Review Article

Indian J Med Res 131, January 2010, pp 17-34

Hepatitis C virus: Molecular biology & current therapeutic options

Suresh D. Sharma

Department of Biochemistry & Molecular Biology, Pennsylvania State University, Pennsylvania, USA

Received July 23, 2009

Hepatitis C virus (HCV) is a small (~55 to 65 nm), spherical, enveloped, hepatotropic RNA virus that
causes acute and chronic hepatitis in humans. Persistent virus infection with HCV often leads to cirrhosis
and hepatocellular carcinoma (HCC). At present there is neither a selective antiviral therapy nor a
preventive vaccine. The only available treatment option is a long-acting pegylated-interferon-alpha, given
in combination with nucleoside analog ribavirin, which is not very effective. Molecular studies of HCV
began with the successful cloning of its genome in 1989. For many years, research to develop therapeutics
was stalled by the inability to grow virus in tissue culture. A major milestone was achieved with the recent
development of a robust cell culture system for HCV propagation. HCV proteins assemble and form
replication complexes on modified host membranes, called as membranous webs. Even though HCV is
detected and targeted by host immune mechanisms, it establishes and maintains a life-long persistent
infection. HCV has evolved multiple strategies to survive and persist in hostile cellular environments;
and the viral population is known to rapidly change during the course of a natural infection thereby
escaping immune surveillance. Rapid mutations also help virus to survive by selecting for the variants
which are resistant to antiviral drugs. Although precise mechanisms regulating HCV entry into hepatic
cells via receptors remain unknown, HCV also has the capability of direct cell-to-cell transmission.
The extremely complex and incompletely understood nature of the HCV lifecycle has complicated the
discovery of new therapies. A complete understanding of the functional roles played by the HCV proteins
during HCV lifecycle is vital for developing a successful cure. This review deals with current status of
efforts in addressing these daunting tasks and challenges in developing therapeutics against chronic and
rapidly changing hepatitis C virus.

Key words HCV - hepatocellular carcinoma (HCC) - novel HCV therapeutics - pegylated-Interferon - ribavirin - structural and
nonstructural proteins

Introduction a major cause of concern and a huge burden on public

Hepatitis C is a complex liver disease. Its medical
importance and the need to rapidly identify new
therapeutic approaches has resulted in intensive study
of its causative agent, hepatitis C virus (HCV). Humans
are the only known natural hosts of HCV. Even after
two decades since its discovery, HCV continues to be
health systems worldwide. The WHO estimates that
a minimum of 3 per cent of the world’s population is
chronically infected with HCV1,2.
HCV is a prototype member of the Hepacivirus
genus (from the Greek hepar, hepatos, liver) and is
further classified into at least seven major genotypes

17
18 INDIAN J MED RES, january 2010

that differ by about 30 per cent in their nucleotide

sequence. These genotypes (1, 2, 3, 4, 5, 6 & 7) show
differences with regard to their worldwide distribution,
transmission and disease progression3, 4. These
genotypes have been further classified into sub-types
(a, b, c, d, etc). In fact, HCV circulates in infected
individuals as a population of diverse but closely
related variants referred to as “quasispecies”.
HCV is most commonly spread by direct
contact with infected blood and blood products.
Availability of injectable therapies and drugs has
had a remarkable influence on HCV epidemiology.
The incubation period of HCV, though ranging up
to several months, averages 6-8 wk. HCV infection
is often asymptomatic, making it a very difficult to
detect it at an early stage. This is the major reason
why early treatment is difficult. Therefore, hepatitis C
is often referred to as a “silent disease”. In a majority
of infected people, virus infection does not resolve
naturally. Neutralizing antibodies appear to be
produced during the course of a natural infection, yet
the virus mutates to escape surveillance5. When liver
fails to clear the virus, the individuals become chronic
carriers. However, within this chronically infected Fig. 1. what happens when infected with hepatitis C virus? Acute
population the disease outcomes vary, it can be mild liver infection is followed by a chronic infection in almost 80 per
(minimal inflammation of the liver) or severe and can cent of the infected population152. Even among them the disease
lead to scar tissue formation (Fig. 1). Chronic infection outcome can be very different (mild to severe). Chronic infection
eventually causes cirrhosis leading to hepatocellular eventually causes cirrhosis leading to hepatocellular carcinoma36.
carcinoma (HCC) and ultimately death. Currently,
there is no vaccine to prevent hepatitis C. in cell culture but could also generate viral particles.
HCVcc (for HCV grown in cell culture) has allowed
Liver steatosis occurs in more than 50 per cent of
researchers for the first time to study the complete life
the patients with chronic hepatitis C (CHC) infection.
cycle of HCV. Yet, owing to the limited host range of
Some individuals with CHC may report non specific
HCV, the development of a small animal model (to
symptoms such as fatigue, muscle aches, nausea and
study viral replication and pathogenesis) is still a big
pain in the right upper quadrant. Antibodies directed
challenge in the field.
against several HCV proteins can be detected in
chronic patients. A variety of autoimmune and HCV virions exhibit a wide range of densities,
immunecomplex-mediated diseases have also been although the most infectious fraction has a density of
associated with chronic HCV infection, autoimmune 1.15-1.17 g/ml8,9. Present inside the outer envelope,
thyroiditis being one of them6. there is a (30-35 nm) inner core which encapsulates
the single-strand viral RNA (positive-sense), which
Molecular Biology of HCV
is approximately 9.6 kb (Fig. 2). The HCV genome
For many years the failure to replicate and produce does not enter the cell nucleus. HCV- RNA replication
authentic infectious hepatitis C virus in cell culture occurs in the cytoplasm of hepatocytes. The genomic
remained a major obstacle for innovative and cost- organization of HCV is shown schematically in
effective therapy. A breakthrough in the field came Fig. 3. The viral-RNA genome harbours a single ORF
with the development of a complete in vitro cell culture (open reading frame) which is flanked by 5’ and 3’
system for HCV (JFH1) in 20057,8. JFH1 viral genome non translated RNA segments (NTRs). The cis-acting
(genotype 2a), cloned from a Japanese patient with replication elements or CREs are located in both the 5'
fulminant hepatitis could not only replicate efficiently and 3' NTRs and in the NS5B coding sequence10. RNA
 Sharma: New therapeutic approaches for HCV 19
Fig. 2. Hepatitis C virus particle structure: The HCV core protein
interacts with viral genomic RNA to form the nucleocapsid51.
Two membrane-associated envelope glycoproteins, E1 and E2 are
embedded in a lipid envelope which is derived from the host8,164.
structures are known to participate and play vital roles
in the multiplication of RNA viruses.
The 5'- and the 3'-NTRs of the genome are highly
conserved and contain control elements for translation
of the viral polyprotein and replication. The 5′ UTR (+)
is ~341 nucleotides in length and contains an internal
ribosomal entry site (IRES). The HCV IRES is folded
into four stem-loop motifs which are called as I, II,
III and IV. The IRES is required for cap-independent
translation of viral RNA, which is carried out by host
cell ribosome. The domain IIId of the IRES constitutes
the key anchoring site for the 40S subunit11. The IRES
domains III-IV have also been shown to be an activator
of protein kinase R (PKR)12. However, this activation
does not interfere with cap-independent translation of
HCV viral proteins. HCV core protein was reported
to interact with the 5’-NTR of plus-strand RNA13.
However, recent work with JHF1 viral RNA suggested
that its 5’-NTR (+) does not contain RNA packaging
signals14 and authors further speculate that it may reside
in the RNA region encoding the replicase.
The 3'-UTR (+) is around ~200nt and is involved
in RNA replication. Three different domains can be
recognized in this UTR: (i) a poly (U/UC) tract with
an average length of 80 nucleotides (nt), (ii) a variable
region, and (iii) a virtually invariant 98-nt X-tail
region made up of 3 stem-loops (3’SLI, 3’SLII and
3’SLIII). The 3'-UTR can robustly stimulate IRES-
dependent translation in human hepatoma cell lines15.
Recent studies have recognized that various stem-
loop structures exist in the negative strand 3’-NTR.
This region is recognized by the viral polymerase as
the initiation site for plus-strand synthesis of the HCV
genome16. A recent study identified a cellular factor
called Far-upstream element (FUSE) binding protein
(FBP) which binds to 3'NTR by interacting with the
poly(U) tract17. The importance of long-range RNA-
RNA interactions in the modulation of HCV lifecycle
has been well documented. Within the 3’-end of the
non-structural protein 5B (NS5B) coding sequence, a
cis-acting replication element (CRE) was discovered18.
This CRE is called as SL9266 (or 5BSL3.2) and its
disruption blocks RNA replication19. Mutual long-
range binding with both 5' and 3' sequences is
suggested to stabilize the CRE at the core of a complex
pseudoknot10.
Non coding RNA molecules or microRNAs
(miR) are important in the control of gene expression
and regulation. MicroRNA, miR-122 is specifically
expressed and is found to be abundant in the human
liver20. A recent discovery showed binding of a miRNA
(miR-122) to the 5’-UTR of HCV. Sequestration of
miR-122 in liver cell lines strongly reduced HCV
translation, whereas its addition stimulated translation
via direct interaction of miR-122 with two sites in the
5'-UTR21. These studies have generated a lot of interest
in the role of miR-122 in HCV multiplication and its
potential as a therapeutic target. A role for proteasome
alpha-subunit PSMA7 in regulating HCV IRES-
mediated translation has also been demonstrated22.
These host factors require further scrutiny to be
considered as candidates for drug targets.
HCV Structural Proteins
HCV encodes a single polyprotein (NH2-C-E1-
E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH)
which is approximately 3010 amino acids (Fig. 3). The
structural proteins (core, E1 and E2) and the p7 protein
are released from the polyprotein after cleavage by
host endoplasmic reticulum (ER) signal peptidase(s).
The non structural proteins (NS2, NS3, NS4A, NS4B,
NS5A, and NS5B) are cleaved by viral proteases NS2-
3 and NS3-4A. This proteolytic processing of the
polyprotein during and after translation by host and
viral proteases yield at least 10 mature viral proteins.
Core: HCV core is a multifunctional protein which
is highly basic in nature23. It forms the structural
component of the virus particle. Core has been
implicated in the development of hepatocellular
20 INDIAN J MED RES, january 2010
Fig. 3. A schematic representation of HCV genome, structural and
nonstructural proteins: The viral RNA consists of a 5' untranslated
region (UTR) containing the internal ribosome entry site (IRES),
followed by the genomic region for structural and nonstructural
genes and the 3' UTR. HCV is translated as a polyprotein that is
proteolytically processed by host and viral proteases59. The structural
proteins (C, core; E1, envelope protein 1; E2, envelope protein
1) are located at the amino-terminal end while the nonstructural
proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) are located
at the carboxyl-terminal end70. The p7 protein (ion channel or
viroporin) is located at the junction of structural and nonstructural
proteins59.
steatosis and oncogenesis. The core protein is generated
from a polyprotein encoded by the viral genome and
is processed by cellular proteases in the endoplasmic
reticulum (ER)24. A recent study showed that core
protein can self-assemble in HCV-like particles (HCV-
LPs) in ER-membranes25. A region of core protein
(spanning amino acids 112 to 152) is essential for
association, not only with the ER but also with the outer
mitochondrial membrane26. The HCV core protein is
known to pass from the ER into mitochondria and is
involved with Ca2+ regulation and apoptotic signals27.
The HCV core protein was shown to affect the steady
state levels of a subset of mitochondrial proteins,
including prohibitin (functions as a chaperon of
mitochondrial proteins)28. This interaction of core with
the chaperon protein resulted in an increased oxidative
stress due to perturbation of normal interactions
between cytochrome c oxidase and prohibitin.
