A clinical textbook

Hepatology 2018

6. HCV virology

Bernd Kupfer

Hepatitis C virus (HCV) is a major cause of progressive liver disease with an estimated 185 million people infected worldwide, 350,000 of whom die each year from liver damage associated with the infection. HCV infection leads to chronic infection in up to 80% of infected individuals. The main complications of HCV are severe liver fibrosis and cirrhosis, and 30–50% of individuals with cirrhosis go on to develop hepatocellular carcinoma (Tong 1995, Poynard 1997). As a consequence, chronic HCV infection is the major reason for liver transplantation in high-income countries.

History

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, as HAV and HBV were excluded from being the cause of approximately 65% of posttransfusion hepatitis, these cases were termed “non-A, non-B hepatitis” (NANBH) (Feinstone 1975). Inoculation 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 NANBH and the limited availability of chimpanzees prevented further characterisation 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 characterised (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).

Before HCV was identified, a limited number of patients with NANBH were successfully treated by long-term administration of interferon α. However, it was only after the molecular characterisation of HCV that it became possible to develop target-specific therapeutics as well as laboratory tests for diagnosis and monitoring of both HCV infection and treatment response.

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 genus hepacivirus within the Flaviviridae family. This viral family contains four genera, flavivirus, pestivirus, hepacivirus, and pegivirus (Stapleton 2011). Novel hepaciviruses have been described from primates, bats, bank voles, horses, and dogs enabling researchers to possibly develop new model systems for the analysis of the molecular biology and the pathogenesis of HCV (Kapoor 2013, Drexler 2013, Lauck 2013).

Comparisons of HCV nucleotide sequences derived from individuals from different geographical regions revealed the presence of at least seven major HCV genotypes with a large number of subtypes within each genotype (Smith 2014). HCV strains belonging to the major genotypes 1, 2, 4, and 5 are found in sub-Saharan Africa whereas genotypes 3 and 6 are detected with extremely high diversity in South East Asia. This suggests that these geographical areas could be the origin of the different HCV genotypes. The emergence of different HCV genotypes in North America and Europe and other non-tropical countries appears to represent more recent epidemics introduced from the sites of the original HCV endemics (Simmonds 2001, Ndjomou 2003). In a recent study more than 1300 (nearly) complete HCV coding region sequences were analysed in order to validate new genotype and subtype assignments (Smith 2014). This revealed the presence of at least 7 different HCV genotypes and 67 subtypes. Genomes assigned to the newly described HCV genotype 7 could be detected in human subjects from Central Africa (Murphy 2015). The fast growing number of full-length HCV genome sequences will probably lead to even higher numbers of HCV genotypes. Moreover, it has been reported that inter-subtype as well as inter-genotype HCV recombinants occur (Shi 2012). Although these recombination variants still appear to be rare, this phenomenon may be relevant in patients treated with genotype-specific regimen.

Viral structure

Structural analyses of HCV virions are very limited since the virus is difficult to cultivate in cell culture systems, a prerequisite for yielding sufficient virions for electron microscopy. Moreover, serum-derived virus particles are associated with serum low-density lipoproteins (Thomssen 1992), which makes it difficult to isolate virions from serum/plasma of infected subjects by ultracentrifugation. Visualisation of HCV virus-like particles via electron microscopy succeeds 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 suggested that HCV has a diameter of 55–65 nm confirming the prediction of the NANBH agent by ultra-filtration (Bradley 1985). In a recent study with highly purified HCV, heterogeneous viral particles with diameters between 50 and 80 nm were observed (Catanese 2013). 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 hypothesis is that HCV enters hepatocytes via the LDL receptor (Agnello 1999, Nahmias 2006). However, in a more recent study it was demonstrated that involvement of the LDL receptor led to non-productive HCV infection (Albecka 2012).

The design and optimisation of subgenomic and genomic HCV replicons in the human hepatoma cell line Huh7 offered for the first time the possibility to investigate HCV RNA replication in a standardised manner (Lohmann 1999, Ikeda 2002, Blight 2002). However, despite the high level of HCV gene expression, no infectious viral particles are produced with that replication system. Therefore, it cannot be used for structural analysis of cell-free virions.

