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Tan SL, editor. Hepatitis C Viruses: Genomes and Molecular Biology. Norfolk (UK): Horizon Bioscience; 2006.

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Hepatitis C Viruses: Genomes and Molecular Biology.

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Chapter 1HCV Genome and Life Cycle

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Hepatitis C virus (HCV) infection afflicts more than 170 million people worldwide, with the great majority of patients with acute hepatitis C developing chronic HCV infection. It can ultimately result in liver cirrhosis, hepatic failure or hepatocellular carcinoma, which are responsible for hundreds of thousands of deaths each year. Despite the discovery of HCV over 15 years ago, our knowledge of the HCV lifecycle has been limited by our inability to grow the virus in cell culture, as well as by the lack of small-animal models of HCV infection. Nevertheless, data accumulated through the use of multiple in vitro and in vivo study systems have provided a general picture of the biology of HCV, although sometimes with contradictory results. Herein, we summarize our current understanding of the HCV genome and how its structure and encoded gene products, in a complex interplay with host cell factors, might orchestrate a productive viral lifecycle while evading the scrutiny of the host immune system. The recently developed robust in vitro HCV infection systems should help fill in some of the gaps in understanding the HCV lifecycle in the next few years.

HCV Genome Organization and Function

The Flaviviridae family is divided into three genera: flavivirus, pestivirus, and hepacivirus. Flaviviruses include yellow fever virus, dengue fever virus, Japanese encephalitis virus, and Tick-borne encephalitis virus. Pestiviruses include bovine viral diarrhea virus, classical swine fever virus and Border disease virus. HCV, with at least 6 genotypes and numerous subtypes, is a member of the hepacivirus genus, which includes tamarin virus and GB virus B (GBV-B) and is closely related to human virus GB virus C (GBV-C) (Lindenbach and Rice, 2001).

The members of the Flaviviridae family share a number of basic structural and virological characteristics. They are all enveloped in a lipid bilayer in which two or more envelope proteins (E) are anchored. The envelope surrounds the nucleocapsid, which is composed of multiple copies of a small basic protein (core or C), and contains the RNA genome. The Flaviviridae genome is a positive-strand RNA molecule ranging in size from 9.6 to 12.3 thousand nucleotides (nt), with an open reading frame (ORF) encoding a polyprotein of 3000 amino acids (aa) or more.

The structural proteins are encoded by the N-terminal part of the ORF, whereas the remaining portion of the ORF codes for the nonstructural proteins (Fig. 1). Sequence motif-conserved RNA protease-helicase and RNA-dependant RNA polymerase (RdRp) are found at similar locations in the polyproteins of all of the Flaviviridae (Miller and Purcell, 1990). In addition, all Flaviviridae share similar polyprotein hydropathic profile, with flaviviruses and hepaciviruses being closer to each other than to pestiviruses (Choo et al., 1991). The ORF is flanked in 5′ and 3′ by untranslated regions (UTR) of 95–555 and 114–624 nt in length, respectively, which play an important role in polyprotein translation and RNA replication (Fig. 1) (Thurner et al., 2004).

Fig. 1. Organization of Flaviviridae genomes.

Fig. 1

Organization of Flaviviridae genomes. The figure shows, from top to bottom, the genomes of HCV (hepacivirus), pestivirus, and yellow fever virus (flavivirus). NS: non structural.

Flaviviridae bind to one or more cellular receptors organized as a receptor complex and appear to trigger receptor-mediated endocytosis. Fusion of the virion envelope with cellular membranes delivers the nucleocapsid to the cytoplasm. After decapsidation, translation of the viral genome occurs in the cytoplasm, leading to the production of a precursor polyprotein, which is then cleaved by both cellular and viral proteases into structural and nonstructural proteins. Replication of the viral genome is carried out by the viral replication complex which is associated with cellular membranes and resistant to actinomycin D. Viral replication occurs in the cytoplasm via the synthesis of full-length negative-strand RNA intermediates. Progeny virions are assembled from cytoplasmic vesicles formed by budding through intracellular membranes. Finally, mature virions are released into the extracellular milieu by exocytosis.

Despite the above-mentioned similarities with the members of other Flaviviridae genera, HCV does exhibit a number of virological, epidemiological as well as pathophysiological differences. Flavivirus translation is cap-dependent, i.e. mediated by a type I cap structure located in the 5′ UTR (m7GpppAmp), followed by the conserved AG sequence and a relatively short stretch upstream of the polyprotein coding region (Brinton and Dispoto, 1988). In contrast, the HCV 5′ UTR is not capped and, like that of pestiviruses and GB viruses, folds into a complex secondary RNA structure forming, together with a portion of the core-coding domain, an internal ribosome entry site (IRES) that mediates direct binding of ribosomal subunits and cellular factors and subsequent translation (see Chapter 2). Whereas the flavivirus 3′ UTR is highly structured, the HCV 3′ UTR is relatively short, less structured and contains a poly-uridyl tract that varies in length.

HCV has a narrow host specificity and tissue tropism. HCV is transmitted exclusively through direct blood-to-blood contacts between humans. Flaviviruses are principally vectored by mosquitoes or ticks and can infect a broad range of vertebrate animals, with humans being a dead-end host that does not participate in the perpetuation of virus transmission. No known pestivirus can infect humans and no known insect vector has been identified. Infections caused by flaviviruses are acute-limited in vertebrate animals, whereas HCV has a high chronicity rate in humans (50%–80%, depending on the age at infection). Strong and adapted humoral and cellular immune responses have been shown to be involved in flavivirus and pestivirus infection recovery and protection. However, HCV infection induces an immune response that fails to prevent chronicity in most cases and does not confer protection against reinfection with homologous and heterologous strains in the chimpanzee model (Farci et al., 1997).

HCV Genome Structure and Organization

The structural organization of HCV genome is schematically depicted in Fig. 2.

Fig. 2. HCV genome organization (top) and polyprotein processing (bottom).

Fig. 2

HCV genome organization (top) and polyprotein processing (bottom). The 5′ UTR consists of four highly structured domains and contains the IRES. The 3′ UTR consists of stable stem-loop structures (more...)

