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24 (2000) 253–269
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Review Article
Hepatitis C Virus: Genome Organization, Viral
Proteins and Implications in Disease Pathogenesis
Çağla EROĞLU, Ergün PINARBAŞI
Bilkent University, Department of Molecular Biology and Genetics 06533 Bilkent, Ankara-TURKEY
Received: 21.09.1998
Abstract: The hepatitis C virus (HCV) infection is a significant cause of morbidity and mortality
worldwide. Infection with HCV becomes chronic in more than 80% of cases and it accounts for 20%
of all cases of acute hepatitis. The hepatitis C virus was first identified by the molecular cloning of the
virus genome in 1989. It is an enveloped, positive strand RNA virus with a genome size of around 9.5
kilobases. The single-stranded RNA genome of the virus contains a large open reading frame that
encodes a large polyprotein of 3,010 to 3,033 amino acids shown to be processed by a combination
of host and viral proteinases to produce at least ten proteins post-translationally. The proteins that are
closer to the amino terminal of the polyprotein are termed structural and the rest, closer to the
carboxy terminal, are called nonstructural (NS) proteins.
Hepatit C Virusu: Genom Organizasyonu, Viral Proteinler ve Hastalık
Patogenezindeki Yeri
Özet: Hepatit C virüsünün (HCV) yol açtığı infeksiyon tüm dünyada önemli bir hastalık ve ölüm
nedenidir. Vakaların yüzde 80’ninden çoğunda HCV infeksiyonu kronikleşir ve bu tüm akut hepatit
vakalarının yüzde 20’sini oluşturur. Hepatit C virüsü ilk olarak 1989 yılında moleküler klonlama
yöntemi ile tanımlandı. Bu zarflı, pozitif iplikli, RNA virüsü 9.5 kilobazlık bir genoma sahiptir. Virüsün
tek iplikli RNA genomu 3.010 ila 3033 amino asitlik büyük bir poli-protein kodlayan geniş bir açık
okuma çerçevesi içerir. Bu poli-protein, sentezlendikten sonra, hücresel ve viral proteinazlar tarafından
en azından on ayrı proteine bölünür. Poli-proteinin amino ucuna yakın olan proteinler yapısal ve diğer,
karboksil ucuna yakın olan proteinler ise yapısal olmayan diye adlandırılırlar.
Introduction
The HCV virus infection is an important cause of morbidity and mortality worldwide.
Infection with HCV becomes chronic in more than 80% of cases. This disease is estimated to
affect around 100 million people worldwide and is characterized by a mild and often
undiagnosed acute illness which evolves into a persistent infection and eventually to liver failure
and cirrhosis. Epidemiological data also suggest a link between the chronic infection and the
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development of hepatocellular carcinoma. The viral genome and antigens have been detected in
affected liver cells (1). The degeneration of the infected hepatocytes may be caused either
directly by the cytopathic effect of the virus or indiretctly by immune responses of the host
mainly through the function of the cytotoxic T cell lymphocytes (CTL). Continuous occurrence of
degeneration followed by regeneration of hepatocytes, together with the fibrotic changes of the
liver tissues, would then result in liver cirrhosis, from which hepatocellular carcinoma eventually
arises in some cases.
The risk groups besides the parental route of infection can be listed as:
• Intravenous drug abusers
• Hemophiliacs
• Recipients of unscreened blod transfusions
There are three phases of HCV infection, namely acute, silent and reactivated. The acute
phase lasts from the onset of the disease until 2-3 years thereafter, and the silent phase which
follows lasts for 10-15 years. In majority of cases the infection gives rise to an acute illness and
68% of these cases develop into chronic hepatitis (2).
Several diagnostic tests have been developed with two means, serological (using recombinant
antigens), and molecular (PCR being used to determine the extent of the virus variation). The
drawback with the serological studies is that the infection cannot be detected in the early stages,
however this system is easy to use and rather cheaper. The most reliable method is PCR however
this technique is expensive and more sophisticated equipment is needed. False positive and false
negative results are the main problems of inexperienced laboratories.