Infectious HCV particles have been achieved in cell culture by using recombinant systems (Heller 2005, Lindenbach 2005, Wakita 2005, Zhong 2005, Yu 2007). However, even in these in vitro systems the limited production of viral particles prevents 3D structural analysis (Yu 2007). Nevertheless, it has been shown by cryoelectron microscopy (cryoEM) and negative-stain transmission electron microscopy that HCV virions isolated from cell culture have a spherical shape with a diameter of approximately 50 to 55 nm (Heller 2005, Wakita 2005, Yu 2007) confirming earlier results that measured the size of putative native HCV particles from the serum of infected individuals (Prince 1996). The outer surface of the viral envelope seems to be smooth. Size and morphology are therefore very similar to other members of the Flaviviridae family such as the dengue virus and the West Nile virus (Yu 2007). Modifying a baculovirus system (Jeong 2004, Qiao 2004) the same authors were able to produce large quantities of HCV-like particles (HCV-LP) in insect cells (Yu 2007). Analysing the HCV-LPs by cryoEM it was demonstrated that the HCV E1 protein is present in the outer surface of the LPs. In a recent study, analysing viral particles derived from cultivated primary hepatocytes spike projections were observed in the outer surface of HCV (Catanese 2013). These spikes could be the key structures for viral adsorption and entry of HCV to the host hepatocytes.

Using 3D modelling of the HCV-LPs together with genomic comparison of HCV and well-characterised flaviviruses it was assumed that 90 copies of a block of two heterodimers of HCV proteins E1 and E2 form the outer layer of the virions with a diameter of approximately 50 nm (Yu 2007). This outer layer surrounds the lipid bilayer that contains the viral nucleocapsid consisting of the HCV core (C) protein. An inner spherical structure with a diameter of approximately 30–35 nm has been observed (Wakita 2005) representing the nucleocapsid that harbours the genomic viral RNA (Takahashi 1992).

Association of HCV particles with a set of lipoproteins in human sera suggests the existence of so-called lipoviral particles (LVP) in vivo (Lindenbach 2013).

Genome organisation

The genome of the hepatitis C virus consists of one 9.6 kb single-stranded RNA molecule with positive polarity. Similar to other positive-strand RNA viruses, the genomic RNA of hepatitis C virus serves as messenger RNA (mRNA) for the translation of viral proteins. The linear molecule contains a single open reading frame (ORF) coding for a precursor polyprotein of approximately 3000 amino acid residues (Figure 1). During viral replication the polyprotein is cleaved by viral as well as host enzymes into three structural proteins (core, E1, E2) and seven non-structural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B). An additional protein (termed F [frameshift] or ARF [alternate reading frame]) is predicted as a result of ribosomal frameshifting during translation within the core region of the genomic RNA (Xu 2001, Walewski 2001, Varaklioti 2002, Branch 2005). Detection of anti-F protein antibodies in the serum of HCV positive subjects indicates that the protein is indeed expressed during infection in vivo (Walewski 2001, Komurian-Pradel 2004).

The structural genes encoding the viral core protein and the viral envelope proteins E1 and E2 are located at the 5’ terminus of the open reading frame followed downstream by the coding regions for the non-structural proteins p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B (Figure 1). The structural proteins are essential components of the HCV virions, whereas the non-structural proteins are not associated with virions but are involved in RNA replication and virion morphogenesis.

The ORF is flanked by 5’ and 3’ non-translated regions (NTR; also called untranslated regions, UTR or noncoding regions, NCR) containing nucleotide sequences relevant for the regulation of viral replication. Both NTRs harbour highly conserved regions compared to the protein encoding regions of the HCV genome. The high grade of conservation of the NTRs makes them candidates i) for improved molecular diagnostics, ii) as targets for antiviral therapeutics, and iii) as targets for an anti-HCV vaccine.

Figure 1. Genome organisation and polyprotein processing. A) Nucleotide positions correspond to the HCV strain H77 genotype 1a, accession number NC_004102.
nt, nucleotide; NTR, non-translated region. B) Cleavage sites within the HCV precursor polyprotein for the signal peptide peptidase (SPP) and the viral proteases NS2/NS3 and NS3/NS4A, respectively

The 5’NTR is approximately 340 nucleotides long with a complex secondary structure of four distinct domains (I-IV) (Fukushi 1994, Honda 1999). The first 125 nucleotides of the 5’NTR spanning domains I and II have been shown to be essential for viral RNA replication (Friebe 2001, Kim 2002). Domains II-IV build an internal ribosome entry side (IRES) involved in ribosome binding and subsequent cap-independent initiation of translation (Tsukiyama-Kohara 1992, Wang 1993).