5′ Untranslated Region

The HCV 5′ UTR contains 341 nt located upstream of the ORF translation initiation codon. It is the most conserved region of the genome (nt sequence identity is 60% with GBV-B and approximately 50% with pestiviruses (Choo et al., 1991; Han et al., 1991). The 5′ UTR contains four highly structured domains, numbered I to IV, containing numerous stem-loops and a pseudoknot (Brown et al., 1992; Wang et al., 1995). Domains II, III and IV together with the first 12 to 30 nt of the core-coding region constitute the IRES (Honda et al., 1996). Structural characterization by electron microscopy (EM) indicated that domains II, III and IV form distinct regions within the molecule, with a fl exible hinge between domains II and III (Beales et al., 2001). The HCV IRES has the capacity to form a stable pre-initiation complex by directly binding the 40S ribosomal subunit without the need of canonical translation initiation factors, an event that likely constitutes the fi rst step of HCV polyprotein translation.

Several reports suggested a tissue compartmentalization of IRES sequences (Laskus et al., 2000; Lerat et al., 2000; Nakajima et al., 1996; Shimizu et al., 1997). Infection of lymphoid cell lines with HCV genotype 1a H77 strain led to the selection of a quasispecies with nucleotide substitutions within the 5′ UTR relative to the inoculum that conferred a 2- to 2.5-fold increase in translation effi ciency in human lymphoid cell lines relative to monocyte, granulocyte or monocyte cell lines (Lerat et al., 2000). Furthermore, different translation effi ciencies of HCV quasispecies variants isolated from different cell types in the same patient were observed, suggesting cell type-specifi c IRES interactions with cellular factors may also modulate polyprotein translation (Forton et al., 2004; Laporte et al., 2000; Lerat et al., 2000).

3′ Untranlated Region

The 3′ UTR contains approximately 225 nt. It is organized in three regions including, from 5′ to 3′, a variable region of approximately 30–40 nt, a long poly(U)-poly(U/UC) tract, and a highly conserved 3′-terminal stretch of 98 nt (3′ X region) that includes three stem-loop structures SL1, SL2 and SL3 (Kolykhalov et al., 1996; Tanaka et al., 1995; Tanaka et al., 1996). The 3′ UTR interacts with the NS5B RdRp and with two of the four stable stem-loop structures located at the 3′ end of the NS5B-coding sequence (Cheng et al., 1999; Lee et al., 2004). The 3′ X region and the 52 upstream nt of the poly(U/C) tract were found to be essential for RNA replication, whereas the remaining sequence of the 3′ UTR appears to enhance viral replication (Friebe and Bartenschlager, 2002; Ito and Lai, 1997; Yi and Lemon, 2003a; Yi and Lemon, 2003b).

Characteristics and Functions of HCV Proteins

The HCV ORF contains 9024 to 9111 nt depending on the genotype. The ORF encodes at least 11 proteins, including 3 structural proteins (C or core, E1 and E2), a small protein, p7, whose function has not yet been definitively defined, 6 nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B), and the so-called “F” protein which results from a frameshift in the core coding region (Fig. 2; Table 1). The characteristics and functions of HCV proteins are extensively described elsewhere in this book. Here, we provide a brief overview of the viral gene products and their roles in the HCV lifecycle.

Table 1. HCV proteins and their functions in the viral life cycle.

Table 1

HCV proteins and their functions in the viral life cycle. Adapted from Bartenschlager et al., 2004.

Structural Proteins

Core Protein

The HCV core protein is a highly basic, RNA-binding protein, which presumably forms the viral capsid (see Chapter 3). The HCV core protein is released as a 191 aa precursor of 23-kDa (P23). Although proteins of various sizes (17 to 23 kDa) were detectable, the 21-kDa core protein (P21) appeared to be the predominant form (Yasui et al., 1998). The core protein contains three distinct predicted domains : an N-terminal hydrophilic domain of 120 aa (domain D1), a C-terminal hydrophobic domain of about 50 aa (domain D2), and the last 20 or so aa that serve as a signal peptide for the downstream envelope protein E1 (Grakoui et al., 1993c; Harada et al., 1991; Santolini et al., 1994). Domain D1 contains numerous positive charges. It is principally involved in RNA binding and nuclear localization, as suggested by the presence of three predicted nuclear localization signals (NLS) (Chang et al., 1994; Suzuki et al., 1995; Suzuki et al., 2005). Domain D2 is responsible for core protein association with endoplasmic reticulum (ER) membranes, outer mitochondria membranes and lipid droplets (Schwer et al., 2004; Suzuki et al., 2005).

Both membrane-bound and membrane-free core proteins appear to exist as dimeric or multimeric forms. When expressed in various in vitro systems, including cell-free or mammalian, bacterial, insect or yeast cell culture models, the HCV core protein can form nucleocapsid-like particles (NLPs) (Baumert et al., 1998; Blanchard et al., 2002; Blanchard et al., 2003; Dash et al., 1997; Ezelle et al., 2002; Iacovacci et al., 1997; Klein et al., 2004; Mizuno et al., 1995; Pietschmann et al., 2002; Serafino et al., 1997; Shimizu et al., 1996). The region between aa 82 and 102 of hydrophilic domain D1 contains a tryptophan-rich sequence and has been suggested to allow the P21 core protein to interact with itself, a property not borne by the precursor P23 (Nolandt et al., 1997). On the other hand, the 75 N-terminal residues of the core protein appear sufficient for NLP assembly in a bacterial system (Majeau et al., 2004). Recently, two clusters of basic residues located in the 68 N-terminal nt were shown to play a critical role in capsid assembly in a cell-free system, whereas the region between aa 82 and 102 did not play a major role (Klein et al., 2005). The critical residues for capsid assembly remain to be precisely identified.

In addition to its role in viral capsid formation, the core protein has been suggested to directly interact with a number of cellular proteins and pathways that may be important in the viral lifecycle (McLauchlan, 2000). The HCV core protein has pro- and anti-apoptotic functions (Chou et al., 2005; Kountouras et al., 2003; Meyer et al., 2005), stimulates hepatocyte growth in Huh-7 cell line by transcriptional upregulation of growth-related genes (Fukutomi et al., 2005), and has been implicated in tissue injury and fibrosis progression (Nunez et al., 2004). The HCV core protein could also regulate the activity of cellular genes, including c-myc and c-fos, and alter the transcription of other viral promoters (Ray et al., 1995; Shih et al., 1993). It induces hepatocellular carcinoma when expressed in transgenic mice (Moriya et al., 1998; Moriya et al., 1997). It could also induce the formation of lipid droplets and may play a direct role in steatosis formation (Barba et al., 1997; Moriya et al., 1998; Moriya et al., 1997).