Being a rapidly evolving RNA virus, extensive variations in the HCV genotype have been
observed. Several isolates of HCV have been sequenced and distinct genotypes have been
proposed on the basis of differences both in the coding and the non-coding regions of the virus
(3-5). Differences between nucleotide sequences of various HCV strains were found to be mostly
in the HVR1 and the 3’UTR. In contrast, the nucleic acid sequence of 5’UTR is not highly
divergent as mentioned previously. So a new classification system was proposed due to the
differences in the 5’UTR of the different HCV groups, and a phylogenic tree was constructed
where HCV variants were classified into six equally divergent main groups of sequences, many
of which contained more closely related groups within them. The main types havee been
numbered 1 to 6 and the subtypes have been lettered a, b and c in order of discovery (6, 7).
Some genotypes of HCV such as 1a, 1b, 2a and 2b, have been reported to show a broad
worldwide distribution whereas others such as 5a and 6a have only been found in specific
geographical areas. Genotype 1b seems to be the most common variant worldwide with a high
incidence in Japan and Western Europe. The prototype 1a is relatively rare in these areas but
the most common from in America. Type 4 is the principal from in the Middle East and Central
Africa (8, 9). However, to date no significant genotyping or subtyping studies have been
reported for Turkey. Unconfirmed information was mentioned in several articles stating that
HCV 1b is the major subtype for the Turkish population, but this statement needs to be
confirmed by large scale studies (3).
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At present, the only therapeutic approach to HCV is long-term treatment with alpha
interferon, alone or in combination with ribavirin (10). This treatment is effective only for a
minority of patients, and the development of a more effective therapeutic agent and/or of a
prophylactic immunogen still remains an important public health issue. This is implicated in the
increasing number of adult liver transplants (11).
The Molecular Biology of the Hepatitis C Virus
The hepatitis C virus (HCV) which is the major causative agent of non-A non-B hepatitis
(NANBH) was first identified by the molecular cloning of the virus genome in 1989 (12). It is
an enveloped, positive-strand RNA virus with a genome size of around 9.5 kbs (13, 14). By the
analysis of sequence similarity and hydropathy profile, the genome of the HCV is shown to be
related with that of flaviviruses and pestiviruses and therefore this virus is placed in a new
monotypic genus in the flaviviridae family (3).
The single-stranded RNA genome of the virus contains a large open reading frame of 9,030
to 9,099 that encodes a large polyprotein of 3,010 to 3,033 amino acids (13, 15-17). This
polyprotein product is shown to be processed by a combination of host and viral proteinases to
produce at least ten proteins post-translationally. The proteins that are closer to the amino
terminal of the polyprotein are termed structural and the rest, closer to the carboxy terminal,
are called nonstructural (NS) proteins (18, 19). As a result four domains are evident including
the two untranslated reginos (UTRs), one at the 5’ and the other at the 3’ of the genome (20).
The structural region includes four proteins: the core protein (C), two putative envelope proteins
which are glycosylated and called E1 and E2 and a short protein called p7. These N-terminal
proteins are thought to be involved in the forming of the viral particle. The nonstructural region
on the other hand encodes for proteins NS2, NS3, NS4 and NS5. Some of these proteins are
shown to be cleaved into smaller units and are thought to be involved in the replication of the
virus in the cell (19, 20). The HCV gene order has been determined to be 5’-C-E1-E2-p7NS2(p23)-NS3-NS4A-NS4B-NS5A-NS5B-3’. Polyprotein cleavages in the structural (C/E1,
E1/E2, E2/p7 and p27/NS2) are catalyzed by a host signal peptidase localized in the endoplasmic
reticulum (ER) (21, 22). Cleavage at the C/E1, E1/E2 and NS2/NS3 sites are co-translational,
whereas those at the E2/p7 and p7/NS2 occur post-translationally and generate two precursors
for E2: E2-NS2 and E2-p7 (17). The nonstructural proteins are processed by two virus
proteinases, the NS2 (p23) and NS3 (23, 24, 25, 26).
