Book on hepatitis from page 87 to 95

Book on hepatitis from page 86 to 95

86  Hepatology 2012
Taxonomy and genotypes
HCV is a small-enveloped virus with one single-stranded positive-sense RNA
molecule of approximately 9.6 kb. It is a member of the Flaviviridae family. This
viral family contains three genera, flavivirus, pestivirus, and hepacivirus. To date,
only two members of the hepacivirus genus have been identified, HCV and GB
virus B (GBV-B), a virus that had been initially detected together with the then-unclassified virus GB virus A (GBV-A) in a surgeon with active hepatitis (Thiel
2005, Ohba 1996, Simons 1995). However, the natural hosts for GBV-B and GBV-C  seem to be monkeys of the Saguinus  species (tamarins). Analyses of viral
sequences and phylogenetic comparisons support HCV’s membership in a distinct
genus from flavivirus or pestivirus (Choo 1991). The error-prone RNA polymerase
of HCV together with the high replication rate of the virus is responsible for the
large interpatient genetic diversity of HCV strains. Moreover, the extent of viral
diversification of HCV strains within a single HCV-positive individual increases
significantly over time, resulting in the development of quasispecies (Bukh 1995).
Comparisons of HCV nucleotide sequences derived from individuals from
different geographical regions revealed the presence of six major HCV genotypes
with a large number of subtypes within each genotype (Simmonds 2004, Simmonds
2005). Sequence divergence of genotypes and subtypes is 20% and 30%,
respectively. HCV strains belonging to the major genotypes 1, 2, 4, and 5 are found
in sub-Saharan Africa whereas genotypes 3 and 6 are detected with extremely high
diversity in South East Asia. This suggests that these geographical areas could be
the origin of the different HCV genotypes. The emergence of different HCV
genotypes in North America and Europe and other non-tropical countries appears to
represent more recent epidemics introduced from the countries of the original HCV
endemics (Simmonds 2001, Ndjomou 2003). Besides epidemiological aspects,
determination of the HCV genotype plays an important role for the initiation of anti-HCV treatment since the response of different genotypes varies significantly with
regard to specific antiviral drug regimens, e.g., genotype 1 is most resistant to the
current therapy of the combination of pegylated interferon α and ribavirin (Manns
2001, Fried 2002).
Viral structure
Structural analyses of HCV virions are very limited since the virus is difficult to
cultivate in cell culture systems, a prerequisite for yielding sufficient virions for
electron microscopy. Moreover, serum-derived virus particles are associated with
serum low-density lipoproteins (Thomssen 1992), which makes it difficult to isolate
virions from serum/plasma of infected subjects by centrifugation. Visualization of
HCV virus-like particles via electron microscopy succeeded only rarely (Kaito
1994, Shimizu 1996a, Prince 1996) and it was a point of controversy if the detected
structures really were HCV virions. Nevertheless, these studies suggest that HCV
has a diameter of 55-65 nm confirming size prediction of the NANBH agent by
ultra-filtration (Bradley 1985). Various forms of HCV virions appear to exist in the
blood of infected individuals: virions bound to very low density lipoproteins
(VLDL), virions bound to low density lipoproteins (LDL), virions complexed with
immunoglobulins, and free circulating virions (Bradley 1991, Thomssen 1992,
HCV Virology  87
Thomssen 1993, Agnello 1999, Andre 2002). The reasons for the close association
of a major portion of circulating virions with LDL and VLDL remain unexplained.
One possible explanation is that HCV theoretically enters hepatocytes via the LDL
receptor (Agnello 1999, Nahmias 2006). Moreover, it is speculated that the
association with LDL and/or VLDL protects the virus against neutralization by
HCV-specific antibodies.
The design and optimization of subgenomic and genomic HCV replicons in the
human hepatoma cell line Huh7 offered for the first time the possibility to
investigate HCV RNA replication in a standardized manner (Lohmann 1999, Ikeda
2002, Blight 2002). However, despite the high level of HCV gene expression, no
infectious viral particles are actually produced. Therefore, it cannot be used for
structural analysis of free virions.
