Chapter 6

HCV Virology

B. Kupfer

History

Hepatitis C virus (HCV) is a major cause of progressive liver disease with approximately 170 million people infected worldwide. HCV induces chronic infection in up to 80% of infected individuals. The main complications of HCV infection are severe liver fibrosis and cirrhosis, and 30-50% of individuals with cirrhosis develop hepatocellular carcinoma (Tong 1995; Poynard 1997).

Until 1975, only two hepatitis viruses had been identified, the "infectious hepatitis virus" (hepatitis A virus, HAV) and the "serum hepatitis virus" (hepatitis B virus, HBV). However, other viruses were excluded from being the cause of approximately 65% of post-transfusion hepatitis. Therefore, these hepatitis cases were termed "non-A, non-B hepatitis" (NANBH) (Feinstone 1975). Innoculation of chimpanzees (Pan troglodytes) with blood products derived from humans with NANB hepatitis led to persistent increases of serum alanine aminotransferase (ALT) indicating that an infectious agent was the cause of the disease (Alter 1978; Hollinger 1978).

Subsequently, it was demonstrated that the NANBH agent could be inactivated by chloroform (Feinstone 1983). Moreover, it was reported that the infectious agent was able to pass through 80 nm membrane filters (Bradley 1985). Taken together these findings suggested that the NANBH causing agent would be a small virus with a lipid envelope. However, the lack of a suitable cell culture system for cultivation of the NANBH agent and the limited availability of chimpanzees prevented further characterization of the causative agent of NANBH for several years. In 1989, using a newly developed cloning strategy for nucleic acids derived from plasma of NANBH infected chimpanzees the genome of the major causative agent for NANBH was characterized (Choo 1989). cDNA clone 5-1-1 encoded immunological epitopes that interacted with sera from individuals with NANBH (Choo 1989; Kuo 1989). The corresponding infectious virus causing the majority of NANBH was subsequently termed hepatitis C virus (HCV).

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 and classified, 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 of a separate genus distinct from the generas 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-infected individual increases significantly over time resulting in the development of different 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). The 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 the 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 alfa 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 a sufficient number of 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 to 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; 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 Huh-7 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 produced using this otherwise powerful tool. Thus, it cannot be used for structural analysis of free virions.

Only recently have infectious HCV particles 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). Very recently, it was shown by cryo-electron 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 bi-layer which itself 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...

more...
Hepatology 2009
501 pages, PDF, 9 MB

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]) has been predicted as a result of ribosomal frameshifting during translation within the core-region of the genomic RNA (Xu 2001; Walewski 2001; Varaklioti 2002; Branch 2005). Detection of anti-F-protein antibodies in the serum of HCV-infected subjects indicates that the protein is expressed during infection in vivo (Walewski 2001; Komurian-Pradel 2004). 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 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 termed 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 anti-HCV vaccines. The 5'NTR is approximately 341 nucleotides long and it has a complex secondary structure with 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 different 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 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 following 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. 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 Table 1. Overview of the size of HCV proteins. aa, amino acid; MW, molecular weight; kd, kilodalton; ref. seq., reference sequence (HCV strain H77; accession number NC_004102). 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 (191 aa) 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). 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 analyzed further. E1 and E2. Downstream of the core-coding region the HCV RNA genome two envelope glycoproteins are encoded, E1 (gp35; 192 aa) and E2 (gp70; 363 aa). 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 posttranslationally (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 their 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 (SR-BI) (Scarselli 2002; Kapadia 2007), 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 amino acids (position 91-97). The high variability of the HVRs reflects exposition 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 humoural immune response leads to viral escape via epitope alterations. This makes the development of vaccines inducing 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 which is localized 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 for design of antiviral treatments. The C-terminal portion of NS3 (442 aa) has ATPase/helicase activity, i.e., it catalyses 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 nonstructural 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, respectively. In addition, a nucleotide-binding motif (aa 129-134) has been identified (Einav 2004). Although 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, whereas 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 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-infected 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. 