The 3’NTR consists of three functionally distinct regions: a variable region, a poly U/UC tract of variable length, and the highly conserved X tail at the 3’ terminus of the HCV genome (Tanaka 1995, Kolykhalov 1996, Blight 1997). The 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 a homouridine stretch of 26 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 circularisation (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 (Figure 1B). 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 and NS3 (Santolini 1995) and the NS3 serine protease cleaving the remaining functional proteins (Bartenschlager 1993, Eckart 1993, Grakoui 1993a, Tomei 1993).

The positions of viral nucleotide and amino acid residues correspond to the HCV strain H77 genotype 1a, accession number NC_004102. Some parameters characterising HCV proteins are summarised in Table 1.

Core. The core-encoding sequence starts at codon AUG at nt position 342 of the H77 genome, the start codon for translation of the entire HCV polyprotein. During translation the polyprotein is transferred to the endoplasmic reticulum (ER) where the core protein (aa 191) is excised by a cellular signal peptidase (SP). The C terminus of the resulting core precursor still contains the signal sequence for ER membrane translocation of the E1 ectodomain (aa 174–191). This protein region is further processed by the cellular intramembrane signal peptide peptidase (SPP) leading to removal of the E1 signal peptide sequence (Hüssy 1996, McLauchlan 2002, Weihofen 2002).

The multifunctional core protein has a molecular weight of 21 kilodalton (kd). In vivo, the mature core molecules are believed to form homo-multimers located mainly at the ER membrane (Matsumoto 1996). They have a structural function since they form the viral capsid that contains the HCV genome. In addition, the core protein has regulatory functions including particle assembly, viral RNA binding, and regulation of RNA translation (Ait-Goughoulte 2006, Santolini 1994). Moreover, protein expression analyses indicate that the core protein may be involved in many other cellular reactions such as cell signalling, apoptosis, lipid metabolism, and carcinogenesis (Tellinghuisen 2002). However, these preliminary findings need to be analysed further.

Table 1. Overview of the size of HCV proteins*
Protein No. of aa aa position in ref. seq. MW of protein
Core immature 191 1–191 23 kd
Core mature 174 1–174 21 kd
F protein or ARF protein 126–161 ~ 16–17 kd
E1 192 192–383 35 kd
E2 363 384–746 70 kd
p7 63 747–809 7 kd
NS2 217 810–1026 21 kd
NS3 631 1027–1657 70 kd
NS4A 54 1658–1711 4 kd
NS4B 261 1712–1972 27 kd
NS5A 448 1973–2420 56 kd
NS5B 591 2421–3011 66 kd
* aa, amino acid; MW, molecular weight; kd, kilodalton; ref. seq., reference sequence (HCV strain H77; accession number NC_004102)

E1 and E2. Downstream of the core coding region of the HCV RNA genome two envelope glycoproteins are encoded, E1 (gp35, aa 192) and E2 (gp70, aa 363). During translation at the ER both proteins are cleaved from the precursor polyprotein by a cellular SP. Inside the lumen of the ER both polypeptides experience post-translational N-linked glycosylation (Duvet 2002). The glycoproteins E1 and E2 harbour 6 and 11 putative N-glycosylation sites, respectively. Recent findings suggest that HCV E2 contains further 6–7 putative sites for O-linked glycosylation (Bräutigam 2012).

E1 and E2 are type I transmembrane proteins with large hydrophilic ectodomains and short transmembrane domains (TMD) of 30 aa. The TMD is responsible for anchoring of the envelope proteins in the membrane of the ER and ER retention (Cocquerel 1998, Duvet 1998, Cocquerel 1999, Cocquerel 2001). Moreover, the same domains have been reported to contribute to the formation of E1-E2 heterodimers (Op de Beeck 2000). The E1-E2 complex is involved in adsorption of the virus to its putative receptors tetraspanin CD81 and low-density lipoprotein (LDL) 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 as involved in viral entry. These candidates include the scavenger receptor B type I (Scarselli 2002, Kapadia 2007), the tight junction proteins claudin-1 (Evans 2007) and occludin (Ploss 2009), the C-type lectins L-SIGN and DC-SIGN (Gardner 2003, Lozach 2003, Pöhlmann 2003) and heparan sulfate (Barth 2003).