At present, little is known about the molecular mechanisms of HCV replication. However it
is thought that it resembles the replication of the other positive-straded RNA viruses; that is,
following the entry and uncoating in the cytoplasm of the host cell, the viral genome acts as a
template for the synthesis of the complementary (minus) RNA molecule. The minus-strand then
in turn serves as a template for the synthesis of the progeny positive-stranded RNA molecules.
It has been impossible to detect any DNA intermediates in the serum or liver of the affected
individuals. Moreover, it has been shown that the region NS5 encodes a protein (will be
discussed in detail later) that has been demonstrated to have RNA-dependent RNA polymerase
activity (27). Antigenomic (minus) RNA strands have been detected in the serum and in the liver
of some patients (28-30), which indicates the presence of RNA intermediates in replication (19).
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As antigenomic strand synthesis should start at the 3’terminus, some 6-8 basepairs repeating
sequences that are found both at the 5’ and 3’ UTRs may be involved in secondary structure
formation or cyclization of the RNA genome (31).
The complete 5’ UTR comprises 341 nucleotides (14, 20), but many shorter sequences have
been detected and reported (13, 15). This region is thought to be involved in the regulation of
the translation and the replication of the genome. Studies on ribonuclease sensitivity analysis and
thermodynamic secondary structure modelling, (32, 33) have revealed the fact that a large
conserved stem-loop structureis present in the proximal part of the 5’ UTR which might act as
an internal ribosomal entry site (IRES) (34). HCV polyprotein translation seems to be cap
independent and initiate at the IRES within the 5’-UTR proximal to the initiator AUG codon of
the polyprotein. The translation initiation seems to be inhibited by the presence of a 27
nucleotides sequence that is capable of forming a stable hairpin structure (20).
Evidence that HCV does not replicate efficiently in cell culture systems has been a leitmotif
in the HCV literature. All published reports indicate that the level of HCV propagation is
extremely low in cultured cells, requiring the use of very sensitive detection methods such as
reverse transcription-PCR (RT-PCR) to monitor infection and replication (35). No system
developed to date allows the detection of HCV polyprotein processing so other expression and
transfection systems have been utulized exclusively.
Structural Proteins of HCV
Core Protein (C or p22)
The first structural protein on the amino terminus of the the polyprotein is the HCV core
protein (p22). Unlike the envelope proteins E1 and E2 it lacks potential N-glycosylation sites. Its
N-terminal region is highly basic while the C terminus is hydrophobic. The amino acid sequence
is well conserved among different HCV isolates (36) which suggests the importance of this
protein for the survival of the virus. Also because of this property, both recombinant core
proteins and synthetic peptides, presenting linear core epitopes, can be used for efficient
detection of antibodies in most patients’ sera (19). HCV core protein is a multifunctional protein.
First it was shown that HCV core protein binds to cellular membranes, RNA molecules, and the
60S ribosomal subunit, and the RNA and ribosome binding domains have been mapped to its N
terminus. More recent data revealed that the core protein can form dimeric and multimeric
complexes (37). The ability of the protein to be multimerized and bind to RNA at the same time
indicates that this protein plays an important role in the assembly of the HCV nucleocapsid.
Apart from this primary function of the core protein in the encapsulation of the virus, it also
plays important roles in gene expression regulation. A lot of data have been accumulated
indicating that HCV core protein is a trans-acting regulatory protein. The 22-kDa core protein
trans-suppressed CAT gene expression under the control of tummor suppressor gene p53 and
universal cyclin-dependent kinase inhibitor p21 promoters (38, 41, 42). It was reported to
regulate the replication and the expression of the HBV genome (43) and to trans-activate c-myc
oncogene (39). It also enhanced the H,ras oncogene activity in immortalizing rat embryo
fibroblast (40). A very recent study (44) showed that the HCV core protein interacts with the
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cytoplasmic tail of the Lymphotoxin-β. This result implies than HCV core protein is a potential
modulator of the host immune system and it is involved in a mechanism for viral evasion of host
defenses, perhaps allowing for viral persistence.