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 to be a receptor for HCV is the tetraspanin CD81 (Pileri 1998). CD81 is a ubiquitous 25 kd molecule expressed on the surface of a large variety of cells including hepatocytes and PBMC. Experimental binding of anti-CD81 antibodies to CD81 were reported to inhibit HCV entry into Huh-7 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 in HCV cell entry were used to 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, 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 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). Very recently, 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). After the complex procedure of binding to the different host 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 factors that probably interact with the viral glycoproteins the precise mechanisms of interaction remain to be investigated more in depth. The fact that some human cell lines are not susceptible to HCV infection despite expressing SR-BI, CD81, and CLDN1 indicates that other cellular factors are involved in viral entry (Evans 2007). 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 build 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 to 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 needs NS4A as a cofactor (Failla 1994; Tanji 1995; Bartenschlager 1995; Lin 1995; Tomei 1996). HCV RNA replication. The complex process of HCV RNA replication is poorly understood. The key enzyme for viral RNA replication is NS5B an RNA-dependent RNA polymerase (RdRp) of HCV (Behrens 1996). In addition, several cellular as well as viral factors have been reported to be part of the HCV RNA replication complex. One important viral factor for the formation of the replication complex appears to be NS4B which is able to induce an ER-derived membranous web containing most of the non-structural HCV proteins including NS5B (Egger 2002). This web could serve as the platform for the next steps of viral RNA replication. The RdRp uses the previously released genomic positive-stranded HCV RNA as a template for the synthesis of an intermediate minus-stranded RNA. In order to facilitate synthesis of minus-strand RNA the NS3 helicase is assumed to unwind putative secondary structures of the template RNA (Jin 1995; Kim 1995). In turn, again with the assistance of the NS3 helicase the newly synthesized antisense RNA molecule serves as the template for the synthesis of numerous positive-stranded RNA. The resulting sense RNA could subsequently be used as genomic RNA for HCV progeny as well as for polyprotein translation. Assembly and release. After the viral proteins, glycoproteins, and the genomic HCV RNA have been synthesized these single components have to be arranged in order to produce infectious virions. As is the case for all other steps in the HCV lifecycle viral assembly is a multi-step procedure involving most viral components along with many cellular factors. Investigation of viral assembly and particle release is still in its infancy since the development of in vitro models for the production and release of infectious HCV occurred only recently. Previously, it was reported that core protein molecules were able to self-assemble in vitro, yielding nucleocapsid-like particles. Very recent findings suggest that viral assembly takes place within the endoplasmic reticulum (Gastaminza 2008) and that lipid droplets (LD) are involved in particle formation (Moradpour 1996; Barba 1997; Miyanari 2007; Shavinskaya 2007; Appel 2008). It appears that LD-associated core protein targets viral non-structural proteins and the HCV RNA replication complex including positive and negative stranded RNA from the endoplasmic reticulum to the LD (Miyanari 2007). Beside the core protein, LD-associated NS5A seems to play a key role in the formation of infectious viral particles (Appel 2008). Moreover, E2 molecules are detected in close proximity to LD-associated membranes. Finally, spherical virus-like particles associated with membranes can be seen very close to the LD. Using specific antibodies the virus-like particles were shown to contain core protein as well as E2 glycoprotein molecules indicating that these structures may represent infectious HCV (Miyanari 2007). However, the precise mechanisms for the formation and release of infectious HCV particles are still unknown. Model systems for HCV research For a long time HCV research was restricted due to a lack of small animal models and efficient cell culture systems. The development of the first HCV replicon system 10 years after the identification of the hepatitis C virus offered the opportunity to investigate the molecular biology of HCV infection in a standardized manner (Lohmann 1999). Using total RNA derived from the explanted liver of an individual chronically infected with HCV genotype 1b, the authors amplified and cloned the entire HCV ORF sequence in two overlapping fragments. The flanking NTRs were amplified and cloned