Three hypervariable regions have been identified within the coding region of E2. These regions, termed hypervariable region 1 (HVR1), 2 (HVR2) and 3 (HVR3), have a sequence variability of up to 80% in their amino acid sequences (Weiner 1991, Kato 2001, Troesch 2006). The high variability of the HVRs reflects exposure of these domains to HCV-specific antibodies. In fact, E2-HVR1 has been shown to be the most important target for neutralising antibodies (Farci 1996, Shimizu 1996b). However, the combination of viral mutation with the selective pressure of the humoral immune response leads to viral escape via epitope alterations (Pantua 2013). Moreover, association of virions with lipoproteins and the presence of a glycan shield on the surface of the viral glycoproteins reduce the effectivity of neutralising antibodies, respectively (Voisset 2006, Helle 2010). This makes the development of vaccines that induce effective neutralising antibodies challenging.

The p7 protein. The small p7 protein (63 aa) is located between the E2 and NS2 regions of the polyprotein precursor. During translation the cellular SP cleaves the E2/p7 as well as the p7/NS2 junction. The functional p7 is a membrane protein localised in the endoplasmic reticulum where it forms an ion channel (Haqshenas 2007, Pavlovic 2003, Griffin 2003). The p7 protein is not essential for RNA replication since replicons lacking the p7 gene replicate efficiently (Lohmann 1999, Blight 2000), however it has been suggested that p7 plays an essential role for virus assembly, formation of infectious virions, and secretion (Sakai 2003, Haqshenas 2007, Gentzsch 2013).

NS2. The non-structural protein 2 (p21, 217 aa), together with the N-terminal portion of the NS3 protein, form the NS2/NS3 cysteine protease which autocatalyses the 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. After cleavage from the NS3, the protease domain of NS2 seems to play an essential role in the early stage of virion assembly and morphogenesis (Jones 2007), probably through physical interactions with the E1-E2 glycoprotein and NS3/NS4A complexes (Stapleford 2011). Moreover, it was demonstrated that NS2 interacts with different host factors. The binding of NS2 to the liver-specific pro-apoptotic CIDE-B protein (Erdtmann 2003) leads to inhibition of CIDE-B-induced apoptosis. Furthermore, the HCV NS2 protein seems to inhibit cell growth and induces cell cycle arrest in the S phase through down-regulation of cyclin A expression (Yang 2006). Finally, it seems that HCV NS2 is involved in the inhibition of cellular IFN β production (Kaukinen 2013), weakening the unspecific antiviral cellular response.

NS3. The non-structural protein 3 (p70; 631 aa) is cleaved at its N terminus by the NS2/NS3 autoprotease. The C terminal portion of NS3 (442 aa) has ATPase/helicase activity, i.e., it catalyses the binding and unwinding of the viral RNA genome during viral replication (Jin 1995, Kim 1995). However, later 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. 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/NS4A protease function is essential for viral infectivity it is a promising target in the design of antiviral treatments. In 2011 two potent NS3/NS4A inhibitors, boceprevir (Malcolm 2006) and telaprevir (Perni 2006), were approved by FDA and EMA to be used in combination with IFN α and ribavirin. However, several resistance-associated mutations within the HCV NS3/NS4A coding region have been observed. Meanwhile two additional HCV protease inhibitors, paritaprevir and simeprevir have been approved treatment of HCV genotypes 1 and 4, respectively and additional drugs are awaiting approval.

NS4A. The HCV non-structural protein 4A (p4; 54 aa) is a 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; 217 aa) is an integral membrane protein that forms oligomers localised in the endoplasmic reticulum (Yu 2006). 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 HCV therapeutics or vaccines. In addition, a nucleotide-binding motif (129–134 aa) has been identified (Einav 2004). Moreover, NS4B has the capability of RNA binding (Einav 2008). It has already been demonstrated that the protein induces an ER-derived membranous web that may serve as a platform for HCV RNA replication (Egger 2002). In summary, NS4B appears to be the central viral protein responsible for the formation of the HCV RNA replication complex (Blight 2011).