Core has many intriguing regulatory functions
with one of the most important being recruitment of
non structural proteins to the lipid droplet-associated
membranes29. Lipid droplets (LDs) are intracellular
organelles involved in lipid storage and also take
part in intracellular vesicular trafficking30. HCV
makes use of lipid droplets for replication. In infected
Huh-7 cells, the core protein is associated with the
surface of lipid droplets31. Besides its structural and
regulatory function, core plays an imperative role in
the pathogeneses of liver steatosis. Upregulation of
de novo fatty acid biosynthesis by HCV core protein
in Huh7 cells has been reported. Core protein also
interacts with apolipoprotein AII, a component of lipid
droplets. HCV core protein is targeted to lipid droplets
by its domain 2 (D2) and this association with lipid
droplets is required for virus production. Disrupting
the association of core protein with lipid droplets is
deleterious for HCVcc production and this interaction
is thought to contribute to steatosis, via deposition
of triglycerides in the liver30. These data are further
supported by studies showing that the expression of
core protein can lead to the development of steatosis in
transgenic mice28.
Clinical studies have reported that virus-induced
steatosis is very severe with HCV genotype 3 than with
other genotypes32. Interestingly, core protein derived
from genotype 3a induced higher fatty acid synthase
activity than core protein derived from genotype 1b.
However, no genetic or functional differences were
observed between genotype 3a core proteins from
patients with and without HCV-induced steatosis, thus,
suggesting a possible role of other viral proteins in
the development of hepatocellular steatosis33. HCV-
subgenomic replicon systems (which allow HCV-RNA
to replicate autonomously), have shown subcellular
localization of core to be both cytoplasmic and
nuclear26,34. A study with chronically HCV-infected liver
also revealed a similar distribution, with core localized
to both cytoplasm and the nucleus35. In the cytoplasm
the core protein is mostly localized to the endoplasmic
reticulum (ER). HCV core protein, circulating 'free' in
non enveloped state has also been detected in HCV-
infected patients36.
Transgenic mice expressing the core protein
develop HCC, indicating a direct part played by the
core protein in this process37. A study found two
mutations in the core gene (36G/C and 209A), which
were linked with increased HCC risk38. The core gene
sequence data may provide useful information about
HCC risk and more studies should be performed to
develop it further. However, it is important to consider
the type of genotype involved, as it is clear that gene
expression profile in hepatocytes is dependent on the
HCV-core-genotype sequence39. Tumour suppressor
protein promyelocytic leukaemia (PML) is known
to be involved in antiviral response. Interestingly, a
recent study suggests a potential mechanism for the
development of liver cancer via the HCV-core mediated
inactivation of the PML tumour suppressor pathway40.
 Sharma: New therapeutic approaches for HCV 21
ARFPs: Alternate reading frame proteins or ARFPs are
encoded by the +1 reading frame of the viral genome
which overlaps with the core protein coding sequence.
These additional proteins of unknown function are also
called as core+1 or F1 and were recently identified41.
Since then multiple mechanisms have been proposed
for the expression of ARFPs, which include (i) frame
shifting (ii) form transcriptional slippage, or (iii) from
internal initiation in the +1 open reading frame (ORF)
of the core protein coding sequence. ARFPs have been
shown to be associated with the ER and mitochondria.
Interestingly, these proteins are labile and very short
lived42, 43. Bases on the ability of ARFPs to bind the
proteasome subunit alpha3, it was suggested that it
may regulate protein degradation in cells44. However,
the functional role of ARFPs in the HCV lifecycle is
not clear yet.
Envelope glycoproteins: The HCV envelope
glycoproteins, E1 and E2 are structural components of
the virion. They constitute the outer coat of fine spike-
like projections of the HCV particle (Fig. 2)9. They
undergo post-translational modifications (N-linked
addition of carbohydrate chains) while being translated
in the endoplasmic reticulum (ER). Both, E1 and E2
envelope glycoproteins are required for host-cell entry
via receptor binding. Insight into the mechanisms
by which HCV gains entry into host cells is vital to
understand primary HCV infection and re-infection
post-transplantation.
The E2 protein has the binding sites for human
CD81, a tetraspanin expressed on various cell types
including hepatocytes and B lymphocytes45. The
binding of E2 was further mapped to the major
extracellular loop of CD81. CD81 along with
human scavenger receptor SR-BI, and tight junction
molecules claudin-1 (CLDN) and occludin (OCLN)
are the most important receptors that mediate HCV
entry46. In addition, it is thought that HCV may
utilize glycosaminoglycans and low density receptors
on hepatocytes as initial attachment factors. Both
CD81 and SR-BI were identified as candidate HCV
receptors based on their physical interaction with a
soluble version of E247,48. The HCVpp system was
subsequently used to prove that they are required
for viral entry49. HCVpp system (also called as HCV
pseudoparticle) generates virus particles, which
display E1-E2 glycoproteins of HCV on their surface.
Cell entry of HCVpp is HCV glycoprotein mediated.
HCVpp are generated by co-transfection of 3 plasmids
into 293T cells: (i) gag-pol genes of HIV or MLV,
(ii) HCVgp, and (iii) a retrovirus genome with LTRs,
packaging signals and a reporter gene. HCVpp system
does not require productive HCV replication and
hence is not restricted to Huh7 or Huh7.5 that support
robust replication50. HCVpp closely resembles the
cell entry properties of genuine HCV virions and was
used to identify CLDN and OCLN. The discovery of
OCLN provides an vital advance towards efforts to
develop small animal models for HCV46.
HCV-like particles (HCV-LPs) were isolated from
insect cells, infected with recombinant baculovirus
expressing HCV structural proteins(Core, E1 and E2)51.
Isolated HCV-LPs were composed of all the structural
proteins (E1, E2 and C). Further analysis of HCV-
LPs by cryoelectron microscopy (CryoEM) revealed
that they are spherical particles with smooth surfaces.
They were found to be consistent with the native HCV
virions isolated from HCV infected patients. HCV-LPs
for the first time allowed 3D structural analysis of HCV
particles51, which further allows studying the complex
mechanisms of HCV assembly and maturation.
A hypervariable domain near the amino terminus
of E2 is the most variable part of the viral polyprotein
and called as HVR-1. It has been shown to be the target
for neutralizing antibodies. It is interesting to note that
HCV can associate with LDL and VLDL from infected
patient serum. Amazingly, HDLs play a very active role
in HCV entry. A complex interplay between HDL, SR-
BI and HCV envelope glycoproteins leads to enhanced
HDL-mediated HCVpp entry in cells48. In addition,
HDL can inhibit HCV-neutralizing antibodies in serum
of acute and chronic HCV-infected patients52, 53. E2 has
also been shown to bind with CD81 receptors which
are expressed on thyroid cell and induce a cascade of
signaling pathway leading to IL-8 release. It is thought
that E2 protein may induce thyroidal inflammation,
thereby triggering thyroiditis by a bystander activation
mechanism6. Finally, E2 protein has been shown to
bind PKR and as a consequence perturb innate immune
pathways.
HCV Non Structural Proteins
The non structural proteins NS2, NS3, NS4A,
NS4B, NS5A and NS5B are thought to be to be
required for replication of the viral genome. The non
structural protein, p7, can form ion channels, required
for the production of infectious virus particles. It is
now recognized that the cross-talk between structural
and the non structural proteins of HCV is required for
efficient virus particle production54.
22 INDIAN J MED RES, january 2010
The p7 ion channel: The HCV p7 is a small 63 amino
acid protein, positioned at the junction of the structural
and non structural proteins. The p7 protein belongs
to a family of viral proteins called as viroporins that
form ion channels. It can oligomerize following its
inclusion into a lipid membrane creating ion channels.
The cleavages mediated by signal peptidases between
p7 and NS2 occurs slowly and are partial at the E2/p7
and p7/NS2 sites55. This results in the formation of an
E2p7NS2 precursor56. It is thought that this precursor
may have a role in the regulation of HCV lifecycle.
Many models have been proposed to explain the
potential significance of these precursor forms, the
simplest being, the necessity for a timely release of
the individual proteins at the appropriate time in the
viral lifecycle. However, the precise role and existence
of these precursors in a natural HCV infection is not
known.
The p7 protein is highly hydrophobic in nature.
It is localized in the ER-membranes when encoded
by a replication-competent genome57. It has two
amphipathic, transmembrane regions, TM1 and TM2
(spanning amino acids 19-32 and 36-58) which are
embedded in the ER-membrane. The N- and C-termini
are exposed to the extracellular environment58. The 3-
dimensional structure of p7-complex was determined
by using single-particle electron microscopy. This
hexameric (42 kDa) protein complex was found to
depict flower-shaped architecture with protruding petals
oriented toward the ER lumen59. The p7 ion channel
protein serves an essential role in the production of
infectious virus particles during HCV lifecycle60. It
appears to be dispensable for viral RNA replication, as
replicons lacking the p7 gene replicate or make viral
RNA efficiently61, 62.
The ion channel blocker, amantadine, was thought
to interfere with the ion channel activity of p7 based
on studies with artificial lipid bilayer system. The p7
protein could form amantadine-sensitive ion channels
in this artificial system63. However, it became clear
after clinical trials, that p7 ion channel function is not
affected by amantadine64. Another study also reported
similar findings, where HCV strains were found
resistant to the ion channel blocker, amantadine65.
Nonetheless, dose-dependent reductions of virus titres
were achieved with iminosugars64,66. The absolute
dependence of HCV on- ion channel function of p7
protein for infectivity makes it an attractive candidate
target for antiviral intervention.
HCV NS2: The non structural protein 2 (NS2) is a
23-kDa transmembrane hydrophobic protein. The
membrane association of NS2 is p7-independent and
occurs co-translationally67,68. NS2 is a membrane-
associated cysteine protease69,70, required for HCVcc
infectivity71. The cleavage between NS2 and NS3
is absolutely required for persistent viral infection
in a chimpanzee72. NS2 followed by only 2 amino
acids of NS3 produces a basal proteolytic activity
in vitro. However, the N-terminal 180 aa of NS3 are
required besides NS2 for a robust protease activity.
Interestingly, all the active site residues (H952, E972
and C993), needed for the catalytic activity of the
NS2/3 cysteine protease, are located entirely in NS273.
This requirement of NS3 remained intriguing until the
recent discovery showing that the zinc binding domain
of NS3 could in fact stimulate the protease activity of
NS2. The functional sub-domains in NS3 essentially
function as its regulatory cofactor73 again highlighting
tight regulation of the proteolytic processing. This
process is undoubtedly vital for virus multiplication.
NS2 interacts with itself forming homo-dimers.
Moreover NS2 has also been shown to interact with all
the other HCV non structural proteins74. Until recently
the only known function for NS2 was its autocleaving
activity at the NS2/3 junction. Expression of NS2 in
Huh7 cells resulted in upregulation of transcriptional
factor sterol regulatory element-binding protein 1c
(SREBP-1c) and fatty acid synthase. These studies
implicate a role of NS2 in promoting steatosis. NS2
protein can be phosphorylated by casein Kinase II on
Ser residue at position 168. This phosphorylation event
appears to regulate the stability of NS2 protein (at least
from genotype 1a). NS2 also appears to be involved
in a particle assembly step which happens post core,
NS5A, and NS3 assembly75. The search for molecules
or inhibitors targeting NS2, should without a doubt,
advance the development of new therapeutics against
HCV.