separately and all fragments were assembled to a modified full-length sequence. Transfection experiments with in vitro transcripts derived from the full-length clones failed to yield viral replication. For this reason, the authors generated two different subgenomic replicons consisting of the 5' IRES, the neomycin phosphotransferase gene causing resistance to the antibiotic neomycin, the IRES derived from the encephalomyocarditis virus (EMCV) and the NS2-3' NTR or NS3-3' NTR sequence, respectively (Figure 3). In vitro transcripts derived from these constructs lacking the genome region coding for the structural HCV proteins were used to transfect the hepatoma cell line Huh-7 (Lohmann 1999). The transcripts are bicistronic, i.e., the first cistron containing the HCV IRES enables the translation of the neomycin phosphotransferase as a tool for efficient selection of successfully transfected cells and the second cistron containing the EMCV IRES directs translation of the HCV-specific proteins. Only some Huh-7 clones can replicate replicon-specific RNA, however, in titres of approximately 108 positive-stranded RNA copies per microgram total RNA. Moreover, all encoded HCV proteins are detected predominantly in the cytoplasm of the transfected Huh-7 cells. The development of this replicon is a milestone in HCV research with regard to the investigation of HCV RNA replication and HCV protein analyses. Figure 3. Structure of subgenomic HCV replicons (Lohmann 1999). This figure illustrates the genetic information of in vitro transcripts used for Huh-7 transfection. A) Full-length transcript derived from the explanted liver of a chronically infected subject. B) Subgenomic replicon lacking the structural genes and the sequence encoding p7. C) Subgenomic replicon lacking C, E1, E2, p7, and NS2 genes. neo, neomycin phosphotransferase gene; E-I, IRES of the encephalomyocarditis virus (EMCV). In more recent years, the methodology has been improved in order to achieve significantly higher replication efficiency. Enhancement of HCV RNA replication was achieved by the use of replicons harbouring cell culture-adapted point mutations or deletions within the NS genes (Blight 2000; Lohmann 2001; Krieger 2001). Further development has led to the generation of selectable full-length HCV replicons, i.e., genomic replicons that also contain genetic information for the structural proteins Core, E1, and E2 (Pietschmann 2002; Blight 2002). This improvement offered the opportunity of investigating the influence of the structural proteins on HCV replication. Thus it has been possible to analyse the intracellular localisation of these proteins. However, using this methodology viral assembly and release has not been achieved. Another important milestone was achieved when a subgenomic replicon based on the HCV genotype 2a strain JFH-1 was generated (Kato 2003). This viral strain derived from a Japanese subject with fulminant hepatitis C (Kato 2001). The corresponding replicons showed higher RNA replication efficiency than previous replicons. Moreover, cell lines distinct from Huh-7, such as HepG2 or HeLa were transfected efficiently with transcripts derived from the JFH-1 replicon (Date 2004; Kato 2005). Transfection of Huh-7 and "cured" Huh-7.5 cells with full-length JFH-1 replicons led for the first time to the production of infectious HCV virions (Zhong 2005; Wakita 2005). The construction of a chimera with the core-NS2 region derived from HCV strain J6 (genotype 2a) and the remaining sequence derived from JFH-1 improved infectivity. In addition, an alternative strategy for the production of infectious HCV particles was developed (Heller 2005). A full-length HCV construct (genotype 1b) was placed between two ribozymes in a plasmid containing a tetracycline-responsive promoter. Huh-7 cells were transfected with those plasmids, resulting in efficient viral replication with HCV RNA titres of up to 107 copies/ml cell culture supernatant. The development of cell culture systems that allow the production of infectious HCV represents a breakthrough for HCV research since it is now possible to investigate the whole viral life-cycle from virus adsorption to virion release. These studies will help to better understand the mechanisms of HCV pathogenesis and they should significantly accelerate the development of HCV-specific antiviral compounds. References Acton SL, Scherer PE, Lodish HF, et al. Expression cloning of SR-BI, a CD36-related class B scavenger receptor. J Biol Chem 1994, 269:21003-21009. Acton S, Rigotti A, Landschulz KT, et al. Identification of scavenger receptor BI as a high-density lipoprotein receptor. Science 1996, 271:518-520. Agnello V, Abel G, Elfahal M, et al. Hepatitis C virus and other Flaviviridae viruses enter cells via low-density lipoprotein receptor. Proc Natl Acad Sci U S A 1999, 96:12766-12771. Ago H, Adashi T, Yoshida A, et al. Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. Structure 1999, 7:417-1426. Ait-Goughoulte M, Hourioux C, Patient R, et al. Core protein cleavage by signal peptide peptidase is required for hepatitis C virus-like particle assembly. J Gen Virol 2006, 87:855-860. Akazawa D, Date T, Morikawa K, et al. CD81 expression is important for the permissiveness of Huh7 cell clones for heterogeneous hepatitis C virus infection. J Virol 81:5036-5045. Alter HJ, Holland PV, Purcell RH, et al. Transmissible agent in non-A, non-B hepatitis. Lancet 1978, 1:459-463. Andre P, Komurian-Pradel F, Deforges S, et al. Characterization of low- and very-low-density hepatitis C virus RNA-containing particles. J Virol 2002, 76:69619-6928. Appel N, Zayas M, Miller S, et al. Essential role of domain III of nonstructural protein 5A for hepatitis C virus infectious particle assembly. PLoS Pathog 4(3): e1000035. Barba G, Harper F, Harada T, et al. Hepatitis C virus core protein shows a cytoplasmic localization and associates to cellular lipid storage droplets. Proc Natl Acad Sci U S A 1997, 94:1200-1205. Bartenschlager R, Ahlborn-Laake L, Mous J, et al. Nonstructural protein 3 of hepatitis C virus encodes a serine-type proteinase required for cleavage at the NS3/4 and NS4/5 junctions. J Virol 1993, 67:3835-3844. Bartenschlager R, Lohmann V, Wilkinson T, et al. Complex formation between the NS3 serine-type proteinase of the hepatitis C virus and NS4A and its importance for polyprotein maturation. J Virol 1995, 69:7519-7528. Barth H, Schäfer C, Adah MI, et al. Cellular binding of hepatitis C virus envelope glycoprotein E2 requires cell surface heparan sulphate. J Biol Chem 2003, 278:41003-41012. Bartosch B, Dubuisson J, Cosset FL. Infectious hepatitis C virus pseudoparticles containing functional E1-E2 envelope protein complexes. J Exp Med 2003a, 197:633-642. Bartosch B, Vitelli A, Granier C, et al. Cell entry of hepatitis C virus requires a set of co-receptors that include CD81 and SR-B1 scavenger receptor. J Biol Chem 2003b, 278:41624-41630. Behrens SE, Tomei L, DeFrancesco R. Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus. EMBO J 1996, 15:12-22. Bertaux C, Dragic T. Different domains of CD81 mediate distinct stages of hepatitis C virus pseudoparticle entry. J Virol 2006, 80:4940-4948. Blanchard E, Belouzard S, Goueslain L, et al. Hepatitis C virus entry depends on clathrin-mediated endocytosis. J Virol 2006, 80:6964-6972. Blight KJ and Rice CM. Secondary structure determination of the conserved 98-base sequence at the 3Žterminus of hepatitis C virus genome RNA. J Virol 1997, 71:7345-7452. Blight KJ, Kolykhalov AA, Rice CM. Efficient initiation of HCV RNA replication in cell culture. Science 2000, 290:1972-1974. Blight KJ, McKeating JA, Rice CM. Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication. J Virol 2002, 76:13001-13014. Bradley DW, McCaustland KA, Cook EH, et al. Post-transfusion non-A, non-B hepatitis in chimpanzees: physiochemical evidence that the tubule-forming agent is a small, enveloped virus. Gastro 1985, 88:773-779. Bradley D, McCaustland K, Krawczynski K, et al. Hepatitis C virus: buoyant density of the factor VIII-derived isolate in sucrose. J Med Virol 1991, 34:206-208. Bradrick SS, Walters RW, Gromeier M. The hepatitis C virus 3'-untranslated region or a poly(A) tract promote efficient translation subsequent to the initiation phase. Nucleic Acids Res 2006, 34:1293-1303. Branch AD, Stumpp DD, Gutierrez JA et al. The hepatitis virus alternate reading frame (ARF) and its family of novel products: the alternate reading frame protein/F-protein, the double-frameshift protein, and others. Semin Liv Dis 2005, 25-105-117. Bressanelli S, Tomei L, Roussel A, et al. Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. Proc Natl Acad Sci USA 1999, 96:13034-13039. Bukh J, Miller RH, Purcell RH. Biology and genetic heterogeneity of hepatitis C virus. Clin Exp Rheumatol 1995, Suppl 13:S3-7. Choo QL, Kuo G, Weiner AJ, et al. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 1989, 244:359-362. Choo QL, Richman KH, Han JH, et al. Genetic organization and diversity of the hepatitis C virus. Proc Natl Acad Sci U S A 1991, 88:2451-2455. Cocquerel L, Meunier JC, Pillez A, et al. A retention signal necessary and sufficient for endoplasmic reticulum localization maps to the transmembrane domain of hepatitis C virus glycoprotein E2. J Virol 1998, 72:2183-2191. Cocquerel L, Duvet S, Meunier JC, et al. The transmembrane domain of hepatitis C virus glycoprotein E1 is a signal for static retention in the endoplasmic reticulum. J Virol 1999, 73:2641-2649. Cocquerel L, Meunier JC, Op de Beeck A, et al. Coexpression of hepatitis C virus envelope proteins E1 and E2 in cix improves the stability of membrane insertion of E2. J Gen Virol 2001, 82:1629-1635. Codran A, Royer C, Jaeck D, et al. Entry of hepatitis C virus pseudotypes into primary human hepatocytes by clathrin-dependent endocytosis. J Gen Virol 2006, 87:2583-2593. Cormier EG, Tsamis F, Kajumo F, et al. CD81 is an entry coreceptor for hepatitis C virus. Proc Natl Acad Sci USA 2004, 101:7270-7274. Date T, Kato T, Miyamoto M, et al. Genotype 2a hepatitis C virus subgenomic replicon can replicate in HepG2 and IMY-N9 cells. J Biol Chem 2004, 279:22371-22376. Duvet S, Cocquerel L, Pillez A, et al. Hepatitis C virus glycoprotein complex localization in the endoplasmic reticulum involves a determinant for retention and not retrieval. J Biol Chem 1998, 273:32088-32095. Duvet S, Op de Beeck A, Cocquerel L, et al. Glycosylation of the hepatitis C virus envelope protein E1 occurs posttranslationally in a mannosylphosphoryldolichol-deficient CHO mutant cell line. Glycobiology 2002, 12.95-101. Eckart MR, Selby M, Maisiarz F, et al. The hepatitis C virus encodes a serine protease involved in processing of the putative non-structural proteins from the viral polyprotein precursor. Biochem Biophys Res Commun 1993, 192:399-406. Egger D, Wölk , Gosert R, et al. Expression of hepatitis C virus proteins induce distinct membrane alterations including candidate viral replication complex. J Virol 2002, 76:5974-5984. Einav