Having a wide variety of functions, the core protein has been shown to be accumulated both
in the cytoplasm and the nucleus. It has been reported that during maturation, the core protein
undergoes two consecutive membrane-dependent cleavage events at amino acid residues 173
and 174 and 191 and 192. As a result two forms of the core protein, C173 (aas 1 to 173) and
C191 (aas 1 to 191) are generated. Core protein products representing both C173 and C191,
produced from either an entire HCV polyprotein or various polyprotein precursors, display a
cytoplasmic localization while the C173 expressed in the absence of C191 is able to translocate
into the nucleus. These observations suggest that an indicent or direct interplay between these
two forms of the core protein, generated by the preferential cleavages, may determine their
subcellular localization (45).
Envelope Proteins, E1 and E2
The other two structural proteins E1 and E2 are putative viral envelope proteins.
Glycoprotein gp33 (E1, also referred as gp35 in some references) contains many potential Nglycosylation sites and glycosylation of this protein has been demonstrated in a cellfree
glycosylation system (46). Transmembrane transport of the gp33 is presumably facilitated by a
N-terminal stretch of 20 hydrophobic amino acids that may function as a signal sequence,
recognized by cellular signalases, thereby cleaving the core protein p22 from the precursor
protein. Between residues 350-390 a stretch of hydrophobic amino acids is present, which could
act as a membrane anchor (47).
As mentioned previously, the processing of the capsid protein and the two membraneassociated glycopproteins E1 and E2 is mediated by host signalase (25, 46). Analysis of the
location of the cleavage sites between structural proteins is based on determination of the Nterminal amino acid sequences of E1 and E2. The first amino acid residue of E1 has been
identified as Tyr192 and the first residue of E2 as His384 (25) or Glu384 depending on the
isolate. E1 and E2 are both preceded by hydrophobic segments (residues 174-191 and 371383 of the polyprotein, respectively) that may be considered signal sequences. The E2 protein
seems to display a complex processing consistent with the generation of multiple E2 species and
forms a stable complex with E1 which is coimmunoprecipitable (25, 48, 49).
Glycoprotein gp72 (alsohefferred as gp70 in some references) is encoded by the E2/p7 and
has a protein backbone of 38 kilodaltons (50). A repeated amino acid sequence has been
observed between residues 471-511; Pro-(X)5- Pro-(X)6- Pro-(X)6- Pro-(X)6- Pro-(X)6- Pro-(X)5Pro. A cysteine residue is present in each of the last four intervening (X) sequences, and their
positions seemed conserved. The HCV gp72 is more heavily N-glycosylated, and the full length
gp72 is not secreted from the cell but remains membrane associated. Presumably this is caused
by the presence of a single transmembrane anchor, as studies showed that C terminal truncated
E2/p7 protein is rapidly secreted.
Comparison of the available E2/p7 sequences revealed the presence of hypervariable regions
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(46, 51, 52) at the N-terminal of the E2 protein. One of these regions, HVR1, is located directly
downstream of the putative cleavage site between E1 and E2/p7 (between residues 380 and
385) and covers the 30 N-terminal residues (384-414) of the E2/p7 protein (53, 54). HVR1
seems to lack a conserved secondary structurend resembles the V3 loop of the human
immunodeficiency virus 1 gp120 (55). Specific antibody reactions where detected against
peptides corresponding to linear epitopes in HVR1, indicating that the N-terminal part of the E2
region encodes antigenically distant variants, subjected to immune selection (55, 56). The
observed hypervariabilty may be a result of sequential mutations leading to escape mutants
(57). Although HVR1 shows hypervariation during most chronic infections, conservation has
also been observed (19). This is presumably due to the absence of selective pressure favoring
new mutants caused by an inadequate immune response against the initial HVR1 epitope. The
variability of the gp72 may have significant implications for the development of a protective
immune response which is important for vaccine strategies.