HCV NS3: The non structural protein 3 (NS3) is a
member of the superfamily 2 DExH/D-box helicases
and its crystal structure has been determined. It is a
67 kDa tri-functional protein with a serine protease,
an RNA helicase and NTPase activities (Fig. 4). The
NS3 enzyme has a chymotrypsin-like serine protease
activity76. Along with its cofactor NS4A is responsible
for cleavage at the NS3/4A, NS4A/4B, NS4B/5A
and NS5A/5B junctions. NS3/4A are localized in ER
cisternae surrounding mitochondria77. However when
co-expressed with p53 (tumour suppressor), it is
 Sharma: New therapeutic approaches for HCV 23
(Helicase domain)
Protease domain Domain I Domain II
1027 1192 1214 1353 1507
Fig. 4. A diagrammatic representation of HCV NS3 protein: The
protease and the helicase domain organization is shown with
boundaries defining these domains (amino acids 1027 to 1658 in
the polyprotein)163,164.
localized along with p53 in the nucleus. NS3 unwinds
both RNA and DNA substrates, although there is no
DNA intermediate involved in HCV lifecycle. It
couples unwinding of RNA strands (with extensive
secondary structures) to ATP hydrolysis. A direct
interaction between NS3 and NS5B occurs through
the protease domain of NS3. RNA unwinding activity
of NS3 helicase is modulated by this interaction with
NS5B polymerase78. In addition to its role in viral poly-
protein processing and HCV multiplication, another
important function of NS3 involves antagonizing host
innate-immune pathways.
The induction of type I interferon (IFN) genes (Type
I IFNs include several IFN-α subtypes and a single
IFN-β subtype) is regulated at the step of transcription
and is best understood for the IFN-β promoter. Innate
immune defense mechanisms activated by alpha/beta
INFs represent an essential first line of protection
against viral infections. Retinoic acid inducible
gene or RIG-I is a cytoplasmic RNA helicase79. It is
an essential pathogen recognition receptor (PRR)
for HCV. RIG-I upon binding viral RNA undergoes
changes in conformation and can then interact with
IFN- β promoter stimulator-1 or IPS-1(IPS-1 is also
known VISA, Cardif or MAVS)80. This interaction of
RIG-I with IPS-1 can signal downstream activation of
IRFs and NFkB to trigger alpha/beta-INFs.
The localization of MAVS to the mitochondria is
critical for its ability to induce IFNs. This function
of MAVS is abolished if the mitochondrial-targeting
domain of MAVS is deleted81. HCV NS3/4A protease
has remarkably evolved to target and cleave ISP-1
(or MAVS at Cys-508), thereby halting alpha/beta
interferon expression81. This activity may be necessary
but does not appear to be sufficient for long-term
viral persistence since there is (cytotoxic) T cell-
mediated clearance of NS3/4A-expressing hepatocytes
in vivo82. Therefore, other HCV proteins are most
likely responsible for interfering with the adaptive
immunity.
A recent study utilizing sub-genomic replicon
demonstrated that besides methylation (which
regulates helicase activity), NS3 undergoes other post-
Domain III translational modifications, such as phosphorylation
1658 and N-terminal acetylation83. The multifunctional
roles played by NS3 in the lifecycle of HCV, makes
it indispensable for virus multiplication. NS3 therefore
represents a very promising target for anti-HCV
therapy.
HCV NS4A: Non structural protein 4A (NS4A) is
a cofactor for NS3 serine protease activity. The N-
terminal portion of 4A is responsible for membrane
association of the NS3-4A complex. NS4A also
associates with other HCV proteins on the ER-derived
membranous webs, where viral replication complex
is assembled. Interestingly, NS4A is localized not
only on the ER, but also on mitochondria84. Thus, in
addition to its essential role in HCV replication it is
also involved in viral pathogenesis by affecting cellular
functions. Further, a study reported altered intracellular
distribution of mitochondria and its damage due to
NS4A expression in Huh7 cells85. The role played by
this small polypeptide during HCV replication is very
significant.
HCV NS4B: The HCV non structural protein 4B (NS4B)
is a 27-KDa polypeptide. It is a highly hydrophobic
integral ER-membrane protein86. It contains four
transmembrane domains and is palmitoylated at two
C-terminal cysteine residues. Palmitoylation of 4B
protein facilitates oligomerization which appears to be
essential for HCV replication87,88. It has an amphipatic
helix at the N-terminal and a C-terminal domain which
are both associated with membranes89. Allelic variation
in the NS4B sequence between closely related HCV
isolates was found to drastically impact HCV replication
in cell culture90. A characteristic feature of Plus-strand
RNA viruses is their ability to induce alterations in
cellular membranes and then utilize it to replicate their
own genomes.
Back in the 1980s, cytoplasmic changes were seen
in hepatocytes of infected chimpanzees with the help of
electron microscope. Sponge-like inclusion composed
of a dense matrix and irregularly arranged membranes
were reported91. Recently, Egger et al92 showed that
NS4B induced a distinct membrane alteration when
expressed in cultured human osteosarcoma (U-2 OS)
cells. Thus, a protein which had no known function
until then was shown to induce a tight structure,
designated membranous web, consisting of vesicles in a
membranous matrix. The immunoelectron microscopy
of Huh7 cells supporting replication of sub-genomic
24 INDIAN J MED RES, january 2010
replicon revealed a loss of the organization and
other morphological alterations of the ER showing
convoluted cisternae and paracrystalline structures77.
The replication of HCV is a very intricate process,
occurring through protein–RNA and protein–protein
interactions. HCV-replication-complex was first
identified in Huh7 cells supporting sub-genomic
replicon in 200393. These membranous webs are made up
of small vesicles (80-180 nm in diameter) embedded in
a membranous matrix and are found closely associated
with the rough endoplasmic reticulum. NS4B co-
localized within this web together with other structural
and NS proteins92. Interestingly, NS4B alone could
induce the formation of membranous web. In vitro
expression studies of NS4B from all major genotypes
have demonstrated the importance of N terminus of
NS4B94. Disrupting the amphipathic-helix in the N
terminus of NS4B abolishes the ability of NS4B to
rearrange membranes. Even before an important role
in formation of membranous web like structures was
discovered for this interesting protein, a role for NS4B in
malignant transformation of NIH3T3 cell in association
with the Ha-ras oncogene had been proposed95. It is an
important protein for HCV lifecycle.
HCV NS5A: The non structural protein 5A (NS5A)
has RNA-binding activity96. NS5A protein can be
found in basally phosphorylated (56 kDa) and hyper-
phosphorylated (58 kDa) forms in cells. P58 form of
NS5A is hyperphosphorylated at additional sites that
remain unmodified in the p56 form by casein kinase
I-alpha97, 98.
The first of its kind, the sub-genomic replicon
utilized a HCV genotype-1b clone, called as Con1.
This clone was engineered such that HCV-structural
genes were replaced by a neomycin resistance gene.
Following transfection of in vitro transcribed RNA
into Huh-7 cells, antibiotic G418-resistant cells could
be obtained in which the sub-genomic RNA replicated
autonomously. Mutations that enhance the capacity of
sub-genomic HCV RNA to replicate in cell culture (Huh-
7 cells) were mapped to the NS5A-coding sequence
and were called as adaptive mutations. The adaptive
mutations alter the phosphorylation state of the NS5A
protein. Loss of hyperphosphorylation (or NS5A-p58
form) stimulates RNA replication of the HCV genotype
1b replcion in Huh-7 cells. Human vesicle-associated
membrane protein (hVAP) subtype A is known to be
a host factor essential for HCV replication by binding
to both NS5A and NS5B. Phosphorylation status of
NS5A is also known to affect the interaction of NS5A
with VAP-A99. NS5A inhibitor (BMS-824) and the
NS3 protease inhibitor block hyperphosphorylation of
NS5A; however, the mechanism of inhibition remains
unknown. Further, the involvement of a protease
inhibitor in inhibiting p58 is quite intriguing100.
NS5A has an amphipathic alpha-helix at its amino
terminus with which it is anchored to the ER membrane.
NS5A is further divided into three-domains which are
separated by linker regions. The crystal structure of
the conserved domain I (DI) was solved recently and
revealed a unique fold. DI has a zinc binding motif
and also forms a nucleic acid-binding domain. NS5A
specifically binds to the G/U rich sequences in the 3’
ends of HCV genomic RNA96. Domains II (DII) and
III (DIII) are more variable among HCV genotypes.
Crystal structures for both DII and DIII are not
available yet. NMR studies have shown DII of NS5A
to be flexible and disordered. NS5A-DII contains
the PKR and the HCV-NS5B binding domains. A
role for NS5A-DIII in virus assembly was shown via
phosphorylation of a serine residue at position 2433
by CKII101. NS5A domain III is not required for RNA
replication as sub-genomic replicon lacking DIII,
replicate in Huh7 cells. However, a recent study with
JFH1 virus showed that DIII of NS5A could influence
RNA replication. Genotype 2 HCV isolates have
a 19 residue insertion near the C terminus of DIII.
Upon its deletion, authors observed a delay in both
RNA replication and particle assembly102 thereby
suggesting a role in viral multiplication.
Full length HCV NS5A has a cytoplasmic
localization. Cleavage by caspase 3 and 6 leads to
genearation of N-terminally truncated NS5A fragments
which get localized within the nucleus. The C-terminal
domain of NS5A can associate with c-Raf kinase103.
This interaction appears to be essential for HCV
replication in Huh7 cells, as sequestration of c-Raf by
truncated NS5A into the nucleus, negatively impacts
HCV replication104. Oxysterol binding protein (OSBP)
can interact with the N-terminal region of DI. OSBP is
co-localized to the golgi with NS5A and this functional
interaction is suggested to play a role in HCV particle
release105. A significant association between variations
in sequences between 2209-2248 nucleotides of NS5A-
gene, and response to interferon treatment has been
proposed. This region of NS5A (amino acids 237−276)
is called as ISDR or interferon sensitivity determining
region. The ISDR was also found to associate with the
antiviral molecule, PKR. Patients with a mutant-type
 Sharma: New therapeutic approaches for HCV 25
NS5A-ISDR had a higher rate of sustained virologic
response (SVR) than those with the wild-type NS5A-
ISDR. However, recent studies have questioned the
existence of an ISDR in NS5A. Existence of an ISDR
is an ongoing debate and controversial. Nevertheless,
NS5A can repress the PKR pathway. NS5A also has
the ability to inhibit IFN-gamma production106. A
remarkable study with NS5A transgenic mice suggested
that NS5A protein could impair both the innate and
adaptive hepatic immune response107. Indeed, NS5A
has attracted tremendous attention in the HCV field
and represents a very promising target for anti-HCV
therapy.
HCV NS5B: The HCV non structural protein 5B
(NS5B) is a RNA dependent RNA polymerase (RDRP)
containing the GDD motif in its active site108. It belongs
to a class of integral membrane proteins termed
tail-anchored proteins. NS5B initiates synthesis of
complementary negative-strand RNA using the HCV
genome (positive polarity) as a template. Subsequently
it generates positive-strand RNA from this negative-
strand RNA template. The NS5B crystal structure
shows the typical fingers, palm and thumb sub-domains.
NS5B is anchored to the ER-derived “membranous
webs" via its C-terminal 21 amino-acid residues109.
Studies with sub-genomic replicon revealed that the
membrane association is indispensable for viral RNA
replication110. In vitro NS5B cannot distinguish between
natural and synthetic templates. NS5A can directly
bind to NS5B and modulate its polymerase (poly A
template/poly U primer) activity111. NS5A (at sub-
stoichometric levels) stimulates replication by NS5B
on templates derived from the 3’ end of the positive
strand112. NS5A stimulates NS5B during elongation;
however the mechanism of action is not clear. It
could either be due to the stimulation of its catalytic
activity or due to a conformational change induced by
NS5A binding112. There appears to be a difference in
polymerase specific activity in vitro, depending on the
type of HCV genotype. The JFH1 NS5B enzyme shows
a 10- fold-higher specific activity when compared to J6
NS5B113. The termination of RNA synthesis in vitro is
not understood well.