Infectious HCV particles have been achieved in cell culture by using recombinant
systems (Heller 2005, Lindenbach 2005, Wakita 2005, Zhong 2005, Yu 2007).
However, even in these in vitro systems the limited production of viral particles
prevents 3D structural analysis (Yu 2007). It was also shown by cryoelectron
microscopy (cryoEM) and negative-stain transmission electron microscopy that
HCV virions isolated from cell culture have a spherical shape with a diameter of
approximately 50 to 55 nm (Heller 2005, Wakita 2005, Yu 2007) confirming earlier
results that measured the size of putative native HCV particles from the serum of
infected individuals (Prince 1996). The outer surface of the viral envelope seems to
be smooth. Size and morphology are therefore very similar to other members of the
Flaviviridae family such as the dengue virus and the West Nile virus (Yu 2007).
Modifying a baculovirus system (Jeong 2004, Qiao 2004) the same authors were
able to produce large quantities of HCV-like particles (HCV-LP) in insect cells (Yu
2007). Analysing the HCV-LPs by cryoEM it was demonstrated that the HCV E1
protein is present in spikes located on the outer surface of the LPs.
Using 3D modeling of the HCV-LPs together with genomic comparison of HCV
and well-characterized flaviviruses it is assumed that 90 copies of a block of two
heterodimers of HCV proteins E1 and E2 form the outer layer of the virions with a
diameter of approximately 50 nm (Yu 2007). This outer layer surrounds the lipid
bilayer that contains the viral nucleocapsid consisting of several molecules of the
HCV core (C) protein. An inner spherical structure with a diameter of
approximately 30-35 nm has been observed (Wakita 2005) suggesting the
nucleocapsid that harbours the viral genome (Takahashi 1992).
Genome organization
The genome of the hepatitis C virus consists of one 9.6 kb single-stranded RNA
molecule with positive polarity. Similar to other positive-strand RNA viruses, the
genomic RNA of hepatitis C virus serves as messenger RNA (mRNA) for the
translation of viral proteins. The linear molecule contains a single open reading
frame (ORF) coding for a precursor polyprotein of approximately 3000 amino acid
residues (Figure 1). During viral replication the polyprotein is cleaved by viral as
well as host enzymes into three structural proteins (core, E1, E2) and seven non-structural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B). An additional
protein (termed F [frameshift] or ARF [alternate reading frame]) is predicted as a
result of ribosomal frameshifting during translation within the core region of the
88  Hepatology 2012
genomic RNA (Xu 2001, Walewski 2001, Varaklioti 2002, Branch 2005). Detection
of anti-F protein antibodies in the serum of HCV-positive subjects indicates that the
protein is expressed during infection in vivo (Walewski 2001, Komurian-Pradel
2004).
The structural genes encoding the viral  core protein and the viral envelope
proteins E1 and E2 are located at the 5’ terminus of the open reading frame
followed downstream by the coding regions for the non-structural proteins p7, NS2,
NS3, NS4A, NS4B, NS5A, and NS5B (Figure 1). The structural proteins are
essential components of the HCV virions, whereas the non-structural proteins are
not associated with virions but are involved in RNA replication and virion
morphogenesis.
The ORF is flanked by 5’ and 3’ nontranslated regions (NTR; also called
untranslated regions, UTR or noncoding regions, NCR) containing nucleotide
sequences relevant for the regulation of viral replication. Both NTRs harbour highly
conserved regions compared to the protein encoding regions of the HCV genome.
The high grade of conservation of the NTRs makes them candidates i) for improved
molecular diagnostics, ii) as targets for antiviral therapeutics, and iii) as targets for
an anti-HCV vaccine.