Escape from the host immunosurveilance by means of mutations in HVR1 might be involved
in the mechanism of persistent infection by HCV, which results in chronic hepatitis and eventually
hepatocellular carcinoma (55, 57). Viral osilate-restricted neutralizing antibodies against HCV
have been demonstrated recently in infected individuals (58, 59) but their molecular target is
presently unknown. Several observations have suggested that the HVR1 could be involved in the
neutralization of the HCV. This assumption is based on the fact that HVR1 is the most variable
region on the HCV genome (46, 53, 54), contains linear epitopes that are recognized by the
patients’ antibodies (55) and mutates rapidly in vivo (60, 61) suggesting that it is under the
selective pressure of the host immune system. This hypothesis is further substantiated by the
lack of variability in the HVR1 observed in an agammaglobulinemic patient over a period of 2.5
years (62). Recent data obtained in vitro suggest that antibodies, present in human sera and
directed against the HVR1 as well as against the E2 protein of HCV, can prevent the binding of
HCV to cells (63, 64). Another recent study was carried out in order to show whether the HVR1
of the E2 protein is a critical neutralizing domain (65). Prevention of HCV infection in
chimpanzees by hyperimmune serum against the hypervariable region 1 has been detected.
The C terminal position of the E2 protein is not absolutely clear at present. The location of
E2 in the HCV open reading frame was predicted to be from amino acids 384 to 729 (16). It
was reported that in an in vitro translation system, the full E2 coding region does not extend
past amino acid 740 (46). More recent data suggested that the N terminal position of NS2 lies
at about amino acid 810 although the precise C-terminal position of E2 is not known (66).
Therefore the residues that the other structural protein p7 is within, was not detected and it
might be expressed with or without E2.
Viral envelope proteins play a role in several aspects of the viral life cycle such as receptor
binding, penetration of host cells, and virus morphogenesis at budding. Expression studies using
recombinant HCV cDNA templates have demonstrated hetero-oligomer formation between the
E1 and E2 proteins detected by immunological means (25, 48, 49, 66, 67). A recent study
reported E1-E2 binding by far-Western blotting and pull-down assay mainly using bacterialexpressed recombinant proteins. The major contribution to the E1-binding of E2 was mapped
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within the region in the vicinity of HVR2, and the “WHY” sequence within the region played a
crucial role in the E1-E2 association. Neither HVR1 nor HVR2 of E2 were necessary for
interaction. By using similar systems it was also demonstrated that the E2-binding of E1 is
mapped to the N-terminal part of E1 (68).
E1 and E2 are candidate antigens for future vaccines to hepatitis C, as the NS1 protein of
flaviviruses are E2 protein of pestiviruses and the counterparts of the E2 protein, have been
reported to produce neutralizing antibodies (69-72). In a study, anti-E2 antibodies were
detected in most of the chronic NANBH patients (93%). The anti-E2 antibodies present in the
patients’ are reacted well to native E2. These anti-E2 antibody may be diagnostically useful (73).
As mentioned previously, before the cellular proteases, such as cellular peptidase areinvolved
in sequential co-translational processing of the cleavage sites on the N-terminus of E1, E2 and
possibly NS2. The NS2-NS3 cleavage is mediated in cis by a zinc requiring metalloproteinase
encoded by the NS2 itself. The region between residues 898-1233 was shown to be essential
for the detection of the second viral protease (21). NS3 acts in cis on its own C-terminus but
the remaining three C-terminal sites can be only processed by NS3 in trans (74). Theres also
homology between NS3 and nucleoside triphosphate-binding helicase enzymes that are
presumably involved in the unwinding of the binding ds RNA replicative intermediate necessary
for genome replication (18).