NS5A. The NS5A protein (p56; 458 aa) is a membrane-associated phosphoprotein that has multiple functions in HCV RNA replication, viral assembly, and virion release. It is phosphorylated by different cellular protein kinases indicating an essential role of NS5A in the HCV replication cycle that is still not fully understood. 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 α sensitivity-determining region (ISDR) that plays a significant role in the response to IFN α-based therapy (Enomoto 1995, Enomoto 1996). An increasing number of mutations within the ISDR showed positive correlation with sustained virological response to IFN α-based treatment. A previous study suggests that NS5A interacts with cytosolic cyclophilin A (CypA) and that this interaction is essential for viral replication (Chatterji 2009). Since inhibitors of CypA, e.g., cyclosporins, already exist, these important findings offer new opportunities for the development of potent anti-HCV therapeutic strategies. Furthermore, HCV NS5A seems to play a key role in preventing oxidative stress-mediated apoptosis keeping the host cell alive, thus enabling the virus to further produce progeny virus (Amako 2013). In addition to the viral enzymes, NS5A is also an interesting target for the development of anti-HCV acting therapeutics, due to its multi-functional properties during different stages of HCV replication. Consequently, three drugs targeting NS5A have been developed and approved to date (daclatasvir, ledipasvir, ombitasvir) and further NS5A inhibitors are to come.

NS5B. The non-structural protein 5B (p66; 591 aa) represents the RNA-dependent RNA polymerase of HCV (Behrens 1996). The hydrophobic domain (21 aa) at the C terminus of NS5B inserts into the membrane of the endoplasmic reticulum, while the active sites of the polymerase are located in the cytoplasm (Schmidt-Mende 2001). During HCV RNA replication NS5B is an essential compound of the HCV replication complex within the NS4B-induced membranous web.

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-strand RNA that then serves as a template for the synthesis of genomic positive-strand 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 proofreading mechanism leading to the conservation of misincorporated ribonucleotides. These enzyme properties together with the high rate of viral replication promote a pronounced intra-patient as well as inter-patient HCV evolution.

Currently, one nucleotidic polymerase inhibitor (sofosbuvir) and one non-nucleosidic polymerase inhibitor (dasabuvir) are approved.

F protein, ARFP. In addition to the ten proteins derived from the long HCV ORF, the F (frameshift) or ARF (alternate reading frame) or core+1 protein has been reported (Walewski 2001, Xu 2001, Varaklioti 2002). As the designations indicate, the ARFP is the result of a –2/+1 ribosomal frameshift between codons 8 and 11 of the core protein-encoding region. The ARFP length varies from 126 to 161 amino acids depending on the corresponding genotype. In vitro studies have shown that ARFP is a short-lived protein located in the cytoplasm (Roussel 2003) primarily associated with the endoplasmic reticulum (Xu 2003). Detection of anti-F protein antibodies in the serum of HCV positive subjects indicates that the protein is expressed during infection in vivo (Walewski 2001, Komurian-Pradel 2004). However, the functions of ARFP in the viral life cycle are still unknown and remain to be elucidated.

Viral life cycle

HCV enters humans via different transmission routes. The most effective mode of transmission is direct blood-to-blood contact, e.g., blood transfusion, needle sharing, organ transplantation, and other invasive procedures. Furthermore, sexual and mother-to-child transmission have also been described as being responsible for HCV infection. After the virus has entered the blood circulation it reaches the basolateral surface of its host cells within the liver, namely the hepatocytes. The not yet fully understood complex mechanisms of virus entry into its target cell and the downstream processes of HCV proliferation are briefly described below.

Adsorption and viral entry

Binding to and entry of HCV into hepatocytes is a very complex multistep process and more and more host factors involved in that process have been identified over the last 18 years. The first candidate as receptor for HCV was the tetraspanin CD81 (Pileri 1998). CD81 is an ubiquitous 25 kd molecule expressed on the surface of a large variety of cells including hepatocytes and PBMCs that is involved in a post- binding step (Cormier 2004, Koutsoudakis 2006, Bertaud 2006). However, further studies have shown that CD81 alone is not sufficient for HCV viral entry and that cofactors such as scavenger receptor B type I (SR-BI) are needed (Bartosch 2003b, Hsu 2003, Scarselli 2002, Kapadia 2007). These findings together with the identification of other host factors involved in HCV cell entry generate the current model for the early steps of HCV infection (Lupberger 2012, Dubuisson 2014).