Figure 1. Genome organization and polyprotein processing. A) Nucleotide positions
correspond to the HCV strain H77 genotype 1a, accession number NC_004102.
nt, nucleotide; NTR, nontranslated region. B) Cleavage sites within the HCV precursor
polyprotein for the cellular signal peptidase the signal peptide peptidase (SPP) and the viral
proteases NS2-NS3 and NS3-NS4A, respectively.
The 5’NTR is approximately 341 nucleotides long with a complex secondary
structure of four distinct domains (I-IV) (Fukushi 1994, Honda 1999). The first 125
nucleotides of the 5’NTR spanning domains I and  II have been shown to be
essential for viral RNA replication (Friebe 2001, Kim 2002). Domains II-IV build
an internal ribosome entry side (IRES) involved in ribosome binding and
subsequent cap-independent initiation of translation (Tsukiyama-Kohara 1992,
Wang 1993).
The 3’NTR consists of three functionally distinct regions: a variable region, a
poly U/UC tract of variable length, and the highly conserved X tail at the 3’
terminus of the HCV genome (Tanaka 1995, Kolykhalov 1996, Blight 1997). The
HCV Virology  89
variable region of approximately 40 nucleotides is not essential for RNA
replication. However, deletion of this sequence led to significantly decreased
replication efficiency (Yanagi 1999, Friebe 2002). The length of the poly U/UC
region varies in different HCV strains ranging from 30 to 80 nucleotides
(Kolykhalov 1996). The minimal length of that region for active RNA replication
has been reported to be 26 homouridine nucleotides in cell culture (Friebe 2002).
The highly conserved 98-nucleotide X tail consists of three stem-loops (SL1-SL3)
(Tanaka 1996, Ito 1997, Blight 1997) and deletions or nucleotide substitutions
within that region are most often lethal (Yanagi 1999, Kolykhalov 2000, Friebe
2002, Yi 2003). Another so-called “kissing-loop” interaction of the 3’X tail SL2 and
a complementary portion of the NS5B encoding region has been described (Friebe
2005). This interaction induces a tertiary RNA structure of the HCV genome that is
essential for HCV replication in cell culture systems (Friebe 2005, You 2008).
Finally, both NTRs appear to work together in a long-range RNA-RNA interaction
possibly resulting in temporary genome circularization (Song 2006).
Genes and proteins
As described above, translation of the HCV polyprotein is initiated through
involvement of some domains in NTRs of the genomic HCV RNA. The resulting
polyprotein consists of ten proteins that are co-translationally or post-translationally
cleaved from the polyprotein. The N-terminal proteins C, E1, E2, and p7 are
processed by a cellular signal peptidase (SP) (Hijikata 1991). The resulting
immature core protein still contains the E1 signal sequence at its C terminus.
Subsequent cleavage of this sequence by a signal peptide peptidase (SPP) leads to
the mature core protein (McLauchlan 2002). The non-structural proteins NS2 to
NS5B of the HCV polyprotein are processed by two virus-encoded proteases (NS2-NS3 and NS3) with the NS2-NS3 cysteine protease cleaving at the junction of NS2-NS3 (Santolini 1995) and the NS3 serine protease cleaving the remaining functional
proteins (Bartenschlager 1993, Eckart 1993, Grakoui 1993a, Tomei 1993).
The positions of viral nucleotide and amino acid residues correspond to the HCV
strain H77 genotype 1a, accession number NC_004102. Some parameters
characterizing HCV proteins are summarised in Table 1.
Core. The core-encoding sequence starts at codon AUG at nt position 342 of the
H77 genome, the start codon for translation of the entire HCV polyprotein. During
translation the polyprotein is transferred to the endoplasmic reticulum (ER) where
the core protein (aa 191) is excised by a cellular signal peptidase (SP). The C
terminus of the resulting core precursor still contains the signal sequence for ER
membrane translocation of the E1 ectodomain (aa 174-191). This protein region is
further processed by the cellular intramembrane signal peptide peptidase (SPP)
leading to removal of the E1 signal peptide sequence (Hüssy 1996, McLauchlan
2002, Weihofen 2002).