E1 and E2 Envelope Glycoproteins

The two envelope glycoproteins, E1 and E2, are essential components of the HCV virion envelope and necessary for viral entry and fusion (Bartosch et al., 2003a; Nielsen et al., 2004) (see Chapter 4). E1 and E2 have molecular weights of 33–35 and 70–72 kDa, respectively, and assemble as noncovalent heterodimers (Deleersnyder et al., 1997). E1 and E2 are type I transmembrane glycoproteins, with N-terminal ectodomains of 160 and 334 aa, respectively, and a short C-terminal transmembrane domain of approximately 30 aa. The E1 and E2 transmembrane domains are composed of two stretches of hydrophobic aa separated by a short polar region containing fully conserved charged residues. They have numerous functions, including membrane anchoring, ER localization and heterodimer assembly (Cocquerel et al., 1998; Cocquerel et al., 2000). The ectodomains of E1 and E2 contain numerous proline and cysteine residues, but intramolecular disulfide bonds have not been observed (Matsuura et al., 1994). E1 and E2 are highly glycosylated, containing up to 5 and 11 glycosylation sites, respectively. In addition, E2 contains hypervariable regions with aa sequences differing up to 80% between HCV genotypes and between subtypes of the same genotype (Weiner et al., 1991). Hypervariable region 1 (HVR1) contains 27 aa and is a major (but not the only) HCV neutralizing epitope (Farci et al., 1996; Zibert et al., 1997). Despite the HVR1 sequence variability, the physicochemical properties of the residues at each position and the overall conformation of HVR1 are highly conserved among all known HCV genotypes, suggesting an important role in the virus lifecycle (Penin et al., 2001).

E2 plays a crucial role in the early steps of infection. Viral attachment is thought to be initiated via E2 interaction with one or several components of the receptor complex (Flint and McKeating, 2000; Rosa et al., 1996). Because HVR1 is a basic region with positively charged residues located at specific sequence positions, it can theoretically interact with negatively charged molecules at the cell surface. This interaction could play a role in host cell recognition and attachment, as well as in cell or tissue compartmentalization (Barth et al., 2003; Bartosch et al., 2003b). In addition, it was recently shown that human serum facilitated infection of Huh7 cells by HCV pseudoparticles, apparently mediated through an interplay between serum high-density lipoproteins (HDL), HVR1 and the scavenger receptor B type I (SR-BI) (Bartosch et al., 2005; Voisset et al., 2005). Less is known about E1, but it is thought to be involved in intra-cytoplasmic virus-membrane fusion (Flint and McKeating, 2000; Rosa et al., 1996).

Frameshift Protein

The F (frameshift) protein or ARFP (alternate reading frame protein) is generated as a result of a −2/+1 ribosomal frameshift in the N-terminal core-encoding region of the HCV polyprotein. Antibodies to peptides from the F protein were detected in chronically infected patients, suggesting that the protein is produced during infection (Walewski et al., 2001). However, the exact translational mechanisms governing the frequency and yield of the F protein during the various phases of HCV infection are completely unknown. Thus, the role of F protein in the HCV lifecycle remains enigmatic but it was proposed to be involved in viral persistence (Baril and Brakier-Gingras, 2005).

Nonstructural Proteins


p7 is a small, 63 aa polypeptide, that has been shown to be an integral membrane protein (Carrere-Kremer et al., 2002). It comprises two transmembrane domains organized in α-helices, connected by a cytoplasmic loop. p7 appears to be essential, because mutations or deletions in its cytoplasmic loop suppressed infectivity of intra-liver transfection of HCV cDNA in chimpanzees (Sakai et al., 2003). In vitro studies suggested that p7 belongs to the viroporin family and could act as a calcium ion channel (Gonzalez and Carrasco, 2003). However, these results remain to be confirmed in vivo.


NS2 is a non-glycosylated transmembrane protein of 21–23 kDa (see Chapter 5). It contains two internal signal sequences at aa positions 839–883 and 928–960, which are responsible for ER membrane association (Santolini et al., 1995; Yamaga and Ou, 2002). NS2, together with the amino-terminal domain of the NS3 protein, the NS2-3 protease, constitutes a zinc-dependent metalloprotease that cleaves the site between NS2 and NS3 (Grakoui et al., 1993b; Grakoui et al., 1993c; Hijikata et al., 1993). NS2 is a short-lived protein that looses its protease activity after self-cleavage from NS3 and is degraded by the proteasome in a phosphorylation-dependent manner by means of protein kinase casein kinase 2 (Franck et al., 2005). In addition to its protease activity, NS2 could interact with host cell proteins, such as the liverspecific pro-apoptotic cell death-inducing DFF45-like effector (CIDE-B), and affect reporter genes controlled by liver and non-liver-specific promoters and enhancers (Dumoulin et al., 2003; Erdtmann et al., 2003). However, the consequences of such interactions within the context of the HCV lifecyle are not clear.


NS3 is a multi-functional viral protein containing a serine protease domain in its N-terminal third and a helicase/NTPase domain in its C-terminal two-thirds. NS4A is a cofactor of NS3 protease activity. NS3-4A also bears additional properties through its interaction with host cell pathways and proteins that may be important in the lifecycle and pathogenesis of infection (see Chapters 6 and 13). Not surprisingly, the NS3-NS4A protease is one of the most popular viral targets for anti-HCV therapeutics (Pawlotsky and McHutchison, 2004; Pawlotsky, 2006).

NS3-NS4A Protease

The NS3-NS4A protease is essential for the HCV lifecycle. It catalyzes HCV polyprotein cleavage at the NS3/NS4A, NS4A/NS4B, NS4B/NS5A and NS5A/NS5B junctions. The 3D structure of the NS3 serine protease domain complexed with NS4A has been determined (Kim et al., 1996; Love et al., 1996; Yan et al., 1998). The catalytic triad is formed by residues His 57, Asp 81 and Ser 139 (Bartenschlager et al., 1993; Grakoui et al., 1993a; Tomei et al., 1993). The central region of NS4A (aa 21–30) acts as a cofactor of NS3 serine protease activity, allowing its stabilization, localization at the ER membrane as well as cleaveage-dependent activation, particularly at the NS4B/NS5A junction (Bartenschlager et al., 1995; Lin et al., 1995; Tanji et al., 1995).

Recently, HCV NS3-NS4A was shown in vitro to antagonize the dsRNA-dependent interferon regulatory factor 3 (IRF-3) pathway, an important mediator of interferon induction in response to a viral infection (Foy et al., 2003). NS3-NS4A also appears to prevent dsRNA signaling via the toll-like receptor 3 upstream of IRF-3 (Li et al., 2005). One potential mechanism includes a blockade of the intracellular double-stranded RNA sensor protein (RIG-I) pathway by NS3-NS4A (Sumpter et al., 2005). Thus, HCV could utilize NS3-4A protease to circumvent the innate immune response at the early stages of infection. In addition, NS3 was also reported to induce malignant transformation of NIH3T3 cells (Sakamuro et al., 1995), suppress actinomycin D-induced apoptosis in murine cell lines (Fujita et al., 1996), and to be involved in hepatocarcinogenesis events (Borowski et al., 1996; Hassan et al., 2005), although the exact mechanisms are not clear.

