environment for the HCV RNA to
replicate and to be translated. Besides the function to protect HCV RNA and
proteins from degradation by the host nucleases and proteases, it can also
concentrate the required factors for the process of RNA replication and translation.

84
During the HCV infection, similar to the infections in other RNA viruses, the
exposed dsRNA of the replication intermediate may induce cellular innate
immune response, such as PKR pathways which may result in the shut-off of
translation machinery by eIF2 α phosphorylation. With the protection provided by
the membrane structure, the chance of exposing dsRNA to endogenous PKR
becomes much lower, and therefore the activity of the required initiation factors
can be preserved. The idea of an elevated concentration locally for HCV RNA
replication and HCV RNA translation rather than a replication mechanism which
affect the entire cell may explain the persistency of the HCV infection.
The HCV RNA can serve as the template for viral translation as well as the
template for the viral negative strand RNA synthesis. The regulation between these
two steps should be highly regulated for the benefit of virus production. Several
cellular factors were known to regulate both steps. It will be interesting to study
how these cellular proteins are involved in and how the RNA is switched from
replication machinery to the translation machinery. The mechanism of switching
between translation and replication of HCV RNA is still unclear; conceivably, it
may be regulated by these “dual-function” proteins, such as SYNCRIP, PTB, or La,
which are involved in both replication and translation. It will be interesting to
determine in the future whether the relative ratio of these proteins may trigger the
switch.
In summary, we propose (Fig. 4-1) that HCV RNA replication takes place in
an ER-derived membranous vesicle. The newly synthesized viral RNA moves
across the membrane to the outside of the vesicle, where protein translation

85
occurs. The viral proteins then are transported to lipid droplets, where viral
assembly takes place (Miyanari et al., 2007). This replication and translation
machinery constitutes the viral “replicasome”, which may reflect the membrane
webs observed previously.

86
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Asset Metadata

Creator Liu, Helene Minyi (author)

Core Title The molecular studies of HCV RNA replication and translation

Contributor Electronically uploaded by the author (provenance)

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Molecular Microbiology & Immunology

Publication Date 05/12/2010

Defense Date 12/16/2008

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

Tag HCV,live cell imaging,membrane structures,OAI-PMH Harvest,RNA replication,RNA translation

Language English

Advisor Lai, Michael (committee chair), Duncan, Roger (committee member), Ou, James (committee member)

Creator Email minyiliu@alumni.usc.edu,minyiliu@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m2250

Unique identifier UC1216845

Identifier etd-Liu-2603 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-237010 (legacy record id),usctheses-m2250 (legacy record id)

Legacy Identifier etd-Liu-2603.pdf

Dmrecord 237010

Document Type Dissertation

Rights Liu, Helene Minyi

Type texts

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

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email uscdl@usc.edu

Abstract (if available)

Abstract Hepatitis C virus RNA replication requires not only the viral replicase but also the host cytoskeletons, membrane structures, and cellular factors. Several hnRNPs, such as polypyrimidin-tract-binding protein (PTB) and La autoantigen were found to regulate both HCV RNA replication and translation. Another hnRNP, SYNCRIP (synaptotamin-Binding, Cytoplasmic RNA-Interacting Protein, or NSAP-1), was found to regulate HCV-IRES dependent translation. In my first part of dissertation, we identified SYNCRIP as a positive regulator of HCV RNA replication.