Nonstructural Proteins (NS)
NS3
The HCV nonstructural 3 protein (NS3) is a 70 kilodalton multifunctional enzyme with three
known catalytic activities segregated in two somewhat independent domains. The essential
machinery of a serine protease is localized in the N-terminal third of the protein and nucleoside
triphosphatase (NPTase) and helicase activities reside in the remaining C-terminal region. There
is no evidence to suggest that the two domains of NS3 are separated by proteolytic processing
in vivo.
The NS3 shows limited amino acid homology with corresponding flavi- and pestivirus
proteins (75). The N terminal third of NS3 resembles a chymotrypsin-like serine protease,
similar to that found in flaviviruses and pestiviruses. Of the Ser-165 in the proposed catalytic
site is replaced with another amino acid, the proteolytic activity of the p72 is abolished (74, 76).
This protease is responsible for some processing steps of the precursor polyprotein into the
mature proteins. All four cleavages occur C terminally of the protease domain (NS3-NS4A,
NS4A-NS4B, NS4B-NS5A and NS4B-NS5A and NS5A-NS5B). These cleavage sites show several
common features, which probably determine the substrate specificity of the NS3 protease (24).
The His-57, Asp-81, and Ser-139 residues of the NS3 protease are conserved among all
sequenced HCV strains and have been proposed to constitute characteristic serine protease
catalytic triad, as in the NS3 protein of flaviviruses and pestiviruses. These three residues are
essential for HCV NS3 proteinase activity as it has been shown by site-directed mutagenesis
experiments (21). Recently it was shown that NS3 protease requires another virus-encoded
protein NS4A, to cleave efficiently the nonstructural proteins (23, 77, 78). In addition to this
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requirement for a protein co-factor, there were reports of NS3 protease sensitivity to divalent
metal ions, behavior that is not expected for a chymotrypsin-like serine protease. A recent report
showed that the NS3 proteinase domain of the HCV is a zinc-containing enzyme (79). Analysis
of the metal content of the purified NS3 protease domain indicated the presence of a single zinc
atom per molecule of enzyme which in turn implied the existence of a metal binding site. This
study and the crystal structure of the NS3 protease domain shows that the role of the zinc atom
is structural rather than catalytical. The zinc atom is coordinated by Cys-97, Cys99 and Cys-145
and the fourth ligand is a water molecule which is attached to the His-149 (79, 80).
The C terminal two-thirds of NS3 also seems to constitute a somewhat independent domain.
It contains conserved sequence motifs characteristic of the NTP-binding proteins and the socalled D-E-A-D superfamily of RNA helicase and several studies have demonstrated RNAdependent NTPase and helicase activities with various recombinant forms of NS3. The NTPase
activities has been characterized for ∼50 kilodalton N terminal NS3 containing only the helicase
domain and for full-length NS3 (81). Similarly, the helicase activity has been characterized by
using a truncated helicase domain and a full-length NS3 complexed with NS4A (81). Despite the
evidence that both domains of NS3 areactive in the absence of one another in vitro, there is no
evidence that they are separated by proteolytic processing in vivo. This may reflect economical
packaging of essential viral replicative components, but it could also mean that there is a
functional interdependence between the two domains. A recent report presents new evidence on
interdomain communications that may indeed help to modulate the various NS3 catalytic
activities (82). A contiguous NS3-NS4A protein precursor was expressed in transiently
transfected COS cells and appeared to autoprocess intracellularly to a noncovalent NS3-NS4A
complex. The complex was purified, and the serine protease, RNA-stimulated NTPase, and RNA
helicase activities were characterized. The binding of polynucleotides to the NS3 helicase domain
not only stimulated the NS3-NS4A NTPase activity but also modulated the helicase and protease
activities and thus showed the interconnection of the two functional domains which seem to
have an independent mode of interaction (82). The multiple functions of this protein suggest
that it is exclusively utilized in the replication of the virus so it can be regarded as a promising
target for the development of anti-HCV drugs.