NS3 Helicase-NTpase

The NS3 helicase-NTPase domain consisting of the 442 C-terminal aa of the NS3 protein is a member of the helicase superfamily-2 (see Chapter 7). Its three-dimentional structure has also been determined (Cho et al., 1998; Kim et al., 1998; Yao et al., 1997). The NS3 helicase-NTPase has several functions, including RNA-stimulated NTPase activity, RNA binding, and unwinding of RNA regions of extensive secondary structure by coupling unwinding and NTP hydrolysis (Gwack et al., 1997; Tai et al., 1996). During RNA replication, the NS3 helicase has been suggested to translocate along the nucleic acid substrate by changing protein conformation, utilizing the energy of NTP hydrolysis. A recent study proposed that the helicase directional movement step is fueled by single-stranded RNA binding energy, while NTP binding allows for a brief period of random movement that prepares the helicase for the next cycle (Levin et al., 2005). In addition, NS3 helicase activity appears to be modulated by the NS3 protease domain and the NS5B RdRp (Zhang et al., 2005).


NS4B is an integral membrane protein of 261 aa with an ER or ER-derived membrane localization (Hugle et al., 2001; Lundin et al., 2003). NS4B is predicted to harbor at least four transmembrane domains and an N-terminal amphipathic helix that are responsible for membrane association (Elazar et al., 2004; Hugle et al., 2001; Lundin et al., 2003). One of the functions of NS4B is to serve as a membrane anchor for the replication complex (see Chapter 8) (Egger et al., 2002; Elazar et al., 2004; Gretton et al., 2005). Additional putative properties include inhibition of cellular syntheses (Florese et al., 2002; Kato et al., 2002), modulation of HCV NS5B RdRp activity (Piccininni et al., 2002), transformation of NIH3T3 cell lines (Park et al., 2000), and induction of interleukin 8 (Kadoya et al., 2005).


NS5A is a 56–58 kDa phosphorylated zinc-metalloprotein that probably plays an important role in virus replication and regulation of cellular pathways (see Chapter 9). The N-terminal region of NS5A (aa 1–30) contains an amphipathic α-helix that is necessary and sufficient for membrane localization in perinuclear membranes as well as for assembly of the replication complex (Brass et al., 2002; Elazar et al., 2003; Penin et al., 2004a). Downstream of this motif, the NS5A protein was predicted to contain three domains, numbered I to III. Domain I, located at the N-terminus, contains an unconventional zinc-binding motif formed by four cysteine residues conserved among the hepacivirus and pestivirus genera (Tellinghuisen et al., 2004). HCV replicon RNA replication was inhibited by mutations in the NS5A sequence (Elazar et al., 2003; Penin et al., 2004b) and abolished by alterations of the zinc-binding site (Tellinghuisen et al., 2004). The recently determined 3-D structure of Domain I suggested the presence of protein, RNA and membrane interaction sites (Moradpour et al., 2005; Tellinghuisen et al., 2005).

The mechanisms by which NS5A regulate HCV replication are not entirely clear. NS5A associates with lipid rafts derived from intracellular membranes through its binding to the C-terminal region of a vesicle-associated membrane-associated protein of 33 kDa (hVAP-33) (Shi et al., 2003; Tu et al., 1999). This interaction appears to be crucial for the formation of the HCV replication complex in connection with lipid rafts (Gao et al., 2004). A recent study in the replicon system proposed a model in which NS5A hyperphosphorylation disrupts the interaction with hVAP- 33 and negatively regulates viral RNA replication (Evans et al., 2004). Another report suggested that the level of NS5A phosphorylation plays an important role in the viral lifecycle by regulating a switch from replication to assembly, whereby hyperphosphorylated forms function to maintain the replication complex in an assembly-incompetent state (Appel et al., 2005). Furthermore, NS5A can interact directly with NS5B, but the mechanism by which NS5A modulates the RdRp activity has not been elucidated (Shimakami et al., 2004). In addition, NS5A was reported to interact with a geranylgeranylated cellular protein (Wang et al., 2005a). This is potentially significant considering that assembly of the viral replication complex has been shown to require geranylgeranylation of one or more host cell proteins (Ye et al., 2003).

Multiple functions have been assigned to NS5A based on its interactions with cellular proteins (Tellinghuisen and Rice, 2002) (see Chapter 9). For instance, NS5A appears to play a role in interferon resistance by binding to and inhibiting PKR, an antiviral effector of interferon-α (Gale et al., 1998). NS5A also bears transcriptional activation functions (Pellerin et al., 2004; Polyak et al., 2001) and appears to be involved in the regulation of cell growth and cellular signaling pathways (Tan and Katze, 2001; Tellinghuisen and Rice, 2002). However, these observations remain to be confirmed in vivo.

NS5B RNA-Dependent RNA Polymerase

NS5B belongs to a class of membrane proteins termed tail-anchored proteins (Ivashkina et al., 2002; Schmidt-Mende et al., 2001) (see Chapter 10). Its C-terminal region (21 residues) forms an α-helical transmembrane domain responsible for post-translational targeting to the cytosolic side of the ER, where the functional protein domain is exposed (Moradpour et al., 2004; Schmidt-Mende et al., 2001). The crystal structure of NS5B revealed that the RdRp has a classical “fingers, palm and thumb” structure formed by its 530 N-terminal aa (Ago et al., 1999; Bressanelli et al., 1999; Lesburg et al., 1999). Interactions between the fingers and thumb subdomains result in a completely encircled catalytic site that ensures synthesis of positive- and negative-strand HCV RNAs (Lesburg et al., 1999). The RdRp is another important target for the development of anti-HCV drugs (Di Marco et al., 2005; Ma et al., 2005; Pawlotsky and McHutchison, 2004; Pawlotsky, 2006).

Interactions between NS5B and cellular components have also been reported. The C-terminus of NS5B can interact with the N-terminus of hVAP-33, and the interaction may play an important role in the formation of the HCV replication complex (Gao et al., 2004; Schmidt-Mende et al., 2001). More recently, NS5B was reported to bind cyclophilin B, a cellular peptidyl-prolyl cis-trans isomerase that apparently regulates HCV replication through modulation of the RNA binding capacity of NS5B (Watashi et al., 2005).