NS5B lacks a “proofreading” function. Due to a
high rate of error-prone replication, complex mutant
swarms are generated. However, HCV must also
maintain highly conserved genomic segments and a
balance between conserved and variable viral elements
is above all important to avoid "error catastrophe".
Over the past few years numerous nucleoside and
non nucleoside inhibitors of the polymerase have
been discovered and demonstrated clinical efficiency
and advanced to clinical trials. NS5B nucleoside
inhibitors (NIs) or analogs (NM-107/NM-283, PSI-
6130/R7128, IDX184, MK-0608) compete with
cellular ribonucleoside triphospahtes act as functional
chain terminators114. Non-nucleoside inhibitors (NNIs)
or analogs (ABT-333, GSK625433, VCH-759, PF-
868554, GS-9190) act by allosteric mechanism. They
bind to allosteric sites on NS5B and thereby inhibit
RNA synthesis114,115.
An important lead was provided by a study which
established that NS5B associates with cyclophilin
A (CypA) via its enzymatic pocket and exploits the
isomerase/chaperone activity of CypA to replicate in
cells116. NS5B is phosphorylated by the protein kinase C-
related kinase 2 (PRK2) in cell culture117. It was further
discovered that inhibitors of NS5B phosphorylation,
HA1077 (fasudil) and Y27632 suppressed the
activation of PRK2. The treatment of liver cell lines
(stably replicating HCV sub-genomic replicon) with
these inhibitors cleared viral RNA suggesting that
PRK2 inhibitors act by suppressing HCV replication
via inhibition of NS5B activity118. However, it would
be interesting to see if resistant mutants emerge over
a period of time. More critical would be the normal
functions affected which are regulated by PRK2.
The continuous generation and selection of
resistant variants allows HCV to escape control by
these inhibitors/antiviral drugs. A study where patients
infected with genotype 1a were treated with ribavirin,
led to the emergence of a Phe-to-Tyr (F415Y) mutation
in the viral polymerase119. There is also evidence for
recombination events leading to genetic variation in
HCV120. Thus, very high rates of genetic variation
ensure the survival of hepatitis C viruses under every
changing hostile cellular environment.
Current Therapy
The very first treatment shown to be effective
against HCV involved systematic administration
of INF-α. INFs are produced naturally by host’s
immune system in response to a viral infection. INF
was actually discovered in 1957, during studies with
influenza virus121. Through the years HCV treatment has
improved with the development of modified pegylated-
interferon (PEG-INF), which is a stabilized version,
has longer biological half-life. A SVR is defined as
negative HCV RNA, six months after treatment. INF is
known to activate several direct and indirect antiviral
26 INDIAN J MED RES, january 2010

mechanisms (such as viral RNA degradation, halt viral genes involved in the IFN-α pathway could possibly
translation). However, the antiviral mechanism of IFN explain some of these leading questions. This insight
in vivo remains unknown. and further research exploiting a relationship between
therapy response and host genetic polymorphisms in
Ribavirin was first used in treating children with
the IFN-α pathway may lead to the development of
respiratory syncytial virus infection122. It was discovered
new therapeutic strategies129.
more than 30 yr ago and is effective against HCV, only
when administered along with interferon in combination Current State of Antivirals
therapy. Ribavirin (1-beta-D-ribofuranosyl-1, 2, 4-
At present, there is no approved (HCV specific)
triazole-3-carboxamide) is a purine-analog with broad-
antiviral therapy. However, search for “specific
spectrum antiviral activity123.Administration of ribavirin
targeted antiviral therapy for HCV” (STAT-C) has been
in combination with PEG-INF-alfa-2a (40kDa) to HCV
ongoing for a long time. Advances in the model systems
infected patient’s results in an increased SVR rate124,125.
such as: (i) sub-genomic and genomic replicons, (ii)
The mechanisms or pathways involved, leading to
HCVpp, (iii) HCVcc-JHF1, (iv) HCV-LPs to study
anti-HCV effects are not fully understood yet. Some
HCV have further improved our understanding of
of the proposed mechanisms include: modulation
HCV lifecycle and potential therapeutic targets. The
of interferon-stimulated gene expression, inhibition
latest development being, the ability to generate HCV
of host inosine monophosphate dehydrogenase (by
particles that are infectious both in cell culture and
ribavirin monophosphate), modulation of the host
in vivo with the Con1 wild type genome54. We are
immune responses, direct inhibition of the viral RNA
approaching a very productive as well as challenging
polymerase (by ribavirin triphosphate) and lethal
phase in the field of HCV virology. Wide varieties
mutagenesis of HCV-RNA genomes caused via
of inhibitors or directly acting antiviral agents are at
incorporation of ribavirin triphosphate by NS5B126, 127.
different phases of development and some are currently
Thus, ribavirin can be a substrate for the HCV RNA
in clinical trials. These inhibitors target HCV receptors,
polymerase although surprisingly it is not very effective
HCV-IRES, NS3/4A, NS5A and NS5B. In addition
in monotherapy128. Therefore, pegylated interferon
molecules targeting host factors which play a role in
(PEG-INF) and ribavirin combination has become the
HCV replication, are also being pursued.
current “gold standard” regimen or treatment of choice
for hepatitis C. HCV enters into hepatocytes by interacting with cell
surface receptors. These initial events in virus lifecycle
Unfortunately, both these compounds are toxic
offer an attractive target for antiviral development. The
and their administration causes side effects such as
lectin cyanovirin-N (CV-N) was originally discovered
headache, fever, severe depression, myalgia, arthralgia
against HIV and subsequently also shown to be a potent
and haemolytic anaemia. Moreover, the cost of therapy
inhibitor of HCV. It binds to envelope glycoproteins and
is very high. Several months of therapy is required for
inhibits the interaction between E2 and CD81130. This
eradication of chronic HCV infection, despite which
suggests that targeting the glycans of viral envelope
SVR is often not achieved. In summary, the existing
proteins holds potential in the development of antiviral
treatment is not very effective and causes significant
therapies. Another interesting class of inhibitors such as
side effects. Development of better and improved
celgosivir, n-butyl deoxynojirimycin and N-(n-nonyl)
treatment is required urgently and represents a major
deoxynojirimycin target host alpha-glucosidase enzyme
challenge for virologists
and may offer alternative treatment options131,132.
In addition, responses to treatment vary widely Alpha-glucosidase enzyme plays a critical role in HCV
depending on the genotype of HCV. Genotypes 1 and maturation by initiating the processing of the N-linked
4 are the most difficult-to-treat. Only 40-50 per cent of oligosaccharides of viral envelope glycoproteins133.
the patients achieve SVR with genotype 1 infections. Thus their inhibitors can affect HCV morphogenesis.
Age is also an important factor as older patients exhibit This was further confirmed by using HCV-LPs, in
lower response to therapy than younger patients. the presence of alpha-glucosidase inhibitors, viral
Moreover, current available treatment options for non glycoproteins were misfolded, retained in the ER and
responders are very few. The factors determining the also impaired in their interaction with calnexin134. Thus,
triumph or failure of the antiviral therapy are not well this novel class of inhibitors can target host-directed
understood. A clear understanding of the function of glycosylation and may prevent the appearance of drug
 Sharma: New therapeutic approaches for HCV 27
resistance when given in combination with the current
standard of care132. Celgosivir (rapidly converted to
castanospermine) is currently undergoing clinical
trials.
A study where phosphatidylinositol 4-kinase type
III-alpha (PI4KIII-α) was knocked down prevented
infection by HCVpp or by HCVcc (JFH-1). In
addition, the susceptibility to HCVpp infection and
replication in cells was dependent on the presence of
phosphatidylinositol 4-kinase type III-beta (PI4KIII-
β)135. Another study showed that PI4K-III-α is involved
in events vital for membrane alterations, which
are essential for the formation of HCV replication
complexes136. PI4K-III-α also plays an essential role
in membrane alterations leading to the formation of
HCV replication complexes. PI4KIII-α and β are very
important host factors for HCV replication. These
kinases are potential therapeutic targets.
Other recent studies have demonstrated that iridoids
(such as aglycones of shanzhiside methyl ester: loganin,
verbenalin) and amphipathic DNA polymers (APs) are
novel antiviral molecules which inhibit HCV entry.
Inhibitors loganin and verbenalin were shown to have
anti-HCV entry and anti-infectivity activities137. APs
inhibit HCV internalization owing to their amphipathic
nature. Thus APs target the cell entry step of HCV.
A recent study showed that CD81 has no role in the
cell-to-cell spread of HCVcc. In addition, this mode of
transmission appears to be shielded from neutralizing
antibodies. This study highlights the urgent need to
develop strategies aimed at disrupting direct cell-to-
cell HCV transmission as targeting cell-free virus
alone may not be enough to halt a chronic infection
and a dual approach is required138.
The significance of targeting NS3-4A protease
is clearly evident from its vital function, required
to process viral polyprotein. Targeting NS3-4A has
additional advantage, since NS3-4A protease also
functions to inhibit host innate response by cleaving
IFN-β promoter stimulator-1 (IPS-1) and TRIF139. The
latter activity of NS3 affects signaling of the RIG-I
and Toll-like-receptor-3 pathways, thereby perturbing
the interferon response. A specific inhibitor of NS3,
telaprevir (VX-950) was put to clinical test recently
with some success. The current thought in the field
being that a combination therapy along with inhibitors
which simultaneously inhibit multiple steps in the
virus lifecycle (protease and replicase functions) may
yield better SVR. Indeed, recent clinical trials where
PEG-IFN and ribavirin were administered along with
telaprevir, showed significantly improved SVR rates in
patients with genotype 1, which happens to be the most
difficult to treat140. However, there were higher rates of
discontinuation in the treatment because of the adverse
effects such as pruritus, rash, and anaemia.
Another study which involved administration of
interferon with telaprevir highlighted the importance
of retaining ribavirin during the treatment. As response
rates were lowest when ribavirin was not present
in the treatment141. There is supporting evidence of
a decreased SVR and relapse when ribavirin was
prematurely discontinued or ribavirin doses were
frequently missed. However, the major drawback of
ribavirin is side effects. Further research is required to
advance, refine and develop non-toxic versions of these
inhibitors. There is some progress in this direction and
several ribavirin analogues (such as 4-iodo-1-beta- D-
ribofuranosylpyrazole-3-carboxamide, 4-propynyl-
1-beta-D-ribofuranosylpyrazole-3-carboxamide, and
4-phenylethynyl-1-beta-D-ribofuranosylpyrazole-3-
carboxamide) are being evaluated and could offer an
alternative and better therapeutic option142.
NS5A plays a vital role in HCV replication and
is emerging as an important target for the therapy of
HCV infection. BMS-824 is a small molecule which
targets NS5A and inhibits hyperphosphorylation or p58
formation100. Inhibitors that disrupt phosphorylation
with a concomitant decrease in virus replication are
indeed very attractive and should be developed further.
Another molecule, zinc mesoporphyrin (a synthetic
heme analogue with a central zinc of the mesoporphyin
macrocycle) causes a dramatic downregulation of the
NS5A protein by enhancing its polyubiquitination
and proteasome-dependent degradation. Zinc
mesoporphyrin holds immense potential as a novel
drug to treat HCV infection143.
HCV polymerase is an attractive target owing to its
essential role in viral RNA replication. It is beyond any
doubt the most validated target for the development
of antiviral therapy. Valopicitabine (NM-283) was the
first NS5B analogue to demonstrate proof of concept
for 5B nucleoside inhibitors in the clinic. Currently
only Roche-1728 NI is under active development and
clinical trials144. Idenix NI IDX-184 is under phase I
trial in patients infected with genotype 1. Another NI
from Merck, MK-0608 is in preclinical trials. The
NIs can be utilized by the polymerase once converted
to the 5’-triphosphate form. NNIs on the other hand
28 INDIAN J MED RES, january 2010
are non-competitive inhibitors which target the viral
polymerase, preferentially in the initiation phase. A
NNI, PF-00868554 (Pfizer) is currently in phase II
clinical trials where it is given along with PEG-INF
and ribavirin. GS-9190 (Gilead) is under development
and currently a study with VCH-759 (ViroChem
Pharma) is ongoing. Several NNIs such as VCH-916
(ViroChem Pharma), GSK-625433 (GlaxoSmithKline)
and ABT-333 (Abbott) have reached phase I clinical
trials.