S, Elazar M, Danieli T, et al. A nucleotide binding motif in hepatitis C virus (HCV) NS4B mediates HCV RNA replication. J Virol 2004, 78:11288-11295. Elazar M, Liu P, Rice CM, et al. An N-terminal amphipathic helix in hepatitis C virus (HCV) NS4B mediates membrane association, correct localization of replication complex proteins, and HCV RNA replication. J Virol 2004, 78:11393-11400. Enomoto N, Sakuma I, Asahina Y, et al. Comparison of full-length sequences of interferon-sensitive and resistant hepatitis C virus 1b. Sensitivity to interferon is conferred by amino acid substitutions in the NS5A region. J Clin Investig 1995, 96:224-230. Enomoto N, Sakuma I, Asahina Y, et al. Mutations in the nonstructural protein 5A gene and response to interferon in patients with chronic hepatitis C virus 1b infection. N Engl J Med 1996, 334:77-81. Evans MJ, von Hahn T, Tscherne DM, et al. Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry. Nature 2007, 446:801-805. Failla C, Tomei L, DeFrancesco R. Both NS3 and NS4A are required for proteolytic processing of hepatitis C virus nonstructural proteins. J Virol 1994, 68:3753-3760. Farci P, Shimoda A, Wong D, et al. Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region 1 of the envelope 2 protein. Proc Nat. Acad Sci USA 1996, 93:15394-15399. Feinstone SM, Kapikian AZ, Purcell RH, et al. Transfusion-associated hepatitis not due to viral hepatitis type A or B. N Engl J Med 1975, 292:767-770. Feinstone SM, Mihalik KB, Purcell AH, et al. Inactivation of hepatitis B virus and non-A, non-B hepatitis by chloroform. Infect Immun 1983, 41:767-770. Flint M, Maidens C, Loomis-Price LD, et al. Characterization of hepatitis C virus E2 glycoprotein interaction with a putative cellular receptor, CD81. J Virol 1999, 73:6235-6244. Friebe P, Lohmann V, Krieger N, et al. Sequences in the 5' nontranslated region of hepatitis C virus required for RNA replication. J Virol 2001, 75:12047-12057. Friebe P and Bartenschlager R. Genetic analysis of sequences in the 3' nontranslated region of hepatitis C virus that are important for RNA replication. J Virol 2002, 76:5326-5338. Friebe P, Boudet J, Simorre JP, et al. Kissing-loop interaction in the 3' end of the hepatitis C virus genome essential of RNA replication. J Virol 2005, 79:380-392. Fried MW, Shiffman ML, Reddy KR, et al. Peginterferon Alfa-2a plus Ribavirin for Chronic Hepatitis C Virus Infection. N Engl J Med 2002, 347:975-982. Fukushi S, Katayama K, Kurihara C, et al. Complete 5' noncoding region is necessary for the efficient internal initiation of hepatitis C virus RNA. Biochem Biophys Res Commun 1994, 199:425-432. Furuse M, Fujita K, Hiiragi T, et al. Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol 1998, 141:1539-1550. Gardner JP, Durso RJ, Arrigale RR, et al. L-SIGN (CD209L) is a liver-specific capture receptor for hepatitis C virus. Proc Nat. Acad Sci USA 2003, 100:4498-4503. Gastaminza P, Cheng G, Wieland S, et al. Cellular determinants of hepatitis C virus assembly, maturation, degradation, and secretion. J Virol 2008, 82:2120-2129. Germi R, Crance JM, Garin D, et al. Cellular glycosaminglycans and low density lipoprotein receptor are involved in hepatitis C virus adsorption. J Med Virol 68:206-215. Grakoui A, McCourt DW, Wychowski C, et al. Characterization of hepatitis C virus-encoded serine proteinase: determination of proteinase-dependent polyprotein cleavage sites. J Virol 1993a, 67:2832-2843. Grakoui A, McCourt DW, Wychowski C, et al. A second hepatitis C virus-encoded proteinase. Proc Natl Acad Sci USA 1993b, 90:10583-10587. Gretton SN, Taylor AI, McLauchlan J. Mobility of the hepatitis C virus NS4B protein on the endoplasmic reticulum membrane and membrane-associated foci. J Gen Virol 2005, 86:1415-1421. Griffin SD, Beales LP, Clarke DS, et al. The p7 protein of hepatitis C virus forms an ion channel that is blocked by the antiviral drug, Amantadine. FEBS Lett 2003, 535:34-38. Grove J, Huby T, Stamataki Z, et al. Scavenger receptor BI and BII expression levels modulate hepatitis C virus infectivity. J Virol 2007, 81: 3162-3169. Haqshenas G, Mackenzie JM, Dong X, et al. Hepatitis C virus p7 protein is localized in the endoplasmic reticulum when it is encoded by a replication-competent genome. J Gen Virol 2007, 88:134-142. Helle F, Dubuisson J. Hepatitis C virus entry into host cells. Cell Mol Life Sci 2008, 65:100-112. Hellen CU, Pestova TV. Translation of hepatitis C virus RNA. J Viral Hepat 1999, 6:79-87. Heller TS, Saito J, Auerbach T, et al. An in vitro model of hepatitis C virion production. Proc Nat. Acad Sci USA 102 (2005), pp. 2579-2583. Heo TH, Chang JH, Lee JW, et al. Incomplete humoral immunity against hepatitis C virus is linked with distinct recognition of putative multiple receptors by E2 envelope glycoprotein. J Immunol 2004, 173:446-455. Hollinger FB, Gitnick GL, Aach RD, et al. Non-A, non-B hepatitis transmission in chimpanzees: a project of the Transfusion-transmitted Viruses Study Group. Intervirology 1978, 10:60-68. Honda M, Beard MR, Ping LH, et al. A phylogenetically conserved stem-loop structure at the 5Ž border of the internal ribosome entry site of hepatitis C virus is required for cap-independent viral translation. J Virol 1999, 73:1165-1174. Hsu M, Zhang J, Flint M, et al. Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles. Proc Natl Acad Sci USA 2003, 100:7271-7276. Hüssy P, Langen H, Mous J, et al. Hepatitis C virus core protein: carboxy-terminal boundaries of two processed species suggest cleavage by a signal peptide peptidase. Virology 1996, 224-93-104. Ito T and Lai MMC. Determination of the secondary structure of and cellular protein binding to the 3'-untranslated region of the hepatitis C virus RNA genome. J Virol 1997, 71:8698-8706. Jennings TA, Chen Y, Sikora D, et al. RNA unwinding activity of the hepatitis C virus NS3 helicase is modulated by the NS5B polymerase. Biochemistry 2008, 47:1126-1135. Jeong SH, Qiao M, Nascimbeni M, et al. Immunization with hepatitis C virus-like particles induces humoral and cellular immune responses in honhuman primates. J Virol 2004, 78:6995-7003. Jin L and Peterson DL. Expression, isolation, and characterization of the hepatitis C virus ATPase/RNA helicase. Arch Biochem Biophys 1995, 323:47-53. Jones CT, Murray CL, Eastman DK, et al. Hepatitis C virus p7 and NS2 proteins are essential for production of infectious virus. J Virol 2007, 81:8374-8383. Kaito M, Watanabe S, Tsukiyama-Kohara K, et al. Hepatitis C virus particle detected by immunoelectron microscopy study. J Gen Virol 1994, 75:1755-1760. Kapadia SB, Barth H, Baumert T, et al. Initiation of hepatitis C virus infection is dependent on cholesterol and cooperativity between CD81 and scavenger receptor B type I. J Virol 2007, 81:374-383. Kato J, Kato N, Yoshida H, et al. Hepatitis C virus NS4A and NS4B proteins suppress translation in vivo. J Med Virol 2002, 66:187-199. Kato N. Molecular virology of hepatitis C virus. Acta Med Okayama 2001, 55:133-159. Kato T, Furusaka A, Miyamoto M, et al. Sequence analysis of hepatitis C virus isolated from a fulminant hepatitis patient. J Med Virol 2001, 64:334-339. Kato T, Date T, Miyamoto M, et al. Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon. Gastroenterology 2003, 125:1808-1817. Kato T, Date T, Miyamoto M, et al. Nonhepatic cell lines HeLa and 293 support efficient replication of hepatitis C virus genotype 2a subgenomic replicon. J Virol 2005, 79:592-596. Kim DW, Gwack Y, Han JH, et al. C-terminal domain of the hepatitis C virus NS3 protein contains an RNA helicase activity. Biochem Biophys Res Commun 1995, 215:160-166. Kim YK, Kim CS, Lee SH, et al. Domains I and II in the 5' nontranslated region of the HCV genome are required for RNA replication. Biochem Biophys Res Commun 2002, 290:105-112. Kolykhalov AA, Feinstone SM, Rice SM. Identification of a highly conserved sequence element at the 3Žterminus of hepatitis C virus genome RNA. J Virol 1996, 70:3363-3371. Kolykhalov AA, Mihalik K, Feinstone SM, et al. Hepatitis C virus-encoded enzymatic activities and conserved RNA elements in the 3' nontranslated region are essential for virus replication in vivo. J Virol 2000, 74:2046-2051. Komurian-Pradel F, Rajoharison A, Berland JL, et al. Antigenic relevance of F protein in chronic hepatitis C virus infection. Hepatology 2004, 40:900-909. Kou YH, Chou SM, Wang YM, et al. Hepatitis C virus NS4A inhibits cap-dependent and the viral IRES-mediated translation through interacting with eukaryotic elongation factor 1A. J Biomed Sci 2006, 13:861-874. Koutsoudakis G, Kaul A, Steinmann E, et al. Characterization of the early steps of hepatitis C virus infection by using luciferase reporter viruses. J Virol 2006, 80:5308-5320. Krieger N, Lohmann V, Bartenschlager R. Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations. J Virol 2001, 75:4614-4624. Kuo G, Choo QL, Alter HJ, et al. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science 1989, 244:362-364. Lavillette D, Tarr AW, Voisset C, et al. Characterization of host-range and cell entry properties of the major genotypes and subtypes of hepatitis C virus. Hepatology 2005, 41:265-274. Lavillette D, Pécheur EI, Donot P, et al. Characterization of fusion determinants points to the involvement of three discrete regions of both E1 and E2 glycoproteins in the membrane fusion process of hepatitis C virus. J Virol 2007, 81:8752-8765. Lesburg CA, Cable MB, Ferrari E, et al. Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site. Nat Struct Biol 1999, 6:937-943. Lin C, Thomson JA, Rice CM. A central region in the hepatitis C virus NS4A protein allows formation of an active NS3-NS4A serine proteinase complex in vivo and in vitro. J Virol 1995, 69:4373-4380. Lindenbach BD, Evans MJ, Syder AJ, et al. Complete replication of hepatitis C virus in cell culture. Science 309 (2005), pp. 623-626. Lohmann V, Körner F, Koch JO, et al. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 1999, 285:110-113. Lohmann V, Körner F, Dobierzewska A, et al. Mutations in hepatitis C virus RNAs conferring cell culture adaptation. J Virol 2001, 75:1437-1449. Lozach PY, Lortat-Jacob H, de Lacroix de Lavalette A, et al. DC-SIGN and L-SIGN are high affinity binding receptors for hepatitis C virus glycoprotein E2. J Biol Chem 2003, 278:20358-20366. Manns MP, McHutchinson JG, Gordon SC, et al. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial. Lancet 2001, 358:958-965. Matsumoto M, Hwang SB, Jeng KS, et al. Homotypic interaction and multimerization of hepatitis C virus core protein. Virology 1996, 218:43-51. McKeating JA, Zhang LQ, Logvinoff C, et al. Diverse hepatitis C virus glycoproteins mediate viral infection in a CD81-dependent manner. J Virol 2004, 78:8496-8505. McLauchlan J, Lemberg MK, Hope G, et al. Intramembrane proteolysis promotes trafficking of hepatitis C virus core protein to lipid droplets. EMBO J 2002, 21:3980-3988. Meertens L, Bertaux C, Cukierman L, et al. The tight junction proteins claudin-1, -6, and -9 are entry cofactors for hepatitis C virus. J Virol 2008, 82:3555-3560. Miyanari Y, Atsuzawa K, Usuda N, et al. The lipid droplet is an important organelle for hepatitis C virus production. Nat Cell Biol 2007, 9:961-969. Molina S, Castet V, Fournier-Wirth C, et al. The low-density lipoprotein receptor plays a role in the infection of primary human hepatocytes by hepatitis C virus. J Hepatol 2007, 46:411-419. Monazahian M, Böhme I, Bonk S, et al. Low-density lipoprotein receptor as a candidate receptor for hepatitis C virus. J Med Virol 1999, 57:223-229. Moradpour D, Englert C, Wakita T, et al. Characterization of cell lines allowing tightly regulated expression of hepatitis C virus core protein. Virology 1996, 222:52-63. NahmiasY, Casali M, Barbe L, et al. Liver endothelial cells promote LDL-R expression and the uptake of HCV-like particles in primary rat and human hepatocytes. Hepatology 2006, 43:257-265. Ndjomou J, Pybus OG, Matz B. Phylogenetic analysis of hepatitis C virus isolates indicates a unique pattern of endemic infection in Cameroon. J Gen Virol 2003, 84:2333-2341. Ohba K, Mizokami M, Lau JYN, et al. Evolutionary relationship of hepatitis C, pesti-, flavi-, plantviruses, and newly discovered GB hepatitis agents. FEBS Lett 1996, 378:232-234. Op de Beeck A, Montserret R, Duvet S, et al. The transmembrane domains of hepatitis C virus envelope glycoprotein E1 and E2 play a major role in heterodimerization. J Biol Chem 2000, 275:31428-31437. Otto GA, Lukavsky PJ, Lancaster AM, et al. Ribosomal proteins mediate the hepatitis C virus IRES-HeLa 40S interaction. RNA 2002, 8:913-923. Pavlovic D, Neville DC, Argaud O, et al. The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives. Proc Nat. Acad Sci USA 2003, 100:6104-6108. Pawlotsky JM, Chevalier S, McHutchinson JG. The hepatitis C virus life cycle as a target for new antiviral therapies. Gastroenterology 2007, 132:1979-1998. Pietschmann T, Lohmann V, Kaul A, et al. Persistent and transient replication of full-length hepatitis C virus genomes in cell culture. J Virol 2002, 76:4008-4021. Pileri P, Uematsu Y, Campagnoli S, et al. Binding of hepatitis C virus to CD81. Science 1998, 282:938-941. Pöhlmann S, Zhang J , Baribaud F, et al. Hepatitis C virus glycoproteins interact with DC-SIGN and DC-SIGNR. J Virol 2003, 77:4070-4080. Poynard T, Bedossa P, Opolon P et al. Natural history of liver fibrosis progression in patients with chronic hepatitis C. Lancet 1997, 349:825-833. Prince AM, Huima-Byron T, Parker TS, et al. Visualization of hepatitis C virions and putative defective interfering particles isolated from low-density lipoproteins. J Viral Hepat 1996, 3:11-17. Qiao M, Ashok M, Bernard KA, et al. Induction of sterilizing immunity against West Nile virus (WNV) by immunization with WNV-like particles produced in insect cells. J Infect Dis 2004, 190:2104-2108. Roussel J, Pillez A, Montpellier C, et al. Characterization of the expression of the hepatitis C virus F protein. J Gen Virol 2003, 84:1751-1759. Sakai A, Claire MS, Faulk K, 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, 100:11646-11651. Santolini E, Migliaccio G, La Monica N. Biosynthesis and biochemical properties of the hepatitis C virus core protein. J Virol 1994, 68:3631-3641. Santolini E, Pacini L, Fipaldini C, et al. The NS2 protein of hepatitis C virus is a transmembrane polypeptide. J Virol 1995, 69:7461-7471. Scarselli E, Ansuini H, Cerino R, et al. The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus. EMBO J 2002, 21:5017-5025. Schmidt-Mende J, Bieck E, Hugle T, et al. Determinants for membrane association of the hepatitis C virus RNA-dependent RNA polymerase. J Biol Chem 2001, 276:44052-44063. Shavinskaya A, Boulant S, Penin F, et al. The lipid droplet binding domain of hepatitis C virus core protein is a major determinant for efficient virus assembly. J Biol Chem 2007, 282:37158-37169. Shimizu YK, Feinstone SM, Kohara M, et al. Hepatitis C virus: detection of intracellular virus particles by electron microscopy. Hepatology 1996a, 23:205-209. Shimizu YK, Igarashi H, Kiyohara T, et al. A hyperimmune serum against a synthetic peptide corresponding to the hypervariable region 1 of hepatitis C virus can prevent viral infection in cell cultures. Virology 1996b, 223:409-412. Simmonds P. The origin and evolution of hepatitis viruses in humans. J Gen Virol 2001, 82:693-712. Simmonds P. Genetic diversity and evolution of hepatitis C virus - 15 years on. J Gen Virol 2004, 85:3173-3188. Simmonds P, Bukh J, Combet C, et al. Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes. Hepatology 2005, 42:962-973. Simons JN, Leary TP, Dawson GJ, et al. Isolation of novel virus-like sequences associated with human hepatitis. Nat Med 1995, 1:564-569. Sizova DV, Kolupaeva VG, Pestova TV, et al. Specific interaction of eukaryotic translation initiation factor 3 with the 5' nontranslated regions of hepatitis C virus and classical swine fever virus RNAs. J Virol 1998, 72:4775-4782. Song Y, Friebe P, Tzima E, et al. The hepatitis C virus RNA 3'-untranslated region strongly enhances translation directed by the internal ribosome entry site. J Virol 2006, 80:1579-11588. Spahn CM, Kieft JS, Grassucci RA, et al. Hepatitis C virus IRES RNA-induced changes in the conformation of the 40s ribosomal subunit. Science 2001, 291:1959-1962. Takahashi K, Kishimoto S, Yoshizawa H, et al. p26 protein and 33-nm particle associated with nucleocapsid of hepatitis C virus recovered from the circulation of infected hosts. Virology 1992, 191:431-434. Tanaka T, Kato N, Cho