The HCV Lifecycle

Cellular Attachment of HCV Virions and Entry

Many efforts have been made to develop models to identify candidate HCV receptors and study viral binding and entry into target cells. Various cellular and in vivo systems utilizing infected blood samples, virus-like particles produced by expression of structural HCV proteins in insect or mammalian cells, liposomes containing E1–E2, as well as pseudotype particles have yielded a considerable amount of data, although they are not always easy to reconcile. Fig. 3 summarizes the hypothetical HCV lifestyle.

Fig. 3. Hypothetical HCV replication cycle.

Fig. 3

Hypothetical HCV replication cycle. HCV particles bind to the host cells via a specific interaction between the HCV envelope glycoproteins and a yet unknown cellular receptor. Bound particles (more...)

HCV Receptors

Several cell surface molecules have been proposed to mediate HCV binding or HCV binding and internalization.


Among all putative HCV receptor molecules, CD81 has been the most extensively studied (Pileri et al., 1998). Human CD81 (target of antiproliferative antibody 1, TAPA-1) is a 25-kDa molecule belonging to the tetraspanin or transmembrane 4 superfamily. It is found at the surface of numerous cell types, where it is thought to assemble as homo- and/or heterodimers by means of a conserved hydrophobic interface. CD81 contains four hydrophobic transmembrane regions (TM1 to TM4) and two extracellular loop domains of 28 and 80 aa, respectively: the small extracellular loop (SEL) and the large extracellular loop (LEL). The LEL is located between TM3 and TM4. It is composed of five α-helices and contains four cysteine residues (Kitadokoro et al., 2001). The SEL is needed for optimal surface expression of the LEL (Masciopinto et al., 2001). The intracellular and transmembrane domains of CD81 are highly conserved among different species. In contrast, the LEL is variable, except between humans and chimpanzees, the only two species permissive to HCV infection (Major et al., 2004; Walker, 1997). The CD81 LEL has been shown to mediate binding of HCV through its envelope glycoprotein E2 (Pileri et al., 1998). The integrity of two disulfide bridges is necessary for the CD81-HCV interaction to occur (Petracca et al., 2000), and the site of interaction appears to involve CD81 residues 163, 186, 188 and 196 (Flint et al., 1999; Meola et al., 2000). The E2 domains involved in CD81 binding remain controversial. Early studies suggested the involvement of aa 480–493 and 544–551 in the truncated soluble form of E2 (Flint et al., 1999), whereas a more recent study pointed to a role for two other domains, including aa 613–618 and a second domain spanning the two HVRs (aa 384–410 and 476–480) (Roccasecca et al., 2003).

Several studies argue that cellular factors other than CD81 are required for HCV infection. The expression of human CD81 in a CD81-deficient human hepatoma cell line restored permissiveness to infection with HCV pseudo-particles, but a murine fibroblast cell line expressing human CD81 remained resistant to HCV entry (Cormier et al., 2004). In addition, expression of human CD81 in transgenic mice did not confer susceptibility to HCV infection (Masciopinto et al., 2002). It is possible that the CD81 molecule could act as a post-attachment entry co-receptor and that other cellular factors act together with CD81 to mediate HCV binding and entry into hepatocytes (Cormier et al., 2004).


The scavenger receptor B type I (SR-BI) has been proposed as another candidate receptor for HCV (Scarselli et al., 2002). SR-BI is a 509-aa glycoprotein with a large extracellular loop anchored to the plasma membrane at both N- and C-termini by means of transmembrane domains with short cytoplasmic extensions (Krieger, 2001). SR-BI is a fatty acylated protein located in lipid raft domains. It is expressed at high levels in hepatocytes and steroidogenic cells (Babitt et al., 1997; Krieger, 2001). The natural ligand of SR-BI is high density lipoproteins (HDL). HDLs are internalized through a non-clathrin-dependent endocytosis process that mediates cholesterol uptake and recycling of HDL apoprotein (Silver et al., 2001). HCV genotypes 1a and 1b recombinant E2 envelope glycoproteins were shown to bind HepG2 cells (a human hepatoma cell line that does not express CD81) by interacting with an 82 kDa glycosylated SR-BI molecule (Scarselli et al., 2002). Binding appeared to be highly specific: tranfection of rodent cells with human or tupaia SR-BI (88 % aa identity with human SR-BI) resulted in E2 binding, whereas neither mouse SR-BI (80 % aa identity) nor the closely related human scavenger receptor CD36 (60 % aa identity) bound E2. The SR-BI LEL appeared to be responsible for HCV binding, and HVR1 was recently suggested to be the E2 envelope region involved in the interaction, which was facilitated by serum HDLs (Bartosch et al., 2003b; Scarselli et al., 2002; Voisset et al., 2005). However, the fact that antibodies directed against SR-BI resulted only in a partial blockade of binding suggests that SR-BI is not the only cell surface molecule involved in HCV binding to hepatocytes (Barth et al., 2005).


The dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN or CD209) and the liver/lymph node-specific intercellular adhesion molecule-3 (ICAM-3)-grabbing integrin (L-SIGN or CD209L) have been proposed as tissue-specific capture receptors for HCV present in various cell types that could play a critical role in viral pathogenesis and tissue tropism (Gardner et al., 2003; Lozach et al., 2004; Lozach et al., 2003; Pohlmann et al., 2003). DC-SIGN is a 44-kDa homotetrameric type II integral membrane protein with a short aminoterminal cytoplasmic domain and a carboxy-terminal C-type (calcium-dependent) lectin domain. DC-SIGN is expressed at a high level on myeloid-lineage dendritic cells. Its interaction with ICAM-3 activates T cells (Geijtenbeek et al., 2000). L-SIGN is abundantly expressed at the surface of endothelial cells of the liver and lymph nodes and shares 77% aa sequence identity with DC-SIGN (Bashirova et al., 2001). A rapid internalization of virus-like particles upon capture of HCV pseudo-particles by both DC-SIGN and L-SIGN, presumably via E2 binding, was reported (Ludwig et al., 2004), although this was not observed in another study (Lozach et al., 2004).


The low-density lipoprotein (LDL) receptor (LDL-R) is an endocytic receptor that transports lipoproteins, mainly the cholesterol-rich LDLs, into cells through receptor-mediated endocytosis (Chung and Wasan, 2004). Virus-like particles complexed with LDLs have been reported to enter into cells via the LDL receptor (Agnello et al., 1999; Monazahian et al., 1999). In support of this view, binding of low-density HCV particles recovered from plasma by sucrose gradient sedimentation correlated with the density of LDL receptors at the surface of MOLT-4 cells and fibroblasts, and the binding was inhibited by LDL but not by soluble CD81 (Wunschmann et al., 2000).