A further advance came with the concept of
administering inhibitors of both, viral and host
proteins. This seems a very intelligent choice for a
rapidly mutating virus like HCV and might be vital to
success in forthcoming therapies. Remarkable work
is being done in this regard with a specific host factor
cyclophilin (Cyp), which belongs to a family of cellular
peptidyl-prolyl isomerases. It is required for HCV
replication. Cyclosporine A inhibits HCV replication by
binding to cyclophilin. It has now been established that
cyclosporine A and other non immunosuppressive Cyp
inhibitors (such as Debio 025, NIM811, and SCY-635)
effectively block HCV replication in vitro116. A recent
study investigated the effects of combining NIM-811
(host inhibitor) with specific inhibitors of viral protein
using replicon system145. Interestingly, a combination
of specific viral inhibitors (viral protease or polymerase
inhibitors) with NIM811 prevented the development of
resistance very effectively. Cyclosporine A derivatives
such as Debio 025 that lack the immunosuppressive
function, are currently in clinical trials146.
HCV has developed complex mechanisms to use the
variety and intricacy of the host lipidome. A promising
candidate for anti-HCV therapy emerged with the recent
development of statins, a drug that interferes with lipid
metabolism. Statins are inhibitors of 3-hydroxy-3-
methylglutaryl coenzyme A (HMG-CoA) reductase.
Statins have been approved clinically for the treatment
of hypercholesterolaemia. These cause a reduction in
intracellular mevulonate, LDL and geranylgeraniol,
thereby inhibiting cholesterol biosynthesis and also
causing a decrease in prenylated proteins147,148. This
drug has also been used to further enhance the antiviral
effects of INF148. An interesting study utilizing replicon
system showed that statins (mevastatin or simvastatin)
in combination with polymerase or protease inhibitors
could effectively clear the cultures from HCV replicons.
In addition, mevastatin was found to prevent selection
of replicons resistant to the non nucleoside inhibitor
HCV-796149. These cell culture studies suggest that a
combination of specific viral inhibitors with statins
may delay or prevent the development of resistance to
viral inhibitors. However, this remains to be tested in
clinical trials.
MicroRNAs (miR) are (~22 nucleotide) naturally
occurring noncoding RNAs. These belong to a family
of small RNA molecules that perform and regulate
critical cellular functions. MicroRNAs serve as
gene regulators controlling the cell cycle and tissue
differentiation150. These are also implicated in apoptosis
and oncogenesis151. The miRNA (miR-122) is required
for HCV replication and development of antiviral
therapies targeting on specific miRNA has great potential
for exploration. However, a better understanding of the
role for miR-122 binding to the HCV is required for
effective design of antiviral therapies. Attempts are
underway utilizing RNAi technology and one such
attempt is the designing of TT-033, which contains three
separate RNAi molecules to shut down replication of
all strains of the hepatitis C virus. This strategy sounds
promising and could potentially stop the generation of
viral escape mutants165.
Another promising area for exploring therapeutic
options are targeted at processing bodies (P bodies).
These P bodies are important for many viruses in their
lifecycles. Recent studies have further highlighted the
importance of miRNAs and P bodies in modulating
host cell interactions with viruses152. An interesting
study established the role of miR-29a and P bodies in
regulating HIV-1 production and infectivity152. A link
between HCV and P bodies has also been observed153.
The RNA helicase DDX3 is a component of P bodies
and is required for HCV RNA replication154. HCV core
protein binds and colocalizes with DDX3 in P bodies155.
The significance of core-DDX3 helicase interaction in
HCV pathogenesis is an active area of research.
There are also reports indicating progress with
therapeutic vaccines for HCV. IC41, a synthetic
peptide vaccine, containing HCV T-cell epitopes has
been recently shown to be safe and is currently in
clinical trial. IC41 can induce HCV-specific INF-
gamma-secreting CD4+ and CD8+ T cells in healthy
individuals. It was shown to induce HCV-specific
Th1/Tc1 responses in a subset of difficult to treat
HCV non-responder patients156. There is also evidence
that broadly neutralizing antibodies against antigenic
regions of E2 can protect Alb-uPA/SCID mouse
(human liver-chimeric), against HCV challenge157.
 Sharma: New therapeutic approaches for HCV 29
Another interesting candidate for a peptide vaccine
is the HCV-specific HLA-A2-restricted NS3 (1073)
epitope158. As with any RNA virus, rapidly mutating
genomes such as HCV pose a huge obstacle to antiviral/
vaccine development and represents a major challenge
to the field. In addition, HCV acquires a lipid envelope,
which originates from host membranes. Thus, the lipid
composition of the HCV envelope resembles that of the
host cell membrane and this clever mimicry may allow
HCV to avoid detection by the immune system159.
Conclusions
Recent advances in our understanding of HCV
structure, genome and its lifecycle have revealed
numerous target sites for potential pharmacological
intervention. These should help in further improving
HCV treatment. Efforts by investigators across the
globe have led to detailed molecular characterization
of the virus, but despite these advances in our
understanding, many fundamental questions still
remain unanswered. For example, we are yet to find
the mechanisms by which HCV replicates in human
liver? What are the molecular mechanisms by which
viral proteins initiate hepatocarcinogenesis? What
are the underlying defense mechanisms that play a
role in the natural resolution of HCV infection in a
very small percentage of people160. The membrane-
associated RNA replication complex presumably
involves various host proteins. However, the precise
host components and mechanisms for replication
are not entirely understood yet. Current efforts to
develop antiviral therapeutics are haunted by the
unique nature of the HCV, i.e., the error-prone nature
of NS5B, generation of resistance mutants which
presents a selective growth advantage. To circumvent
this major hurdle in HCV therapy, the current research
efforts are aimed at combination therapy targeting
multiple proteins both viral and host. Numerous novel
HCV specific inhibitors such as nucleoside and non
nucleoside polymerase inhibitors, protease inhibitors,
as well as non HCV specific compounds with antiviral
activity such as nitazoxanide, cyclophilin inhibitors
(Debio 025), silibinin are under development in clinical
trials. An immunotherapeutic vaccine along with
the current therapy may also be beneficial especially
in immunocompromised patients. In the meantime,
combination therapy utilizing PEG-INF and ribavirin
will remain standard of care. A recent estimate projects
the current treatment market for HCV to be around
US$ 3 billion per year. This market is expected to grow
rapidly and reach around US$ 8 billion by 2010161.
Acknowledgment
Author thanks Dr Craig E. Cameron (Pennsylvania State
University) for encouragement and support.
References
1. Global surveillance and control of hepatitis C. Report of a who
consultation organized in collaboration with the viral hepatitis
prevention board, antwerp, belgium. J Viral hepat 1999; 6 :
35-47.
2. Alter, MJ, Epidemiology of hepatitis C virus infection. World
J Gastroenterol 2007; 13 : 2436-41.
3. Gottwein JM, Scheel TK, Jensen TB, Lademann JB, Prentoe
JC, Knudsen ML, et al, Development and characterization
of hepatitis C virus genotype 1-7 cell culture systems: Role
of cd81 and scavenger receptor class b type i and effect of
antiviral drugs. Hepatol 2009; 49 : 364-77.
4. Kuiken C, Simmonds P, Nomenclature and numbering of the
hepatitis C virus. Methods mol biol (Clifton) NJ 2009; 510 :
33-53.
5. von Hahn T, Yoon JC, Alter H, Rice CM, Rehermann B,
Balfe P, et al. Hepatitis C virus continuously escapes from
neutralizing antibody and t-cell responses during chronic
infection in vivo. Gastroenterology 2007; 132 : 667-78.
6. Akeno N, Blackard JT, Tomer Y. HCV E2 protein binds
directly to thyroid cells and induces IL-8 production: A
new mechanism for HCV induced thyroid autoimmunity. J
Autoimmun 2008; 31 : 339-44.
7. Zhong J, Gastaminza P, Cheng G, Kapadia S, Kato T, Burton
DR, et al. Robust hepatitis C virus infection in vitro. Proc Natl
Acad Sci USA 2005; 102 : 9294-9.
8. Wakita T, Pietschmann T, Kato T, Date T, Miyamoto M, Zhao Z,
et al. Production of infectious hepatitis C virus in tissue culture
from a cloned viral genome. Nat Med 2005; 11 : 791-6.
9. Kaito M, Watanabe S, Tanaka H, Fujita N, Konishi M, Iwasa
M, et al. Morphological identification of hepatitis C virus e1
and E2 envelope glycoproteins on the virion surface using
immunogold electron microscopy. Int J Mol Med 2006; 18 :
673-8.
10. Diviney S, Tuplin A, Struthers M, Armstrong V, Elliott RM,
Simmonds P, et al. A hepatitis C virus cis-acting replication
element forms a long-range RNA-RNA interaction with
upstream rna sequences in ns5b. J Virol 2008; 82 : 9008-22.
11. Lukavsky PJ, Otto GA, Lancaster AM, Sarnow P, Puglisi JD.
Structures of two RNA domains essential for hepatitis C virus
internal ribosome entry site function. Nat Struct Biol 2000; 7 :
1105-10.
12. Shimoike T, McKenna SA, Lindhout DA, and Puglisi JD.
Translational insensitivity to potent activation of pkr by HCV
IRES RNA. Antiviral Res 2009.
13. Fan Z, Yang QR, Twu JS, Sherker AH. Specific in vitro
association between the hepatitis C viral genome and core
protein. J Med Virol 1999; 59 : 131-4.
14. Friebe P, Bartenschlager R. Role of rna structures in genome
terminal sequences of the hepatitis C virus for replication and
assembly. J Virol 2009; 83 : 11989-95.
15. Song Y, Friebe P, Tzima E, Junemann C, Bartenschlager R,
Niepmann M. The hepatitis C virus RNA 3'-untranslated
30 INDIAN J MED RES, january 2010
region strongly enhances translation directed by the internal
ribosome entry site. J Virol 2006; 80 : 11579-88.
16. Ye L, Timani KA, Kong L, Yang X, Liao Q, Wu J. Two cis-
acting elements in negative RNA strand of hepatitis C virus
involved in synthesis of positive RNA strand in vitro. Acta
Virol 2005; 49 : 83-90.
17. Zhang Z, Harris D, Pandey VN. The fuse binding protein is a
cellular factor required for efficient replication of hepatitis C
virus. J Virol 2008; 82 : 5761-73.
18. You S, Stump DD, Branch AD, Rice CM. A cis-acting
replication element in the sequence encoding the ns5b RNA-
dependent RNA polymerase is required for hepatitis C virus
RNA replication. J Virol 2004; 78 : 1352-66.
19. Friebe P, Boudet J, Simorre JP, Bartenschlager R. Kissing-
loop interaction in the 3' end of the hepatitis C virus genome
essential for RNA replication. J Virol 2005; 79 : 380-92.
20. Jopling CL. Regulation of hepatitis C virus by microrna-122.
Biochem Soc Trans 2008; 36 : 1220-3.
21. Henke JI, Goergen D, Zheng J, Song Y, Schuttler CG, Fehr C,
et al. Microrna-122 stimulates translation of hepatitis C virus
RNA. EMBO J 2008; 27 : 3300-10.