The multifunctional core protein has a molecular weight of 21 kilodalton (kd). In
vivo, the mature core molecules are believed to form homo-multimers located
mainly at the ER membrane (Matsumoto 1996). They have a structural function
since they form the viral capsid that contains the HCV genome. In addition, the core
protein has regulatory functions including particle assembly, viral RNA binding,
and regulation of RNA translation (Ait-Goughoulte 2006, Santolini 1994).
90  Hepatology 2012
Moreover, protein expression analyses indicate that the core protein may be
involved in many other cellular reactions such as cell signalling, apoptosis, lipid
metabolism, and carcinogenesis (Tellinghuisen 2002). However, these preliminary
findings need to be analysed further.
Table 1. Overview of the size of HCV proteins.*
Protein  No. of aa  aa position
in ref. seq.
MW of protein
Core immature  191  1-191  23 kd
Core mature  174  1-174  21 kd
F-protein or ARF-protein  126-161    ~ 16-17 kd
E1  192  192-383  35 kd
E2  363  384-746  70 kd
p7  63  747-809  7 kd
NS2  217  810-1026  21 kd
NS3  631  1027-1657  70 kd
NS4A  54  1658-1711  4 kd
NS4B  261  1712-1972  27 kd
NS5A  448  1973-2420  56 kd
NS5B  591  2421-3011  66 kd
* aa, amino acid; MW, molecular weight; kd, kilodalton; ref. seq., reference sequence (HCV
strain H77; accession number NC_004102).
E1 and E2. Downstream of the core coding region of the HCV RNA genome two
envelope glycoproteins are encoded, E1 (gp35, aa 192) and E2 (gp70, aa 363).
During translation at the ER both proteins are cleaved from the precursor
polyprotein by a cellular SP. Inside the lumen of the ER both polypeptides
experience N-linked glycosylation post-translationally (Duvet 2002). Both
glycoproteins E1 and E2 harbour 5 and 11 putative N-glycosylation sites,
respectively.
E1 and E2 are type I transmembrane proteins with a large hydrophilic ectodomain
of approximately 160 and 334 aa and a short transmembrane domain (TMD) of 30
aa. The TMD are responsible for the anchoring of the envelope proteins in the
membrane of the ER and ER retention (Cocquerel 1998, Duvet 1998, Cocquerel
1999, Cocquerel 2001). Moreover, the same domains have been reported to
contribute to the formation of E1-E2 heterodimers (Op de Beeck 2000). The E1-E2
complex is involved in adsorption of the virus to its putative receptors tetraspanin
CD81 and low-density lipoprotein receptor inducing fusion of the viral envelope
with the host cell plasma membrane (Agnello 1999, Flint 1999, Wunschmann
2000). However, the precise mechanism of host cell entry is still not understood
completely. Several other host factors have been identified to be involved in viral
entry. These candidates include the scavenger receptor B type I (Scarselli 2002,
Kapadia 2007), the tight junction proteins claudin-1 (Evans 2007) and occludin
(Ploss 2009), the C-type lectins L-SIGN and DC-SIGN (Gardner 2003, Lozach
2003, Pöhlmann 2003) and heparan sulfate (Barth 2003).
Two hypervariable regions have been identified within the coding region of E2.
These regions termed hypervariable region 1 (HVR1) and 2 (HVR2) differ by up to
80% in their amino acid sequence (Weiner 1991, Kato 2001). The first 27 aa of the
E2 ectodomain represent HVR1, while the HVR2 is formed by a stretch of seven
HCV Virology  91
amino acids (position 91-97). The high variability of the HVRs reflects exposure of
these domains to HCV-specific antibodies. In fact, E2-HVR1 has been shown to be
the most important target for neutralizing antibodies (Farci 1996, Shimizu 1996b).