Asialoglycoprotein Receptor

The asialoglycoprotein receptor (ASGP-R) has been reported to mediate binding and internalization of structural HCV proteins (C-E1-E2±p7) expressed in a baculovirus system. Cotransfection of a non-permissive mouse fibroblast cell line with cDNAs of both ASGP-R subunits (H1 and H2) restored permissiveness (Saunier et al., 2003).


Conservation of positively charged residues in the N-terminus of E2 is in keeping with a possible interaction with heparan sulfate proteoglycans (HSPG) (Barth et al., 2003). E2, in particular its HVR-1, has been shown to bind HSPG with a stronger affinity than other viral envelope glycoproteins, such as human herpes virus 8 or dengue virus envelope proteins. However, glycosaminoglycans are ubiquitously expressed as cell surface molecules. It is conceivable that HSPG could serve as the initial docking site for HCV attachment and the virus is subsequently transferred to another high-affinity receptor (or receptor complex) triggering entry (Barth et al., 2003).

Mechanisms of Cell Entry and Fusion

After attachment, the nucleocapsid of enveloped viruses is released into the cell cytoplasm as a result of a fusion process between viral and cellular membranes. Fusion is mediated by specialized viral proteins and takes place either directly at the plasma membrane or following internalization of the particle into endosomes. The entry process is controlled by viral surface glycoproteins that trigger the changes required for mediating fusion. At least two different classes of fusion proteins (I and II) can be distinguished (Lescar et al., 2001). The flaviviruses enter target cells by receptor-mediated endocytosis and use class II fusion proteins (Lindenbach and Rice, 2001). By analogy, HCV envelope glycoproteins are believed to belong to class II fusion proteins (Yagnik et al., 2000). However, in contrast with other class II fusion proteins, HCV envelope glycoproteins do not appear to require cellular protease cleavage during their transport through the secretory pathway (Op De Beeck et al., 2004). HCV entry into cells is pH-dependent and endocytosis-dependent (Agnello et al., 1999; Bartosch et al., 2003b; Hsu et al., 2003), but the identity of the HCV fusion peptide remains controversial. E1 appeared as a good candidate because sequence analysis suggested the presence of a fusion peptide in its ectodomain (Flint and McKeating, 2000; Rosa et al., 1996). Nevertheless, E2 was shown to share structural homology with class II fusion proteins (Lescar et al., 2001; Yagnik et al., 2000). Crystallographic 3D structure determination and cryo-EM-based studies of both envelope glycoproteins are needed to better understand the mechanisms of HCV fusion.

RNA Translation and Post-Translational Processing

Polyprotein Synthesis

Decapsidation of viral nucleocapsids liberates free positive-strand genomic RNAs into the cell cytoplasm, where they serve, together with newly synthesized RNAs, as messenger RNAs for synthesis of the HCV polyprotein. HCV genome translation is under the control of the IRES, spanning domains II to IV of the 5′ UTR and the first nucleotides of the core-coding region. IRES domain I is not part of the IRES but plays an important role by modulating IRES-dependent translation (Friebe et al., 2001; Luo et al., 2003). The IRES mediates cap-independent internal initiation of HCV polyprotein translation by recruiting both cellular proteins, including eukaryotic initiation factors (eIF) 2 and 3 and viral proteins (Ji et al., 2004; Lukavsky et al., 2000; Otto and Puglisi, 2004). Three distinct translation initiation complexes (40S, 48S and 80S) are generated, as shown by in vitro translation experiments in HeLa S10 cells and rabbit reticulocyte lysates and by ex vivo experiments in mammalian cells (Kong and Sarnow, 2002).

The HCV IRES has the capacity to form a stable pre-initiation complex by directly binding the 40S ribosomal subunit without the need of canonical translation initiation factors (Otto et al., 2002; Spahn et al., 2001). The 40S subunit assembles with eIF3 and this ternary complex joins with eIF2, GTP, and the initiator tRNA to form a 48S particle in which the tRNA is positioned in the P site of the 40S subunit, base-paired to the start codon of the mRNA. Upon hydrolysis of GTP, eIF2 releases the initiator tRNA and dissociates from the complex. A second GTP hydrolysis step involving initiation factor eIF5B then enables the 60S ribosomal subunit to associate, forming a functional 80S ribosome that initiates viral protein synthesis (Ji et al., 2004; Kieft et al., 2001; Otto and Puglisi, 2004; Sizova et al., 1998).

A number of cellular proteins were reported to interact with the 5′ UTR including the polypyrimidine tract-binding protein (PTB) (Ali and Siddiqui, 1995), heterogeneous nuclear ribonucleoprotein L (hnRNP L) (Hahm et al., 1998), La autoantigen (Ali and Siddiqui, 1997), the poly(rC)-binding protein 2 (PCP2) (Spangberg and Schwartz, 1999) and NS1-associated protein 1 (NSAP1) (Kim et al., 2004). The biological significance of these protein-RNA interactions remains unknown. In addition, HCV proteins may affect IRES translational efficiency, including the core protein (Zhang et al., 2002) and non-structural proteins NS4A and NS5B (Kato et al., 2002). The HCV 3′ UTR may also modulate IRES-dependent translation, but this remains controversial (Imbert et al., 2003; Wang et al., 2005b).

Post-Translational Processing

HCV genome translation generates a large precursor polyprotein, which is targeted to the ER membrane for translocation of the E1 ectodomain into the ER lumen, a process mediated by the internal signal sequence located between the core and E1 sequences. Cleavage of the signal sequence by the host signal peptidase yields the immature form of the core protein (P23) (McLauchlan et al., 2002). The signal peptide is further processed by a host signal peptide peptidase (SPP, a presenilintype aspartic protease that resides in the ER membrane) to yield the mature form of the core protein (P21) (Fig. 3) (Penin et al., 2004b). The host signal peptidase also ensures cleavage at the E1–E2 junction in the ER lumen. Additional signal peptidase cleavages at the C-terminal end of E2 and between p7 and NS2 give rise to p7 (Fig. 3). An incomplete cleavage may lead to the production of non-cleaved E2-p7 proteins, the role of which is unknown. E1 and E2 subsequently undergo several maturation steps, including N-glycosylation, conformation and assembly of E1E2 heterodimers (Penin et al., 2004b). Heterogeneous E1E2 aggregates are also produced, but their role in viral particle formation is not known.