22. Kruger M, Beger C, Welch PJ, Barber JR, Manns MP, Wong-
Staal F. Involvement of proteasome alpha-subunit psma7
in hepatitis C virus internal ribosome entry site-mediated
translation. Mol Cell Biol 2001; 21 : 8357-64.
23. Santolini E, Migliaccio G, La Monica N. Biosynthesis and
biochemical properties of the hepatitis C virus core protein. J
Virol 1994; 68 : 3631-41.
24. Targett-Adams P, Hope G, Boulant S, McLauchlan J,
Maturation of hepatitis C virus core protein by signal peptide
peptidase is required for virus production. J Biol Chem 2008;
283 : 16850-9.
25. Ait-Goughoulte M, Hourioux C, Patient R, Trassard S, Brand
D, Roingeard P. Core protein cleavage by signal peptide
peptidase is required for hepatitis C virus-like particle
assembly. J Gen Virol 2006; 87 : 855-60.
26. Suzuki R, Sakamoto S, Tsutsumi T, Rikimaru A, Tanaka K,
Shimoike T, et al. Molecular determinants for subcellular
localization of hepatitis C virus core protein. J Virol 2005; 42. Varaklioti A, Vassilaki N, Georgopoulou U, Mavromara P.
79 : 1271-81.
27. Williamson CD, Colberg-Poley AM. Access of viral proteins
to mitochondria via mitochondria-associated membranes. Rev
Med Virol 2009; 19 : 147-64.
28. Tsutsumi T, Matsuda M, Aizaki H, Moriya K, Miyoshi H,
Fujie H, et al. Proteomics analysis of mitochondrial proteins
reveals overexpression of a mitochondrial protein chaperon,
prohibitin, in cells expressing hepatitis C virus core protein.
Heptatol 2009; 50 : 378-86.
29. Miyanari Y, Atsuzawa K, Usuda N, Watashi K, Hishiki T,
Zayas M, et al. The lipid droplet is an important organelle for
hepatitis C virus production. Nat Cell Biol 2007; 9 : 1089-97.
30. Roingeard P. Hourioux C. Hepatitis C virus core protein, lipid
droplets and steatosis. J Viral Hepat 2008; 15 : 157-64.
31. McLauchlan J. Lipid droplets and hepatitis C virus infection.
Biochim Biophys Acta 2009; 1791 : 552-9.
32. Hourioux C, Patient R, Morin A, Blanchard E, Moreau A,
Trassard S, et al. The genotype 3-specific hepatitis C virus
core protein residue phenylalanine 164 increases steatosis in
an in vitro cellular model. Gut 2007; 56 : 1302-8.
33. Piodi A, Chouteau P, Lerat H, Hezode C, Pawlotsky JM.
Morphological changes in intracellular lipid droplets induced
by different hepatitis C virus genotype core sequences and
relationship with steatosis. Heptatol 2008; 48 : 16-27.
34. Barba G, Harper F, Harada T, Kohara M, Goulinet S, Matsuura
Y, et al. Hepatitis C virus core protein shows a cytoplasmic
localization and associates to cellular lipid storage droplets.
Proc Nat Acad Sci USA 1997; 94 : 1200-5.
35. Lo SY, Masiarz F, Hwang SB, Lai MM, Ou JH. Differential
subcellular localization of hepatitis C virus core gene products.
Virology 1995; 213 : 455-61.
36. Sansonno D, Lauletta G, Nisi L, Gatti P, Pesola F, Pansini N,
et al. Non-enveloped HCV core rotein as constitutive antigen
of cold-precipitable immune complexes in type ii mixed
cryoglobulinaemia. Clin Exp Immunol 2003; 133 : 275-82.
37. Moriya K, Fujie H, Shintani Y, Yotsuyanagi H, Tsutsumi T,
Ishibashi K, et al. The core protein of hepatitis C virus induces
hepatocellular carcinoma in transgenic mice. Nat Med 1998;
4 : 1065-7.
38. Fishman SL, Factor SH, Balestrieri C, Fan X, Dibisceglie AM,
Desai SM, et al. Mutations in the hepatitis C virus core gene
are associated with advanced liver disease and hepatocellular
carcinoma. Clin Cancer Res 2009; 15 : 3205-13.
39. Pazienza V, Clement S, Pugnale P, Conzelmann S, Pascarella
S, Mangia A, et al. Gene expression profile of huh-7 cells
expressing hepatitis C virus genotype 1b or 3a core proteins.
Liver Int 2009; 29 : 661-9.
40. Herzer K, Weyer S, Krammer PH, Galle PR, Hofmann TG,
Hepatitis C virus core protein inhibits tumor suppressor
protein promyelocytic leukemia function in human hepatoma
cells. Cancer Res 2005; 65 : 10830-7.
41. Shesheer Kumar M, Venkateswara Rao K, Mohammed
Habeebullah C, Dashavantha Reddy V. Expression of alternate
reading frame protein (f1) of hepatitis C virus in escherichia
coli and detection of antibodies for f1 in indian patients. Infect
Genet Evol 2008; 8 : 374-7.
Alternate translation occurs within the core coding region of
the hepatitis C viral genome. J Biol Chem 2002; 277 : 17713-
21.
43. Vassilaki N, Mavromara P. Two alternative translation
mechanisms are responsible for the expression of the HCV
arfp/f/core+1 coding open reading frame. J Biol Chem 2003;
278 : 40503-13.
44. Yuksek K, Chen WL, Chien D, Ou JH. Ubiquitin-independent
degradation of hepatitis C virus f protein. J Virol 2009; 83 :
612-21.
45. Pileri P, Uematsu Y, Campagnoli S, Galli G, Falugi F, Petracca
R, et al. Binding of hepatitis C virus to cd81. Science 1998;
282 : 938-41.
46. Ploss A, Evans MJ, Gaysinskaya VA, Panis M, You H, de Jong
YP, et al. Human occludin is a hepatitis C virus entry factor
required for infection of mouse cells. Nature 2009; 457 : 882-
6.
47. Owsianka AM, Timms JM, Tarr AW, Brown RJ, Hickling TP,
Szwejk A, et al. Identification of conserved residues in the E2
 Sharma: New therapeutic approaches for HCV 31
envelope glycoprotein of the hepatitis C virus that are critical
for cd81 binding. J Virol 2006; 80 : 8695-704.
48. Voisset C, Callens N, Blanchard E, Op De Beeck A, Dubuisson
J, Vu-Dac N. High density lipoproteins facilitate hepatitis C
virus entry through the scavenger receptor class b type i. J Biol
Chem 2005; 280 : 7793-9.
49. Cocquerel L, Voisset C, Dubuisson J. Hepatitis C virus entry:
Potential receptors and their biological functions. J Gen Virol
2006; 87 : 1075-84.
50. Voisset C, Dubuisson J. Functional hepatitis C virus envelope
glycoproteins. Biol Cell 2004; 96 : 413-20.
51. Yu X, Qiao M, Atanasov I, Hu Z, Kato T, Liang TJ, et al. Cryo-
electron microscopy and three-dimensional reconstructions of
hepatitis C virus particles. Virology 2007; 367 : 126-34.
52. Voisset C, Op de Beeck A, Horellou P, Dreux M, Gustot
T, Duverlie G, et al. High-density lipoproteins reduce the
neutralizing effect of hepatitis C virus (HCV)-infected patient
antibodies by promoting HCV entry. J Gen Virol 2006; 87 :
2577-81.
53. Bartosch B, Verney G, Dreux M, Donot P, Morice Y, Penin
F, et al. An interplay between hypervariable region 1 of the
hepatitis C virus E2 glycoprotein, the scavenger receptor bi,
and high-density lipoprotein promotes both enhancement
of infection and protection against neutralizing antibodies.
J Virol 2005; 79 : 8217-29.
54. Pietschmann T, Zayas M, Meuleman P, Long G, Appel N,
Koutsoudakis G, et al. Production of infectious genotype 1b
virus particles in cell culture and impairment by replication
enhancing mutations. Pub Libr Sci Pathog 2009; 5 :
e1000475.
55. Lin C, Lindenbach BD, Pragai BM, McCourt DW, Rice
CM. Processing in the hepatitis C virus E2-NS2 region:
Identification of p7 and two distinct E2-specific products with
different c termini. J Virol 1994; 68 : 5063-73.
56. Carrere-Kremer S, Montpellier C, Lorenzo L, Brulin B,
Cocquerel L, Belouzard S, et al. Regulation of hepatitis C
virus polyprotein processing by signal peptidase involves
structural determinants at the p7 sequence junctions. J Biol
Chem 2004; 279 : 41384-92.
57. Haqshenas G, Mackenzie JM, Dong X, Gowans EJ, Hepatitis
C virus p7 protein is localized in the endoplasmic reticulum
when it is encoded by a replication-competent genome. J Gen
Virol 2007; 88 : 134-42.
58. Carrere-Kremer S, Montpellier-Pala C, Cocquerel L,
Wychowski C, Penin F, Dubuisson J, Subcellular localization
and topology of the p7 polypeptide of hepatitis c virus. J Virol
2002; 76 : 3720-30.
59. Luik P, Chew C, Aittoniemi J, Chang J, Wentworth P, Jr.,
Dwek RA, et al. The 3-dimensional structure of a hepatitis C
virus p7 ion channel by electron microscopy. Proc Nat Acad
Sci USA 2009; 106 : 12712-6
60. Sakai A, Claire MS, Faulk K, Govindarajan S, Emerson SU,
Purcell RH, et al. The p7 polypeptide of hepatitis C virus is
critical for infectivity and contains functionally important
genotype-specific sequences. Proc Nat Acad Sci USA 2003;
100 : 11646-51.
61. Lohmann V, Korner F, Koch J, Herian U, Theilmann L,
Bartenschlager R. Replication of subgenomic hepatitis C virus
rnas in a hepatoma cell line. Science 1999; 285 : 110-3.
62. Blight KJ, Kolykhalov AA, Rice CM. Efficient initiation of
HCV RNA replication in cell culture. Science 2000; 290 :
1972-4.
63. Griffin SD, Beales LP, Clarke DS, Worsfold O, Evans SD,
Jaeger J, et al. The p7 protein of hepatitis C virus forms an
ion channel that is blocked by the antiviral drug, amantadine.
FEBS Lett 2003; 535 : 34-8.
64. Steinmann E, Whitfield T, Kallis S, Dwek RA, Zitzmann
N, Pietschmann T, et al. Antiviral effects of amantadine and
iminosugar derivatives against hepatitis c virus. Heptatol
2007; 46 : 330-8.
65. Steinmann E, Penin F, Kallis S, Patel AH, Bartenschlager
R, Pietschmann T. Hepatitis C virus p7 protein is crucial
for assembly and release of infectious virions. Pub Libr Sci
Pathog 2007; 3 : e103.
66. Pavlovic D, Fischer W, Hussey M, Durantel D, Durantel S,
Branza-Nichita N, et al. Long alkylchain iminosugars block
the HCV p7 ion channel. Adv Exp Med Biol 2005; 564 : 3-4.
67. Santolini E, Pacini L, Fipaldini C, Migliaccio G, Monica
N. The NS2 protein of hepatitis C virus is a transmembrane
polypeptide. J Virol 1995; 69 : 7461-71.
68. Yamaga AK, Ou JH. Membrane topology of the hepatitis C
virus NS2 protein. J Biol Chem 2002; 277 : 33228-34.
69. Grakoui A, McCourt DW, Wychowski C, Feinstone SM,
Rice CM. Characterization of the hepatitis C virus-encoded
serine proteinase: Determination of proteinase-dependent
polyprotein cleavage sites. J Virol 1993; 67 : 2832-43.
70. Grakoui A, McCourt DW, Wychowski C, Feinstone SM, Rice
CM. A second hepatitis c virus-encoded proteinase. Proc Natl
Acad Sci USA 1993; 90 : 10583-7.