However, the combination of the mutation of the viral genome with the selective
pressure of the humoral immune response leads to viral escape via epitope
alterations. This makes the development of vaccines that induce neutralizing
antibodies challenging.
The p7 protein. The small p7 protein (63 aa) is located between the E2 and NS2
regions of the polyprotein precursor. During translation the cellular SP cleaves the
E2-p7 as well as the p7-NS2 junction. The functional p7 is a membrane protein
localised in the endoplasmic reticulum where it forms an ion channel (Haqshenas
2007, Pavlovic 2003, Griffin 2003). The p7 protein is not essential for RNA
replication since replicons lacking the p7 gene replicate efficiently (Lohmann 1999,
Blight 2000), however, it has been suggested that p7 plays an essential role for the
formation of infectious virions (Sakai 2003, Haqshenas 2007).
NS2. The non-structural protein 2 (p21, 217 aa) together with the N-terminal
portion of the NS3 protein form the NS2-3 cysteine protease which catalyses
cleavage of the polyprotein precursor between NS2 and NS3 (Grakoui 1993b,
Santolini 1995). The N-terminus of the functional NS2 arises from the cleavage of
the p7-NS2 junction by the cellular SP. Moreover, after cleavage from the NS3 the
protease domain of NS2 seems to play an essential role in the early stage of virion
morphogenesis (Jones 2007).
NS3. The non-structural protein 3 (p70, 631 aa) is cleaved at its N terminus by the
NS2-NS3 protease. The N terminus (189 aa) of the NS3 protein has a serine
protease activity. However, in order to develop full activity of the protease the NS3
protease domain requires a portion of NS4A (Faila 1994, Bartenschlager 1995, Lin
1995, Tanji 1995, Tomei 1996). NS3 together with the NS4A cofactor are
responsible for cleavage of the remaining downstream cleavages of the HCV
polyprotein precursor. Since the NS3 protease function is essential for viral
infectivity it is a promising target in the design of antiviral treatments.
The C-terminal portion of NS3 (442 aa) has ATPase/helicase activity, i.e., it
catalyses the binding and unwinding of the viral RNA genome during viral
replication (Jin 1995, Kim 1995). However, recent findings indicate that other non-structural HCV proteins such as the viral polymerase NS5B may interact
functionally with the NS3 helicase (Jennings 2008). These interactions need to be
investigated further in order to better understand the mechanisms of HCV
replication.
NS4A. The HCV non-structural protein 4A (p4) is a 54 amino acid polypeptide
that acts as a cofactor of the NS3 serine protease (Faila 1994, Bartenschlager 1995,
Lin 1995, Tanji 1995, Tomei 1996). Moreover, this small protein is involved in the
targeting of NS3 to the endoplasmic reticulum resulting in a significant increase of
NS3 stability (Wölk 2000).
NS4B. The NS4B (p27) consists of 217 amino acids. It is an integral membrane
protein localized in the endoplasmic reticulum. The N-terminal domain of the NS4B
has an amphipathic character that targets the protein to the ER. This domain is
crucial in HCV replication (Elazar 2004, Gretton 2005) and therefore an interesting
target for the development of anti-HCV therapeutics or vaccines. In addition, a
nucleotide-binding motif (aa 129-134) has been identified (Einav 2004). Although
92  Hepatology 2012
the function of NS4B is still unknown, it has been demonstrated that the protein
induces a membranous web that may serve as a platform for HCV RNA replication
(Egger 2002).
NS5A. The NS5A protein (p56; 458 aa) is a membrane-associated phosphoprotein
that appears to have multiple functions in viral replication. It is phosphorylated by
different cellular protein kinases indicating an essential but still not understood role
of NS5A in the HCV replication cycle. In addition, NS5A has been found to be
associated with several other cellular proteins (MacDonald 2004) making it difficult
to determine the exact functions of the protein. One important property of NS5A is
that it contains a domain of 40 amino acids, the so-called IFN-a sensitivity-determining region (ISDR) that plays a significant role in the response to IFN-a-based therapy (Enomoto 1995, Enomoto 1996). An increasing number of mutations
within the ISDR showed positive correlation with sustained virological response to
IFN-a-based treatment.