The zinc-dependent NS2-3 auto-protease ensures cis-cleavage of NS3 from NS2 (Fig. 2). NS3 needs to assemble with its cofactor NS4A to catalyze cis-cleavage at the NS3-NS4A junction and trans-cleavage at all downstream junctions including NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B (Fig. 2) (Bartenschlager and Lohmann, 2000; Lindenbach and Rice, 2005). The cleavage sites recognized by the NS3-NS4A protease have in common the following sequence: Asp/GluXXXXCys/Thr-Ser/Ala, with trans cleavages occurring downstream of a cysteine residue and the cis cleavage occurring downstream of a threonine residue.

HCV Replication

The HCV Replication Complex

Infection with a positive-strand RNA virus leads to rearrangements of intracellular membranes, a prerequisite to the formation of a replication complex that associates viral proteins, cellular components and nascent RNA strands. The HCV NS4B protein seems to be sufficient to induce the formation of a membranous web or membrane-associated foci (Egger et al., 2002; Gretton et al., 2005). It is not known whether NS4B recruits cellular proteins responsible for vesicle formation or induces vesicle formation by itself. The membranous web is derived from ER membranes (Bartenschlager et al., 2004). It is rich in cholesterol and fatty acids, the degree of saturation of which (that influences membrane fluidity) modulates HCV replication (Kapadia and Chisari, 2005). HCV replication was shown to occur in detergent-resistant membranes that co-localize with caveolin-2, an essential component of lipid raft domains (Shi et al., 2003). Indeed, lipid rafts are involved in the formation of the replication complex, through protein-protein interactions between hVAP-33 and both NS5A and NS5B HCV proteins (Gao et al., 2004; Shi et al., 2003; Tu et al., 1999). Overall, the membranous web consists of small vesicles embedded in a membranous matrix, forming a membrane-associated multiprotein complex that contains all of the nonstructural HCV proteins (Egger et al., 2002).

Mechanism of HCV Replication

The precise mechanisms of HCV replication are still poorly understood. By analogy with other positive-strand RNA viruses, HCV replication is thought to be semi-conservative and asymmetric with two steps, both of which are catalyzed by the NS5B RdRp. The positive-strand genome RNA serves as a template for the synthesis of a negative-strand intermediate of replication during the first step. In the second step, negative-strand RNA serves as a template to produce numerous strands of positive polarity that will subsequently be used for polyprotein translation, synthesis of new intermediates of replication or packaging into new virus particles (Bartenschlager et al., 2004). The positive-strand RNA progeny is transcribed in a five to ten fold in excess compared to negative-strand RNA. NS5B RpRd was initially thought to catalyze primer-dependent initiation of RNA synthesis, either through elongation of a primer hybridized to the RNA template or through a copy-back mechanism (Behrens et al., 1996). More recently, the HCV RdRp was shown to be capable of initiating de novo RNA synthesis under certain experimental conditions (Zhong et al., 2000).

Initiation of RNA strand synthesis at the 3′-end of the plus and minus strands involves domain I of the 5′ UTR, which can form a G/C-rich stem-loop, the 3′ UTR and a cis-acting replication element (5BSL3.2) consisting of 50 bases located in a large predicted cruciform structure at the 3′ end of the HCV NS5B-coding region (You et al., 2004). Initiation of RNA replication is triggered by an interaction between proteins of the replication complex, the 3′ X region of the 3′ UTR, and 5BSL3.2 that forms a pseudoknot structure with a stem-loop in the 3′ UTR (Astier-Gin et al., 2005; Friebe et al., 2005; You et al., 2004). A phosphorylated form of PTB was found in the replication complex and PTB was shown to interact with two conserved stem-loop structures of the 3′ UTR, an interaction thought to modulate RNA replication (Chang and Luo, 2005; Luo, 1999; Luo, 2004). Importantly, inhibition of PTB expression by means of small interfering RNAs reduced the amount of HCV proteins and RNA in HCV replicon-harboring Huh7 cells (Chang and Luo, 2005).

Virus Assembly and Release

Little is known about HCV assembly and release due to the lack of appropriate study models. Different variants of the HCV core protein, which can exist as dimeric, and probably multimeric forms as well, have been shown to be capable of self assembly in yeast in the absence of viral RNA, generating virus-like particles with an average diameter of 35 nm (Acosta-Rivero et al., 2004a; Acosta-Rivero et al., 2004b). Recent reports suggested that the N-terminal portion of the core protein is sufficient for capsid assembly, in particular the two clusters of basic residues (Klein et al., 2005; Klein et al., 2004; Kunkel et al., 2001; Lorenzo et al., 2001; Majeau et al., 2004). In bacterial systems, HCV core proteins efficiently self-assembled to yield nucleocapsid-like particles with a spherical morphology and a diameter of 60 nm, but the presence of a nucleic acid was required (Kunkel et al., 2001). Overall, particle formation is probably initiated by the interaction of the core protein with genomic RNA; HCV core can indeed bind positive-strand RNA in vitro through stem-loop domains I and III and nt 23-41 (Shimoike et al., 1999; Tanaka et al., 2000). It is tempting to speculate that the core-RNA interaction may play a role in the switch from replication to packaging.

Virus-like particles were produced in mammalian cells by using a chimeric virus replicon allowing high-level expression of HCV structural proteins in BHK-21 cell lines (Blanchard et al., 2002). Budding of virus-like particles of 50 nm in diameter in the dilated ER lumen was observed (Blanchard et al., 2003). Transfection of full-length HCV RNA in HeLa G and HepG2 cell lines led to the formation of virus-like particles with a diameter of 45 to 60 nm, which were synthesized and assembled in the cytoplasm and budded into the ER cisternae to form coated particles (Dash et al., 1997; Mizuno et al., 1995). Indeed, the HCV envelope glycoproteins E1 and E2 associate with ER membranes through their transmembrane domains (Cocquerel et al., 1998), suggesting that virus assembly occurs in the ER. Structural proteins have been detected both in the ER and the Golgi apparatus, suggesting that both compartments are involved in later maturation steps (Serafino et al., 2003). Moreover, the presence of N-glycan residues at the surface of HCV particles is also in keeping with a transit via the Golgi apparatus. The mechanisms underlying exportation of mature virions in the pericellular space have yet to be understood. Newly produced virus particles may leave the host cell by the constitutive secretory pathway.