71. Jones CT, Murray CL, Eastman DK, Tassello J, Rice CM.
Hepatitis C virus p7 and NS2 proteins are essential for
production of infectious virus. J Virol 2007; 81 : 8374-83.
72. Welbourn S, Pause A. The hepatitis C virus NS2/3 protease.
Curr Issu Mol Biol 2007; 9 : 63-9.
73. Schregel V, Jacobi S, Penin F, Tautz N. Hepatitis C virus NS2
is a protease stimulated by cofactor domains in NS3. Proc
Natl Acad Sci USA 2009; 106 : 5342-7.
74. Dimitrova M, Imbert I, Kieny MP, Schuster C. Protein-protein
interactions between hepatitis C virus nonstructural proteins.
J Virol 2003; 77 : 5401-14.
75. Yi M, Ma Y, Yates J, Lemon SM. Trans-complementation
of an NS2 defect in a late step in hepatitis C virus (HCV)
particle assembly and maturation. Pub Libr Sci Pathog 2009;
5 : e1000403.
76. Hahm B, Han DS, Back SH, Song OK, Cho MJ, Kim CJ, et al.
NS3-4A of hepatitis C virus is a chymotrypsin-like protease. J
Virol 1995; 69 : 2534-9.
77. Mottola G, Cardinali G, Ceccacci A, Trozzi C, Bartholomew
L, Torrisi MR, et al. Hepatitis C virus nonstructural proteins
are localized in a modified endoplasmic reticulum of cells
expressing viral subgenomic replicons. Virology 2002; 293 :
31-43.
78. Jennings TA, Chen Y, Sikora D, Harrison MK, Sikora B,
Huang L, et al. RNA unwinding activity of the hepatitis C
virus NS3 helicase is modulated by the ns5b polymerase.
Biochemistry 2008; 47 : 1126-35.
32 INDIAN J MED RES, january 2010
79. Kawai T, Takahashi K, Sato S, Coban C, Kumar H, Kato H, et
al. IPS-1, an adaptor triggering rig-i- and mda5-mediated type
i interferon induction. Nat Immunol 2005; 6 : 981-8.
80. Meylan E, Curran J, Hofmann K, Moradpour D, Binder M,
Bartenschlager R, et al. Cardif is an adaptor protein in the rig-i
antiviral pathway and is targeted by hepatitis C virus. Nature
2005; 437 : 1167-72.
81. Li XD, Sun L, Seth RB, Pineda G, Chen ZJ. Hepatitis C virus
protease NS3/4A cleaves mitochondrial antiviral signaling
protein off the mitochondria to evade innate immunity. Proc
Nat Acad Sci USA 2005; 102 : 17717-22.
82. Ahlen G, Derk E, Weiland M, Jiao J, Rahbin N, Aleman S, et
al. Cleavage of the ips-1/cardif/mavs/visa does not inhibit t
cell-mediated elimination of hepatitis C virus non-structural
3/4A-expressing hepatocytes. Gut 2009; 58 : 560-9.
83. Liefhebber JM, Hensbergen PJ, Deelder AM, Spaan WJ,
van Leeuwen H. Characterization of hepatitis C virus NS3
modifications in the context of replication. J Gen Virol 2009.
84. Selimovic D, Hassan M. Inhibition of hepatitis C virus (HCV)
core protein- induced cell growth by non-structural protein 4A
(ns4A) is mediated by mitochondrial dysregulation. Bosn J
Basic Med Sci 2008; 8 : 4-11.
85. Nomura-Takigawa Y, Nagano-Fujii M, Deng L, Kitazawa S,
Ishido S, Sada K, et al. Non-structural protein 4A of hepatitis
C virus accumulates on mitochondria and renders the cells
prone to undergoing mitochondria-mediated apoptosis. J Gen
Virol 2006; 87 : 1935-45.
86. Hugle T, Fehrmann F, Bieck E, Kohara M, Krausslich HG,
Rice CM, et al. The hepatitis C virus nonstructural protein
4b is an integral endoplasmic reticulum membrane protein.
Virology 2001; 284 : 70-81.
87. Welsch C, Albrecht M, Maydt J, Herrmann E, Welker MW,
Sarrazin C, et al. Structural and functional comparison of
the non-structural protein 4b in flaviviridae. J Mol graphics
modelling 2007; 26 : 546-57.
88. Yu GY, Lee KJ, Gao L, Lai MM. Palmitoylation and
polymerization of hepatitis C virus ns4b protein. J Virol 2006;
80 : 6013-23.
89. Liefhebber JM, Brandt BW, Broer R, Spaan WJ, van Leeuwen
HC. Hepatitis C virus ns4b carboxy terminal domain is a
membrane binding domain. J Virol 2009; 6 : 62-73.
90. Blight KJ, Allelic variation in the hepatitis C virus ns4b
protein dramatically influences RNA replication. J Virol 2007;
81 : 5724-36.
91. Pfeifer U, Thomssen R, Legler K, Bottcher U, Gerlich W,
Weinmann E, et al. Experimental non-a, non-b hepatitis: Four
types of cytoplasmic alteration in hepatocytes of infected
chimpanzees. Virchows Archiv 1980; 33 : 233-43.
92. Egger D, Wolk B, Gosert R, Bianchi L, Blum HE, Moradpour
D, et al. Expression of hepatitis C virus proteins induces
distinct membrane alterations including a candidate viral
replication complex. J Virol 2002; 76 : 5974-84.
93. El-Hage N, Luo G. Replication of hepatitis C virus RNA
occurs in a membrane-bound replication complex containing
nonstructural viral proteins and RNA. J Gen Virol 2003; 84 :
2761-9.
94. Lundin M, Lindstrom H, Gronwall C, Persson MA. Dual
topology of the processed hepatitis C virus protein ns4b is
influenced by the NS5A protein. J Gen Virol 2006; 87 : 3263-
72.
95. Park JS, Yang JM, Min MK. Hepatitis C virus nonstructural
protein ns4b transforms NIH3T3 cells in cooperation with the
ha-ras oncogene. Biochem Biophys Res Commu 2000; 267 :
581-7.
96. Huang L, Hwang J, Sharma SD, Hargittai MR, Chen Y, Arnold
JJ, et al. Hepatitis C virus nonstructural protein 5a (NS5A) is
an RNA-binding protein. J Bio Chem 2005; 280 : 36417-28.
97. Quintavalle M, Sambucini S, Di Pietro C, De Francesco R,
Neddermann P. The alpha isoform of protein kinase CKI is
responsible for hepatitis C virus NS5A hyperphosphorylation.
J Virol 2006; 80 : 11305-12.
98. Quintavalle M, Sambucini S, Summa V, Orsatti L, Talamo
F, De Francesco R, et al. Hepatitis C virus NS5A is a direct
substrate of casein kinase i-alpha, a cellular kinase identified
by inhibitor affinity chromatography using specific NS5A
hyperphosphorylation inhibitors. J Biol Chem 2007; 282 :
5536-44.
99. Evans MJ, Rice CM, Goff SP. Phosphorylation of hepatitis C
virus nonstructural protein 5a modulates its protein interactions
and viral RNA replication. Proc Natl Acad Sci USA 2004;
101 : 13038-43.
100. Lemm JA, O'Boyle D, Liu M, Nower PT, Colonno R,
Deshpande MS, et al. Identification of hepatitis C virus NS5A
inhibitors. J Virol 2009; 84 : 482-91.
101. Tellinghuisen TL, Foss KL, Treadaway J. Regulation of
hepatitis C virion production via phosphorylation of the NS5A
protein. Pub Libr Sci Pathog 2008; 4 : e1000032.
102. Hughes M, Griffin S, Harris M. Domain iii of NS5A contributes
to both RNA replication and assembly of hepatitis C virus
particles. J Gen Virol 2009; 90 : 1329-34.
103. Burckstummer T, Kriegs M, Lupberger J, Pauli EK, Schmittel
S, Hildt E. Raf-1 kinase associates with hepatitis C virus
NS5A and regulates viral replication. FEBS Lett 2006; 580 :
575-80.
104. Sauter D, Himmelsbach K, Kriegs M, Carvajal Yepes M, Hildt
E, Localization determines function: N-terminally truncated
NS5A fragments accumulate in the nucleus and impair HCV
replication. J Hepatol 2009; 50 : 861-71.
105. Amako Y, Sarkeshik A, Hotta H, Yates J, 3rd, Siddiqui A. Role
of oxysterol binding protein in hepatitis C virus infection. J
Virol 2009; 83 : 9237-46.
106. Kanda T, Steele R, Ray R, Ray RB. Inhibition of intrahepatic
IFN-(gamma) production by hepatitis C virus non-structural
protein 5a in transgenic mice. J Virol 2009; 83 : 8463-9.
107. Kriegs M, Burckstummer T, Himmelsbach K, Bruns M, Frelin
L, Ahlen G, et al. The hepatitis C virus non-structural NS5A
protein impairs both the innate and adaptive hepatic immune
response in vivo. J Biol Chem 2009; 284 : 28343-51.
108. Lesburg CA, Cable MB, Ferrari E, Hong Z, Mannarino AF,
Weber PC. Crystal structure of the RNA-dependent RNA
polymerase from hepatitis C virus reveals a fully encircled
active site. Nat Struct Biol 1999; 6 : 937-43.
109. Schmidt-Mende J, Bieck E, Hugle T, Penin F, Rice CM,
Blum HE, et al. Determinants for membrane association of
the hepatitis C virus RNA-dependent RNA polymerase. J Biol
Chem 2001; 276 : 44052-63.
 Sharma: New therapeutic approaches for HCV 33
110. Moradpour D, Brass V, Bieck E, Friebe P, Gosert R, Blum
HE, et al. Membrane association of the RNA-dependent RNA
polymerase is essential for hepatitis C virus RNA replication.
J Virol 2004; 78 : 13278-84.
111. Shirota Y, Luo H, Qin W, Kaneko S, Yamashita T, Kobayashi
K, et al. Hepatitis C virus (HCV) NS5A binds RNA-
dependent RNA polymerase (RDRP) ns5b and modulates
RNA-dependent RNA polymerase activity. J Biol Chem
2002; 277 : 11149-55.
112. Quezada EM, Kane CM. The hepatitis C virus NS5A
stimulates ns5b during in vitro RNA synthesis in a template
specific manner. open Biochem J 2009; 3 : 39-48.
113. Simister P, Schmitt M, Geitmann M, Wicht O, Danielson UH,
Klein R, et al. Structural and functional analysis of hepatitis
C virus strain jfh1 polymerase. J Virol 2009; 83 : 11926-39.
114. Burton JR, Jr., Everson GT. HCV NS5B polymerase
inhibitors. Clin Liver Dis 2009; 13 : 453-65.
115. Wang M, Ng KK, Cherney MM, Chan L, Yannopoulos CG,
Bedard J, et al. Non-nucleoside analogue inhibitors bind to an
allosteric site on HCV ns5b polymerase. Crystal structures and
mechanism of inhibition. J Biol Chem 2003; 278 : 9489-95.
116. Chatterji U, Bobardt M, Selvarajah S, Yang F, Tang H,
Sakamoto N, et al. The isomerase active site of cyclophilin a
is critical for hepatitis C virus replication. J Biol Chem 2009;
284 : 16998-7005.
117. Kim SJ, Kim JH, Kim YG, Lim HS, Oh JW. Protein kinase C-
related kinase 2 regulates hepatitis C virus RNA polymerase
function by phosphorylation. J Biol Chem 2004; 279 : 50031-
41.
118. Kim, SJ, Kim JH, Sun JM, Kim MG, Oh JW. Suppression
of hepatitis C virus replication by protein kinase C-related
kinase 2 inhibitors that block phosphorylation of viral RNA
polymerase. J Viral Hepat 2009; 16 : 697-704.