NS5B.  The non-structural protein 5B (p66; 591 aa) represents the RNA-dependent RNA polymerase of HCV (Behrens 1996). The hydrophobic domain (21
aa) at the C terminus of NS5B inserts into the membrane of the endoplasmic
reticulum, while the active sites of the polymerase are located in the cytoplasm
(Schmidt-Mende 2001).
The cytosolic domains of the viral enzyme form the typical polymerase right-handed structure with “palm”, “fingers”, and “thumb” subdomains (Ago 1999,
Bressanelli 1999, Lesburg 1999). In contrast to mammalian DNA and RNA
polymerases the fingers and thumb subdomains are connected resulting in a fully
enclosed active site for nucleotide triphosphate binding. This unique structure
makes the HCV NS5B polymerase an attractive target for the development of
antiviral drugs.
Using the genomic HCV RNA as a template, the NS5B promotes the synthesis of
minus-stranded RNA that then serves as a template for the synthesis of genomic
positive-stranded RNA by the polymerase.
Similar to other RNA-dependent polymerases, NS5B is an error-prone enzyme
that incorporates wrong ribonucleotides at a  rate of approximately 10
-3
per
nucleotide per generation. Unlike cellular polymerases, the viral NS5B lacks a
proof-reading mechanism leading to the conservation of misincorporated
ribonucleotides. These enzyme properties together with the high rate of viral
replication promote a pronounced intra-patient as well as inter-patient HCV
evolution.
F protein, ARFP. In addition to the ten proteins derived from the long HCV
ORF, the F (frameshift) or ARF (alternate reading frame) or core+1 protein has
been reported (Walewski 2001, Xu 2001, Varaklioti 2002). As the designations
indicate the ARFP is the result of a -2/+1 ribosomal frameshift between codons 8
and 11 of the core protein-encoding region. The ARFP length varies from 126 to
161 amino acids depending on the corresponding genotype. In vitro studies have
shown that ARFP is a short-lived protein located in the cytoplasm (Roussel 2003)
primarily associated with the endoplasmic reticulum (Xu 2003). Detection of anti-F
protein antibodies in the serum of HCV-positive subjects indicates that the protein is
expressed during infection in vivo  (Walewski 2001, Komurian-Pradel 2004).
However, the functions of ARFP in the viral life cycle are still unknown and remain
to be elucidated.
HCV Virology  93
Viral lifecycle
Due to the absence of a small animal model system and efficient in vitro HCV
replication systems it has been difficult to investigate the viral life cycle of HCV.
The recent development of such systems has offered the opportunity to analyse in
detail the different steps of viral replication.
Figure 2. Current model of the HCV lifecycle. Designations of cellular components are in red.
For a detailed illustration of viral translation and RNA replication, see Pawlotsky 2007.
Abbreviations: HCV +ssRNA, single stranded genomic HCV RNA with positive polarity; rough
ER, rough endoplasmic reticulum; PM, plasma membrane. For other abbreviations see text.