Some evidence has been reported about the interference by the viral proteins on the
intracellular signalling pathways. An inspection of the amino acid sequence of the HCV gene
product revealed an arginine-rich domain located in the NS3 region (residues 1487-1500 of the
polyprotein) (16, 83). This sequence strongly resembles the inhibitory sequence of protein
kinase inhibitor, responsible for the inactivation of the c-AMP dependent protein kinase A (PKA)
that plays a crucial role in intracellular signalling, and the consensus sequence surrounding the
phosphoacceptor amino acid in the substrates of PKA. Because of these similarities a synthetic
peptide corresponding to this HCV sequence and a bacterially expressed 43.5 kilodaltons
fragment of NS3 consisting of HCV polyprotein residues 1189 to 1525 potently inhibited PKA
activity in vitro (83). This observation indicates that NS3 or its fragment may act by inhibiting
PKA in a c-AMP-independent manner, thereby interfering with the signal transduction of
infected cell. In a recent study it was demonstrated that the formation of an in vitro complex
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between the NS3 fragment and the catalytic subunit of the PKA and the significance of the
interactions observed for living cells was investigated and interference of the NS3 fragment with
the PKA signalling has been demonstrated (84).
Apoptosis in virus-infected cells, either mediated by a viral protein(s) or by CTL recognizing
the viral antigens, is currently considered a mechanism of clearance of the virus from the host
(reviewed in 85). Suppression of apoptosis on the other hand, is accordingly thought to be a
major mechanism of viral persistence in the affected cell, which might be a crucial step toward
malignant transformation in the case of infection with tumor viruses. These events raised the
possibility that an HCV protein(s) has anti-apoptosis activity to establish persistent infection
which might be an important step toward development of hepatocellular carcinoma. It has
recently been observed that NS3 suppresses actinomycine D-induced apoptosis probably through
the decreased expression of the tumor suppressor gene p53 which is usually found to be
inactivated with mutations in hepatocellular carcinoma. Interaction of the NS3 with the tumor
suppressor p53, which probably abolishes its functions in DNA damage-induced apoptosis of the
liver cells, has been demonstrated recently by the observation that the NS3 protein of the HCV
changes its localization from cytoplasm to nucleus in a p53-induced manner (86). It is still
unknown whether the transport of the NS3 to the nucleus is mediated by the p53 in a direct or
indirect manner although the NS3 protein is probably carried to the nucleus by p53 or a p53induced carrier protein and thereby might interfere with the p53’s functions in gene expression.
NS4
The nonstructural protein 4 is cleaved in two by transacting NS3 serine protease. The NS4A
is a 54 residue protein expressed immediately downstream of NS3 in the viral polyprotein, and
a central stretch of hydrophobic residues in NS4A from an integral structural component of the
NS3 serine protease domain. Previous cell transfection studies suggest that the NS3 serine
protease domain cleaves the NS3-NS4A junction in cis, thus producing a mature non-covalent
NS3-NS4A complex. The binding of NS4A appears to stabilize the NS3 protein and enhance its
ability to transprocess the NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B (23, 24, 76). The minimal
region of NS4A needed to stimulate the activity of NS3 was originally mapped to a central
stretch of about 13 residues (87). These findings derived a physical explanation when the first
X-ray crystal structure appearned for an NS3 protease domain complexed with a synthetic NS4A
peptide that spans essential NS3-binding residues (88). The structure shows that the peptide
forms a β-strand, intercalating within a β-sheet of the NS3 protease. Thus, NS4A contributes
an integral part of the NS3 protease domain and hence, the NS3 protein (82).
NS5
The nonstructural protein 5 is processed into NS5A and NS5B. Recent studies with a purified
NS3 protease domain identified a potential fifth cleavage site, within the NS5A protein.