MJ, et al. A novel sequence found at the 3' terminus of hepatitis C virus genome. Biochem Biophys Res 1995, 215:744-749. Tanaka T, Kato N, Cho MJ, et al. Structure of the 3' terminus of the hepatitis C virus genome. J Virol 1996, 70:3307-3312. Tanji Y, Hijikata M, Satoh S, et al. Hepatitis C virus-encoded nonstructural protein NS4A has versatile functions in viral protein processing. J Virol 1995, 69:1575-1581. Thiel HJ, Collett MS, Gould EA et al. Family Flaviviridae, In: Fauquet CM, Mayo MA, Maniloff J et al., eds. Virus Taxonomy:VIIIth Report of the International Committee on Taxonomy of Viruses. San Diego: Academic Press; 2005:979-996. Thomssen R, Bonk S, Propfe C, et al. Association of hepatitis C virus in human sera with lipoprotein. Med Microbiol Immunol 1992, 181:293-300. Thomssen R, Bonk S, Thiele A. Density heterogeneities of hepatitis C virus in human sera due to the binding of beta-lipoproteins and immunoglobulins. Med Microbiol Immunol 1993, 182:329-334. Timpe JM, Stamataki Z, Jennings A, et al. Hepatitis C virus cell-cell transmission in hepatoma cells in the presence of neutralizing antibodies. Hepatology 2007, 47:17-24. Tomei L, Failla C, Santolini E, et al. NS3 is a Serine protease required for processing of hepatitis C virus polyprotein. J Virol 1993, 67:4017-4026. Tomei L, Failla C, Vitale RL, et al. A central hydrophobic domain of the hepatitis C virus NS4A protein is necessary and sufficient for the activation of the NS3 protease. J Gen Virol 1996, 77:1065-1070. Tong MJ, el-Farra NS, Reikes AR et al. Clinical outcomes after transfusion-associated hepatitis C. N Engl J Med 1995, 332:1463-1466. Tsukiyama-Kohara K, Iizuka N, Kohara M, et al. Internal ribosome entry site within hepatitis C virus RNA. J Virol 1992, 66:1476-1483. Varaklioti A, Vassilaki N, Georgopoulou U, et al. Alternate translation occurs within the core coding region of the hepatitis C viral genome. J Biol Chem 2002, 17713-17721. Wakita T, Pietschmann T, Kato T, et al. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 2005, 11:791-796. Walewski JL, Keller TR, Stump DD, et al. Evidence for a new hepatitis C virus antigen encoded in an overlapping reading frame. RNA 2001, 7:710-721. Wang C, Sarnow P, Siddiqui A. Translation of human hepatitis C virus RNA in cultured cells is mediated by an internal ribosome-binding mechanism. J Virol 1993, 67:3338-3344. Wang H, Shen XT, Ye R, et al. Roles of the polypyrimidine tract and 3' noncoding region of hepatitis C virus RNA in the internal ribosome entry site-mediated translation. Arch Virol 2005, 150:1085-1099. Webb NR, Connell PM, Graf GA, et al. SR-BII, an isoform of the scavenger receptor BI containing an alternate cytoplasmic tail, mediates lipid transfer between high-density lipoprotein and cells. J Biol Chem 1998, 273:15241-15248. Weihofen A, Binns K, Lemberg MK, et al. Identification of signal peptide peptidase, a presenelin-type aspartic protease. Science 2002, 296:2215-2218. Weiner AJ, Brauer MJ, Rosenblatt J, et al. Variable and hypervariable domains are found in the regions of HCV corresponding to the flavivirus envelope and NS1 proteins and the pestivirus envelope glycoproteins. Virology 1991, 180:842-848. Wölk B, Sansonno D, Kräusslich HG, et al. Subcellular localization, stability, and trans-cleavage competence of the hepatitis C virus NS3-NS4A complex expressed in tetracycline-regulated cell lines. J Virol 2000, 74:2293-2304. Wünschmann S, Medh JD, Klinzmann D, et al. Characterization of hepatitis C virus (HCV) and HCV E2 interactions with CD81 and the low-density lipoprotein receptor. J Virol 2000, 74:10055-10062. Xu Z, Choi J, Yen TS, et al. Synthesis of a novel hepatitis C virus protein by ribosomal frameshift. EMBO J 2001, 20:3840-3848. Xu Z, Choi J, Lu W, et al. Hepatitis C virus F protein is a short-lived protein associated with the endoplasmic reticulum. J Virol 2003, 77:1578-1583. Yanagi M, St Claire M, Emerson SU. In vivo analysis of the 3' untranslated region of the hepatitis C virus after in vitro mutagenesis of an infectious cDNA clone. Proc Natl Acad Sci USA 1999, 96:2291-2295. Yi M and Lemon SM. 3' Nontranslated RNA signals required for replication of hepatitis C virus RNA. J Virol 2003, 77:3557-3568. Yi M, Villanueva RA, Thomas DL, et al. Production of infectious genotype 1a hepatitis C virus (Hutchinson strain) in cultured hepatoma cells. Proc Natl Acad Sci USA 2006, 103:2310-2315. You S and Rice CM. 3Ž RNA elements in hepatitis C virus replication: kissing partners and long poly(U). J Virol 2008, 82:184-195. Yu X, Qiao M, Atanasov I, et al. Cryo-electron microscopy and three-dimensional reconstructions of hepatitis C virus particles. Virology 2007, 367:126-134. Zeisel MB, Koutsoudakis G, Schnober EK, et al. Scavenger receptor class B type I is a key host factor for hepatitis C virus infection required for an entry step closely linked to CD81. Hepatology 2007, 46:1722-1731. Zhang J, Yamada O, Yoshida H, et al. Autogenous translational inhibition of core protein: implication for switch from translation to RNA replication in hepatitis C virus. Virology 2002, 293:141-150. Zhang LQ, Randall G, Higginbottom A, et al. CD81 is required for hepatitis C virus glycoprotein-mediated viral infection. J Virol 2004, 78:1448-1455. Zheng A, Yuan F, Li Y, et al. Claudin-6 and claudin-9 function as additional coreceptors for hepatitis C virus. J Virol 2007, 81:12465-12471. Zhong J, Gastaminza P, Cheng G, et al. Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci USA 2005, 102:9294-9299.