Adsorption and viral entry
The most likely candidate as receptor for HCV is the tetraspanin CD81 (Pileri
1998). CD81 is an ubiquitous 25 kd molecule expressed on the surface of a large
variety of cells including hepatocytes and PBMCs. Experimental binding of anti-CD81 antibodies to CD81 were reported to inhibit HCV entry into Huh7 cells and
primary human hepatocytes (Hsu 2003, Bartosch 2003a, Cormier 2004, McKeating
2004, Zhang 2004, Lindenbach 2005, Wakita 2005). Moreover, gene silencing of
CD81 using specific siRNA molecules confirmed the relevance of CD81 in viral
entry (Bartosch 2003b,  Cormier 2004,  Zhang 2004,  Akazawa 2007). Finally,
expression of CD81 in cell lines lacking CD81 made them permissive for HCV
entry (Zhang 2004, Lavillette 2005, Akazawa 2007). However, more recent studies
have shown that CD81 alone is not sufficient for HCV viral entry and that co-factors such as scavenger receptor B type I (SR-BI) are needed (Bartosch 2003b,
Hsu 2003,  Scarselli 2002, Kapadia 2007). Moreover, it appears that CD81 is
involved in a post-HCV binding step (Cormier 2004, Koutsoudakis 2006, Bertaud
2006). These findings together with the identification of other host factors involved
94  Hepatology 2012
in HCV cell entry generate the current model for the early steps of HCV infection
(Helle 2008).
Adsorption of HCV to its target cell is the first step of viral entry. Binding is
possibly initiated by the interaction of the HCV E2 envelope glycoprotein and the
glycosaminglycan heparan sulfate on the surface of host cells (Germi 2002, Barth
2003, Basu 2004, Heo 2004). Moreover, it is assumed that HCV initiates hepatocyte
infection via LDL receptor binding (Agnello 1999, Monazahian 1999, Wünschmann
2000, Nahmias 2006, Molina 2007). This process may be mediated by VLDL or
LDL and is reported to be associated with HCV virions in human sera (Bradley
1991,  Thomssen 1992,  Thomssen  1993). After initial binding the HCV E2
glycoprotein interacts with the SR-BI in cell culture (Scarselli 2002). SR-BI is a
protein expressed on the surface of the majority of mammalian cells. It acts as a
receptor for LDL as well as HDL (Acton 1994, Acton 1996) emphasizing the role of
these compounds for HCV infectivity. Alternative splicing of the SR-BI transcript
leads to the expression of a second isoform of the receptor SR-BII (Webb 1998),
which also may be involved in HCV entry into target cells (Grove 2007). As is the
case for all steps of viral entry the exact mechanism of the HCVE2/SR-BI
interaction remains unknown. In some studies it has been reported that HCV
binding to SR-BI is a prerequisite for the concomitant or subsequent interaction of
the virus with CD81 (Kapadia 2007, Zeisel 2007). The multi-step procedure of
HCV cell entry was shown to be even more complex since a cellular factor termed
claudin-1 (CLDN1) has been newly identified as involved in this process (Evans
2007). CLDN1 is an integral membrane protein that forms a backbone of tight
junctions and is highly expressed in the liver (Furuse 1998). Inhibition assays reveal
that CLDN1 involvement occurs downstream of the HCV-CD81 interaction (Evans
2007). Recent findings suggest that CLDN1 could also act as a compound enabling
cell-to-cell transfer of hepatitis C virus independently of CD81 (Timpe 2007).
Furthermore, it was reported that two other members of the claudin family claudin-6
and claudin-9 may play a role in HCV infection (Zheng 2007, Meertens 2008). The
fact that some human cell lines were not susceptible to HCV infection despite
expressing SR-BI, CD81, and CLDN1 indicates that other cellular factors are
involved in viral entry (Evans 2007). Very recently, a cellular four-transmembrane
domain protein named occludin (OCLN) was identified to represent an additional
cellular factor essential for the susceptibility of cells to HCV infection (Liu 2009,
Ploss 2009). Similar to claudin-1, OCLN is a component of the tight junctions in
hepatocytes. All tested cells expressing SR-BI, CD81, CLDN1, and OCLN were
susceptible to HCV. Although the precise mechanism of HCV uptake in hepatocytes
is still not clarified, these four proteins may represent the complete set of host cell
factors necessary for cell-free HCV entry.