Conserved sequence characteristics preceding the newly identified site show similarities to the
prototypical NS3 serine protease cis recognition sequence. However further studies needed to
infer whether the site is cleaved during viral replication in vivo (82).
Two roteins, a faster migrating form of 56 kilodaltons protein, p56 and a slower migrating
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form 58 kilodaltons of protein, p56 and a slower migrating form 58 kilodaltons of protein, p58
are produced from the NS5A region (21, 89-91) and both are phosho proteins. Recent findings
indicate that both proteins are phosphorylated at the serine residues in the C-terminal region of
the NS5A and that p58 is additionally phosphorylated at serine residues in the central region,
amino acid residues 2194 to 2204 (91). When only the NS5A region of the HCV genome is
expressed in the cultured cells, most of the product is p56 and a trace amount is p58. In the
presence of the NS4A however the production of the p58 is augmented strongly and both forms
of NS5A are produced. In a recent study (92) a N-terminal region of the NS5A protein is shown
to have an important role in the phosphorylation of the central region of NS5A. When that
region was deleted, phosphorylation of the central region of NS5A was no longer augmented by
the NS4A protein. Also it has been demonstrated for the first time that the NS4A and NS5A are
associated with each other. However the physiological role of the phosphorylation of the NS5A
and its association with the NS4A protein still remains to be clarified.
The NS5B is primarily localized at the perinuclear region, suggested by its association with
the nuclear membrane and the endoplasmic reticulum or Golgi complex (93). It is interesting
because these findings suggest that the HCV replication might take place in the membrane
complex which is consistent with the findings on other RNA viruses as their RNA synthesis is
shown to take place in a membrane complex.
It has been suggested (94) that in some isolates a secondary structure of the genomic RNA
exists at the C terminal part of the NS4 region that could possibly form and IRES. This IRES
would be located just upstream of a common in frame ATG codon allowing eariler and increased
production of NS5-encoded replicase. Consequently, this would accelerate the replication rate of
the genomic RNA.
Molecular Biology of Treatment with Interferon
Response to IFN therapy differs among the six HCV genotypes, but is observed at some level
in all HCV genotypes worldwide. Of the two HCV genotypes, HCV-1a and HCV-1b both exhibit
a high level of resistance to IFN therapy. Recently the clinical isolates of the HCV genome others
have been sequenced and correlated mutations within a discrete region of the NS5A protein have
been termed the IFN sensitivity determining region (ISDR), of the HCV-1b with the IFN sensitive
phenotype. These studies demonstrated that the strains closely matching the prototype HCV-1b
NS5A sequence correlated with complete IFN resistance (95, 96).
In a more recent study a possible mechanism underlying this HCV resistance to IFN therapy
has been proposed (97). The IFN-induced cellular antiviral response is mediated in part by the
actions of the Mx proteins, the 2’-5’ oligoadenylate synthetase, RNAse L, and PKR. Induced by
IFN, these antiviral effector proteins block viral gene expression in multiple levels. PKR protein
kinase is the most widely studied among these antiviral proteins. It is known that the PKR
phosphorylates the α subunit of the eukaryotic translation initiation factor 2 (eIF-2α) resulting
in a global cessation of protein synthesis and a concominant block in viral replication within the
infected region. To counteract the deleterious effects of IFN induction and PKR activation, many
viruses have evolved mechanisms to block the activity of the PKR with specific kinase inhibitory
molecules; for example te Tat protein encoded by the HIV virus is a PKR kinase inhibitor. The
262
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large population of IFN resistant HCV indicated indicated that this protein probably utilizes a
similar mechanism. Although the function of NS5A and its role in viral replication had been
unknown, it has been suggested that NS5A, by an ISDR-directed mechanism, may mediate IFN
resistance by interacting with and repressing the IFN induced protein kinase PKR (97).
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