Adsorption of HCV to its target cell is the first step of viral entry. This process may be mediated by VLDL or LDL that is reported to be associated with HCV virions in human sera (Bradley 1991, Thomssen 1992, Thomssen 1993). Dependent on the density of viral particles, HCV binding is thought to be initiated by the interaction of virus-associated apolipoprotein E (ApoE) and the heparan sulfate proteoglycans syndecan-1 and syndecan-4 or SR-BI on the surface of host cells (Dao 2012, Shi 2013, Lefevre 2014, Xu 2015). 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) emphasising 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). SR-BI is capable of binding to HCV-associated lipoproteins as well as the viral glycoprotein E2. For that reason, SR-BI is assumed to represent the bridge step between attachment of HCV and viral entry. This is supported by two studies showing that HCV binding to SR-BI is a prerequisite for 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 identified as being 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). However, CD81 and CLDN1 seem to form a protein complex prior to viral entry. Recent findings suggest that CLDN1 could also act as a compound enabling cell-to-cell transfer of hepatitis C virus independently of CD81 (Timpe 2007). Furthermore, it was reported that two other members of the claudin family, claudin-6 and claudin-9, may play a role in HCV infection (Zheng 2007, Meertens 2008). The observation that some human cell lines were not susceptible to HCV infection despite expressing SR-BI, CD81, and CLDN1 indicated that other cellular factors must be involved in viral entry (Evans 2007). In fact, a cellular four-transmembrane domain protein named occludin (OCLN) was identified to represent an additional cellular factor essential for the susceptibility of cells to HCV infection (Liu 2009, Ploss 2009). Similar to claudin-1, OCLN is a component of the tight junctions in hepatocytes. All tested cells expressing SR-BI, CD81, CLDN1, and OCLN were susceptible to HCV. However, recent work identified E-cadherin as an additional factor that is involved in viral entry (Li 2016). This adhesion protein seems to affect HCV uptake indirectly by triggering the required cell surface distribution of CLDN1 and OCLN, respectively. Although the precise mechanism of HCV uptake in hepatocytes is still not understood, these four proteins may represent the complete minimal set of host cell factors necessary for cell-free HCV entry. Nevertheless, recent studies reported two receptor tyrosine kinases EGFR and ephrin receptor A2 (EphA2), the Niemann–Pick C1-like 1 cholesterol uptake receptor (NPC1L1), transferrin receptor 1 (TfR1), and CD63 as cellular cofactors for HCV adsorption and entry into hepatocytes (Lupberger 2011, Sainz 2012, Martin 2013, Park 2013).

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.

After the complex procedure of binding to the different host membrane factors HCV enters the cell in a pH-dependent manner indicating that the virus is internalised 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 need to continue to be investigated.

Besides the infection of cells through cell-free HCV it has been documented that HCV can also spread via cell-to-cell transmission in vitro (Valli 2006, Valli 2007). This transmission pathway is dependent on several host factors that are also necessary for cell-free HCV infection, including SR-BI, CLDN1, OCLN, EGFR, EphA2, and NPC1L1. However the VLDL pathway, CD81, and TfR1 seem to be dispensable for cell-to-cell transmission in cultivated hepatoma cells (Witteveldt 2009, Barretto 2014). These findings require further investigation in order to analyse the process of cell-to-cell transmission of HCV both in vitro and in vivo. Antiviral treatment strategies must account for the cellular pathways of both cell-free virus and HCV transmitted via cell-to-cell contact. Cell-to-cell spread of HCV is very important particularly since this transmission route remains inaccessible to humoral immune responses as well as extracellular acting anti-HCV therapeutics.

Translation and post-translational 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 non-translated region (NTR) at each terminus. The 5’NTR consists of four distinct domains, I-IV. Domains II-IV form an internal ribosome entry side (IRES) involved in ribosome-binding and subsequent cap-independent initiation of translation (Fukushi 1994, Honda 1999, Tsukiyama-Kohara 1992, Wang 1993). The HCV IRES binds to the 40S ribosomal subunit complexed with eukaryotic initiation factors 1A, 2, and 3 (eIF1A, eIF2 and eIF3), GTP and the initiator tRNA, resulting in the 48S preinitiation complex (Jaafar 2016, 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. Post-translational cleavages lead to the 10 functional viral proteins Core, E1, E2, p7, NS2-NS5B (see Figure 1B). The viral F protein (or ARF protein) originates from a ribosomal frameshift within the first codons of the core-encoding genome region (Walewski 2001, Xu 2001, Varaklioti 2002). Besides several other cellular factors that have been reported to be involved in HCV RNA translation, various viral proteins and genome regions have been shown to enhance or inhibit viral protein synthesis (Zhang 2002, Kato 2002, Wang 2005, Kou 2006, Bradrick 2006, Song 2006).