After the complex procedure of binding to the different host membrane factors
HCV enters the cell in a pH-dependent manner indicating that the virus is
internalized via clathrin-mediated endocytosis (Bartosch 2003b,  Hsu 2003,
Blanchard 2006, Codran 2006). The acidic environment within the endosomes is
assumed to trigger HCV E1-E2 glycoprotein-mediated fusion of the viral envelope
with the endosome membrane (Blanchard 2006, Meertens 2006, Lavillette 2007).
In summary, HCV adsorption and viral entry into the target cell is a very complex
procedure that is not yet fully understood. Despite having identified several host
HCV Virology  95
factors that probably interact with the viral glycoproteins, the precise mechanisms
of interaction need to continue to be investigated.
Besides the infection of cells through cell-free HCV it has been documented that
HCV can also spread via cell-to-cell transmission (Valli 2006, Valli 2007). This
transmission path may differ significantly with regard to the cellular factors needed
for HCV entry into cells. CD81 is dispensable for cell-to-cell transmission in
cultivated hepatoma cells (Witteveldt 2009). These findings require further
investigation in order to analyze the process of cell-to-cell transmission of HCV
both in vitro and in vivo. Antiviral treatment strategies must account for the cellular
pathways of both cell-free virus and HCV transmitted via cell-to-cell contact.
Translation and posttranslational processes
As a result of the fusion of the viral envelope and the endosomic membrane, the
genomic HCV RNA is released into the cytoplasm of the cell. As described above,
the viral genomic RNA possesses a nontranslated region (NTR) at each terminus.
The 5’NTR consists of four distinct domains, I-IV. Domains II-IV form an internal
ribosome entry side (IRES) involved in ribosome-binding and subsequent cap-independent initiation of translation (Fukushi 1994,  Honda 1999,  Tsukiyama-Kohara 1992, Wang 1993). The HCV-IRES binds to the 40S ribosomal subunit
complexed with eukaryotic initiation factors 2 and 3 (eIF2 and eIF3), GTP, and the
initiator tRNA resulting in the 48S preinitiation complex (Spahn 2001, Otto 2002,
Sizova 1998, reviewed in Hellen 1999). Subsequently, the 60S ribosomal subunit
associates with that complex leading to the formation of the translational active
complex for HCV polyprotein synthesis at the endoplasmic reticulum. HCV RNA
contains a large ORF encoding a polyprotein precursor. Posttranslational cleavages
lead to 10 functional viral proteins Core, E1, E2, p7, NS2-NS5B. The viral F protein
(or ARF protein) originates from a ribosomal frameshift within the first codons of
the core-encoding genome region (Walewski 2001, Xu 2001, Varaklioti 2002).
Besides several other cellular factors that have been reported to be involved in HCV
RNA translation, various viral proteins and genome regions have been shown to
enhance or inhibit viral protein synthesis (Zhang 2002, Kato 2002, Wang 2005, Kou
2006, Bradrick 2006, Song 2006).
The precursor polyprotein is processed by at least four distinct peptidases. The
cellular signal peptidase (SP) cleaves the N-terminal viral proteins immature core
protein, E1, E2, and p7 (Hijikata 1991), while the cellular signal peptide peptidase
(SPP) is responsible for the cleavage of the E1 signal sequence from the C-terminus
of the immature core protein, resulting in the mature form of the core (McLauchlan
2002). The E1 and E2 proteins remain within the lumen of the ER where they are
subsequently N-glycosylated with E1 having 5 and E2 harbouring 11 putative N-glycosylation sites (Duvet 2002).
In addition to the two cellular peptidases HCV encodes two viral enzymes
responsible for cleavage of the non-structural proteins NS2 to NS5B within the
HCV polyprotein precursor. The zinc-dependent NS2-NS3 cysteine protease
consisting of the NS2 protein and the N-terminal portion of NS3 autocatalytically
cleaves the junction between NS2 and NS3 (Santolini 1995), whereas the NS3
serine protease cleaves the remaining functional proteins (Bartenschlager 1993,
Eckart 1993, Grakoui 1993a, Tomei 1993). However, for its peptidase activity NS3