119. Young KC, Lindsay KL, Lee KJ, Liu WC, He JW, Milstein
SL, et al. Identification of a ribavirin-resistant ns5b mutation
of hepatitis C virus during ribavirin monotherapy. Heptatol
2003; 38 : 869-78.
120. Sentandreu V, Jimenez-Hernandez N, Torres-Puente M,
Bracho MA, Valero A, Gosalbes MJ, et al. Evidence of
recombination in intrapatient populations of hepatitis C
virus. Pub Libr Sci ONE 2008; 3 : e3239.
121. Lindenmann J, Burke DC, Isaacs A. Studies on the production,
mode of action and properties of interferon. Br J Exp Pathol
1957; 38 : 551-62.
122. Feld JJ, Hoofnagle JH. Mechanism of action of interferon
and ribavirin in treatment of hepatitis C. Nature 2005; 436 :
967-72.
123. Potter CW, Phair JP, Vodinelich L, Fenton R, Jennings R.
Antiviral, immunosuppressive and antitumour effects of
ribavirin. Nature 1976; 259 : 496-7.
124. NIH consensus statement on management of hepatitis C:
2002. NIH Consens State Sci Statements 2002; 19 : 1-46.
125. McHutchison JG, Gordon SC, Schiff ER, Shiffman ML,
Lee WM, Rustgi VK, et al. Interferon alfa-2b alone or in
combination with ribavirin as initial treatment for chronic
hepatitis C. Hepatitis interventional therapy group. N Engl J
Med 1998; 339 : 1485-92.
126. Hong Z, Cameron CE. Pleiotropic mechanisms of ribavirin
antiviral activities. Prog Drug Res 2002; 59 : 41-69.
127. Crotty S, Maag D, Arnold JJ, Zhong W, Lau JY, Hong Z, et
al. The broad-spectrum antiviral ribonucleoside ribavirin is
an RNA virus mutagen. Nat Med 2000; 6 : 1375-9.
128. Lee JH, von Wagner M, Roth WK, Teuber G, Sarrazin C,
Zeuzem S. Effect of ribavirin on virus load and quasispecies
distribution in patients infected with hepatitis C virus. J
Hepatol 1998; 29 : 29-35.
129. Welzel TM, Morgan TR, Bonkovsky HL, Naishadham D,
Pfeiffer RM, Wright EC, et al. Variants in interferon-alpha
pathway genes and response to pegylated interferon-alpha2a
plus ribavirin for treatment of chronic hepatitis C virus
infection in the hepatitis Cantiviral long-term treatment
against cirrhosis trial. Heptatol 2009; 49 : 1847-58.
130. Helle F, Wychowski C, Vu-Dac N, Gustafson KR, Voisset
C, Dubuisson J. Cyanovirin-n inhibits hepatitis C virus entry
by binding to envelope protein glycans. J Biol Chem 2006;
281 : 25177-83.
131. Ouzounov S, Mehta A, Dwek RA, Block TM, Jordan
R. The combination of interferon alpha-2b and n-butyl
deoxynojirimycin has a greater than additive antiviral effect
upon production of infectious bovine viral diarrhea virus
(BVDV) in vitro: Implications for hepatitis C virus (HCV)
therapy. Antiviral research 2002; 55 : 425-35.
132. Durantel D. Celgosivir, an alpha-glucosidase i inhibitor for
the potential treatment of HCV infection. Curr Opin Investig
Drugs 2009; 10 : 860-70.
133. Zitzmann N, Mehta AS, Carrouee S, Butters TD, Platt FM,
McCauley J, et al. Imino sugars inhibit the formation and
secretion of bovine viral diarrhea virus, a pestivirus model of
hepatitis C virus: Implications for the development of broad
spectrum anti-hepatitis virus agents. Proc Natl Acad Sci USA
1999; 96 : 11878-82.
134. Chapel C, Garcia C, Roingeard P, Zitzmann N, Dubuisson J,
Dwek RA, et al. Antiviral effect of alpha-glucosidase inhibitors
on viral morphogenesis and binding properties of hepatitis C
virus-like particles. J Gen Virol 2006; 87 : 861-71.
135. Trotard M, Lepere-Douard C, Regeard M, Piquet-Pellorce C,
Lavillette D, Cosset FL, et al. Kinases required in hepatitis C
virus entry and replication highlighted by small interference
RNA screening. FASEB J 2009; 23 : 3780-9.
136. Berger KL, Cooper JD, Heaton NS, Yoon R, Oakland
TE, Jordan TX, et al. Roles for endocytic trafficking and
phosphatidylinositol 4-kinase iii alpha in hepatitis C virus
replication. Proc Natl Acad Sci USA 2009; 106 : 7577-82.
137. Zhang H, Rothwangl K, Mesecar AD, Sabahi A, Rong L,
Fong HH. Lamiridosins, hepatitis C virus entry inhibitors
from lamium album. J Nat Prod 2009; (PMID: 19904996).
138. Witteveldt J, Evans MJ, Bitzegeio J, Koutsoudakis G,
Owsianka AM, Angus AG, et al. Cd81 is dispensable for
hepatitis C virus cell-to-cell transmission in hepatoma cells.
J Gen Virol 2009; 90 : 48-58.
139. Ferreon JC, Ferreon AC, Li K, Lemon SM. Molecular
determinants of TRIF proteolysis mediated by the hepatitis C
virus NS3/4A protease. J Biol chem 2005; 280 : 20483-92.
140. McHutchison JG, Everson GT, Gordon SC, Jacobson
IM, Sulkowski M, Kauffman R, et al. Telaprevir with
34 INDIAN J MED RES, january 2010
peginterferon and ribavirin for chronic HCV genotype 1
infection. N Engl J Med 2009; 360 : 1827-38.
141. Hezode C, Forestier N, Dusheiko G, Ferenci P, Pol S, Goeser T,
et al. Telaprevir and peginterferon with or without ribavirin for
chronic HCV infection. N Engl J Med 2009; 360 : 1839-50.
142. Moriyama K, Suzuki T, Negishi K, Graci JD, Thompson CN,
Cameron CE, et al. Effects of introduction of hydrophobic
group on ribavirin base on mutation induction and anti-RNA
viral activity. J Medicinal Chem 2008; 51 : 159-66.
143. Hou W, Tian Q, Zheng J, Bonkovsky HL. Zinc
mesoporphyrin induces rapid proteasomal degradation of
hepatitis c nonstructural 5a protein in human hepatoma cells.
Gastroenterology, 2009.
144. Gardelli C, Attenni B, Donghi M, Meppen M, Pacini B, Harper
S, et al. Phosphoramidate prodrugs of 2'-c-methylcytidine
for therapy of hepatitis C virus infection. J Medicinal Chem
2009; 52 : 5394-407.
145. Mathy JE, Ma S, Compton T, Lin K. Combinations of
cyclophilin inhibitor nim811 with hepatitis C virus NS3-4A
protease or ns5b polymerase inhibitors enhance antiviral
activity and suppress the emergence of resistance. Antimicrob
Agents Chemother 2008; 52 : 3267-75.
146. Coelmont L, Kaptein S, Paeshuyse J, Vliegen I, Dumont
JM, Vuagniaux G, et al. Debio 025, a cyclophilin binding
molecule, is highly efficient in clearing hepatitis C virus
(HCV) replicon-containing cells when used alone or in
combination with specifically targeted antiviral therapy for
HCV (stat-c) inhibitors. Antimicrob Agents Chemother 2009;
53 : 967-76.
147. Bader T, Fazili J, Madhoun M, Aston C, Hughes D, Rizvi S,
et al. Fluvastatin inhibits hepatitis c replication in humans.
Am J Gastroenterol 2008; 103 : 1383-9.
148. Ikeda M, Kato N. Life style-related diseases of the digestive
system: Cell culture system for the screening of anti-hepatitis
C virus (HCV) reagents: Suppression of HCV replication by
statins and synergistic action with interferon. J Pharmacol
Sci 2007; 105 : 145-50.
149. Delang L, Paeshuyse J, Vliegen I, Leyssen P, Obeid S, Durantel
D, et al. Statins potentiate the in vitro anti-hepatitis C virus
activity of selective hepatitis C virus inhibitors and delay or
prevent resistance development. Heptatol 2009; 50 : 6-16.
150. Makeyev EV, Zhang J, Carrasco MA, Maniatis T. The
microrna mir-124 promotes neuronal differentiation by
triggering brain-specific alternative pre-mrna splicing. Mol
Cell 2007; 27 : 435-48.
151. Fabbri M, Ivan M, Cimmino A, Negrini M, Calin GA.
Regulatory mechanisms of micrornas involvement in cancer.
Expert Opin Biol Ther 2007; 7 : 1009-19.
Reprint requests: Dr Suresh D. Sharma, The Pennsylvania State University, 201 Althouse Laboratory University Park, PA 16802, USA
e-mail: sds20@psu.edu
152. Nathans R, Chu CY, Serquina AK, Lu CC, Cao H, Rana
TM. Cellular microrna and p bodies modulate host-hiv-1
interactions. Mol Cell 2009; 34 : 696-709.
153. Mamiya N, Worman HJ. Hepatitis C virus core protein binds
to a dead box RNA helicase. J Biol Chem 1999; 274 : 15751-
6.
154. Ariumi Y, Kuroki M, Abe K, Dansako H, Ikeda M, Wakita T,
et al, Ddx3 dead-box rna helicase is required for hepatitis C
virus rna replication. J Virol 2007; 81 : 13922-6.
155. Owsianka AM, Patel AH. Hepatitis C virus core protein
interacts with a human dead box protein ddx3. Virology
1999; 257 : 330-40.
156. Klade CS, Wedemeyer H, Berg T, Hinrichsen H, Cholewinska
G, Zeuzem S, et al. Therapeutic vaccination of chronic
hepatitis C nonresponder patients with the peptide vaccine
IC41. Gastroenterol 2008; 134 : 1385-95.
157. Law M, Maruyama T, Lewis J, Giang E, Tarr AW, Stamataki
Z, et al. Broadly neutralizing antibodies protect against
hepatitis C virus quasispecies challenge. Nat Med 2008; 14 :
25-7.
158. Fytili P, Dalekos GN, Schlaphoff V, Suneetha PV, Sarrazin
C, Zauner W, et al. Cross-genotype-reactivity of the
immunodominant HCV cd8 t-cell epitope NS3-1073. Vaccine
2008; 26 : 3818-26.
159. van der Meer-Janssen YP, van Galen J, Batenburg JJ, Helms
JB. Lipids in host-pathogen interactions: Pathogens exploit
the complexity of the host cell lipidome. Prog Lipid Res
2009.
160. Meyer MF, Lehmann M, Cornberg M, Wiegand J, Manns MP,
Klade C, et al. Clearance of low levels of HCV viremia in the
absence of a strong adaptive immune response. Virology J
2007; 4 : 58.
161. Smith RE. Hepatitis C virus therapies. Nat Rev Drug Discov
2006; 5 : 715-6.
162. Rudolf KFB, Anna MP. Hepatitis C Viral NS3-4A protease
activity is enhanced by the NS3 helicase. J Biol Chem 2008;
283 : 29929-37.
163. Frick DN. The hepatitis C virus NS3 protein: a model RNA
helicase and potential drug target. Curr Issues Mol Biol 2007;
9 : 1-20.
164. Shimoike TS, Mimori HT, Matsuura Y, Miyamura. T.
Interaction of hepatitis C virus core protein with viral sense
RNA and suppression of its translation. J Virol 1999; 73 :
9718-25.
165. http://www.tacerebio.com/Technology/technology.html,
accessed in June 2009.