Structure of HCV Virions

HCV is thought to adopt a classical icosahedral scaffold in which glycoproteins E1 and E2 are anchored to the host cell-derived double-layer lipid envelope. Within the envelope is the nucleocapsid which is likely composed of multiple copies of the core protein, forming an internal icosahedral viral coat that encapsidates the viral genomic positive-strand RNA. EM and immuno-EM (IEM) studies of bona fide HCV particles have been hampered by the low amount of viruses in blood and tissues, the failure to efficiently propagate HCV in cell culture, the poor sensitivity of these methods, and antibody cross-reactivity. Visualization of HCV virions or virus-like particles was therefore made essentially from in vitro or non-human in vivo models.

Infection of primary cells or stable cell lines of hepatic or lymphoid origin with sera from HCV-infected patients revealed the presence of spherical virus-like particles (Iacovacci et al., 1997; Serafino et al., 1997; Shimizu et al., 1996). Transfection of Huh7 cells with full-length HCV genomes did not lead to virion production (Pietschmann et al., 2002), but virus-like particles were generated after transfection of HepG2 or Hela G cells (Dash et al., 1997; Mizuno et al., 1995). HCV virus-like particles could also be produced in mammalian cells, by means of recombinant Semliki Forest virus (SFV) or vesicular stomatitis virus (VSV) replicons expressing genes encoding the structural HCV proteins (Blanchard et al., 2003; Ezelle et al., 2002), and in insect cells infected with a recombinant baculovirus expressing HCV structural proteins (Baumert et al., 1998; Luckow and Summers, 1988; Maillard et al., 2001).

Morphology of HCV Particles

Early filtration studies performed in sera from chimpanzees with non-A, non-B hepatitis suggested that the diameter of the causal agent was in the order of 30–60 nm (He et al., 1987; Yuasa et al., 1991). EM and IEM analysis of particles recovered from the blood and liver of infected chimpanzees and patients revealed the presence of spherical particles of 33–70 nm (Bosman et al., 1998; Ishida et al., 2001; Jacob et al., 1990; Kaito et al., 1994; Li et al., 1995; Petit et al., 2003). Detergent treatment of infectious sera yielded 30–40 nm icosahedron-shaped particles containing both the HCV core protein and HCV RNA (Takahashi et al., 1992). Virus-like particles of 45–60 nm was observed in the supernatant of primary cells or stable cell cultures treated with infectious sera and of cell lines transfected with the full-length HCV ORF (Dash et al., 1997; Iacovacci et al., 1997; Mizuno et al., 1995; Serafino et al., 1997; Shimizu et al., 1996). HCV-like particles of 20–60 nm in diameter were also produced by the expression of HCV structural proteins in cell-free systems (Klein et al., 2004), SFV replicons (Blanchard et al., 2002; Blanchard et al., 2003), VSV vectors in rodent BHK-21 cells (Ezelle et al., 2002), bacterial systems (Kunkel et al., 2001; Lorenzo et al., 2001), baculovirus vectors in insect cells (Baumert et al., 1998; Xiang et al., 2002) and yeast expression vectors (Acosta-Rivero et al., 2001; Acosta-Rivero et al., 2004b; Falcon et al., 1999).

The recently developed cell culture system is capable of producing large amounts of infectious HCV virions (Lindenbach et al., 2005b; Wakita et al., 2005; Zhong et al., 2005). Two types of viral particles could be visualized in IEM: particles of 30–35 nm in diameter likely to correspond to the viral nucleocapsids, and particles of 50–60 nm in diameter likely to be the infectious virions (Fig. 4) (Wakita et al., 2005).

Fig. 4. HCV viral particle produced in a tissue culture system from a cloned viral genome (Wakita et al.

Fig. 4

HCV viral particle produced in a tissue culture system from a cloned viral genome (Wakita et al., 2005). Viral particles were generated after transfection of the human (more...)

Circulating Forms of HCV Virions

Plasma Compartmentalization of HCV Particles

HCV was initially reported to have a lower buoyant density than other members of the Flaviviridae family on 20–60% isopycnic sucrose density gradients (1.05 to 1.07 g/ml vs 1.15 to 1.25 g/ml, respectively) (Lindenbach and Rice, 2001; Trestard et al., 1998; Yoshikura et al., 1996). Ultracentrifugation of sera from patients with acute and chronic HCV infection revealed the presence of two populations of HCV particles with a broad range of densities, from 1.06 to 1.25 g/ml. Low-density HCV particles were shown to be principally associated with lipids and lipoproteins and to contain the infectious virus, whereas high-density HCV particles were largely associated with immunoglobulins in the form of immune complexes and supposedly less infectious (Aiyama et al., 1996; Andre et al., 2002; Dienstag et al., 1979; Hijikata et al., 1993; Thomssen et al., 1992). Interestingly, the respective proportions of high- and low-density fractions in infected patients' blood were reported to fluctuate over the course of infection and according to the stage of liver disease (Choo et al., 1995; Hijikata et al., 1993; Kanto et al., 1994; Kanto et al., 1995; Petit et al., 2003).

Non-Enveloped Nucleocapsids

The existence of non-enveloped HCV nucleocapsids during natural infection and their role in the pathophysiology of HCV infection has been debated. Lipo-viroparticles (LVPs) rich in HCV RNA, HCV core protein, triglycerides and apoproteins (especially apoB and apoE) were recently described as large spherical particles of 100 nm, the delipidation of which yielded capsid-like structures (Andre et al., 2002). Non-enveloped nucleocapsids were detected in the serum of infected patients and in hepatocytes from patients and experimentally infected chimpanzees (Falcon et al., 2003a; Falcon et al., 2003b; Maillard et al., 2001). Non-enveloped HCV particles recovered from the plasma of infected individuals had a buoyant density of 1.27 to 1.34 g/ml (Maillard et al., 2001). They were heterogeneous in size, with a diameter of 38–62 nm in EM, and were recently shown to exhibit Fcγ receptor-like activity and bind non-immune IgG (Maillard et al., 2001; Maillard et al., 2004). Whether or not non-enveloped nucleocapsids are infectious remains to be established.


The development of novel anti-HCV therapeutic agents has been stymied by the lack of an efficient in vitro viral infection system and a suitable animal model. Although significant progress has been made through genetic and biochemical approaches in dissecting the molecular processes of HCV replication, our understanding of the viral entry and virion production steps remains rudimentary. Furthermore, HCV exists as “quasispecies” in patients due to its high mutation rate and thus viral resistance will likely be a problem for the emerging small-molecule HCV inhibitors (Pawlotsky, 2003; Pawlotsky, 2006). The recent development of a robust cell culture system for HCV infection may unravel new aspects of HCV replication, which in turn will facilitate the development of specific antivirals that target each stage in the virus life cycle.


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