The precursor polyprotein is processed by at least four distinct peptidases. The cellular signal peptidase (SP) cleaves the N-terminal viral proteins’ immature core protein, E1, E2, and p7 (Hijikata 1991), while the cellular signal peptide peptidase (SPP) is responsible for the cleavage of the E1 signal sequence from the C-terminus of the immature core protein, resulting in the mature form of the core (McLauchlan 2002). The E1 and E2 proteins remain within the lumen of the ER where they are subsequently N-glycosylated, with E1 having 5 N-glycosylation sites 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 (MW) containing most of the non-structural HCV proteins including NS5B (Egger 2002). Further analyses revealed that the MW consists of rough ER, endosomes, mitochondria and cytosolic lipid droplets. The main MW-structures associated with HCV replicase activity are ER-derived protrusions called double membrane vesicles (DMV) which are inducible primarily by HCV NS5A (Romero-Brey 2012). Accordingly, DMV are proposed to be the cytosolic subsites of downstream processes during HCV RNA replication.

HCV NS5B uses the previously released genomic positive-strand HCV RNA as a template for the synthesis of an intermediate minus-strand RNA. After the viral polymerase has bound to its template, the NS3 helicase is assumed to unwind putative secondary structures of the template RNA in order to facilitate the synthesis of minus-strand RNA (Jin 1995, Kim 1995). In turn, again with the assistance of the NS3 helicase, the newly synthesised antisense RNA molecule serves as the template for the synthesis of numerous plus-strand RNA. The resulting sense RNA may be used subsequently as genomic RNA for HCV progeny as well as for further polyprotein translation.

Using a single molecule HCV RNA detection assay it was shown recently that low level synthesis of single stranded (+) HCV RNA as well as (-) HCV RNA occurs within a few hours of infection and prior to formation of robust replication complexes (Shulla 2015). This indicates that initial HCV RNA replication may ensure sustained infection of the host cell independently of the continuous integrity of the infecting HCV RNA molecule.

Assembly and release

Viral assembly represents the steps of arranging structural viral (glyco)proteins, and the genomic HCV RNA in order to form infectious viral particles (reviewed in Lindenbach 2013).

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. Previously it was reported that core protein molecules were able to self-assemble in vitro, yielding nucleocapsid-like particles. More recent findings suggest that viral assembly takes place within the ER (Gastaminza 2008) and that cytosolic lipid droplets (cLD) are involved in particle formation (Moradpour 1996, Barba 1997, Miyanari 2007, Shavinskaya 2007, Appel 2008). As one of the first steps of viral assembly it appears that newly synthesised HCV core molecules are relocated from the ER to cLD, where it homodimerises.

HCV NS5A is assumed to play a key role in discharging genomic HCV RNA from replication or translation to core-cLD-complexes (Appel 2008, Masaki 2008, Benga 2010).

Recent studies suggest that HCV NS2 as well as p7 may be coordinators of virion assembly via multiple interactions with several viral as well as host proteins, respectively (Jirasko 2010, Guo 2015). NS2 interacts with the viroporin p7. The resulting NS2-p7 complex is anchored in the ER-membrane with other domains localised in the cytosol. Subsequent cytosolic interaction of NS2-p7 with the NS3-NS4A enzyme complex is proposed to lead to detraction of core molecules from cLD to the site of budding into the ER (Counihan 2011) as well as to the packaging of genomic RNA. Finally, the NS2-p7 complex is presumably responsible for the transport from ER membrane-bound glycoproteins E1-E2 to the site of viral assembly. As a consequence all required components for HCV particle formation are now in close proximity and budding of the assembled structures into the ER occurs.

During the subsequent cellular secretory processes, HCV particles experience maturation. This includes post-translational glycan modification as well as refolding by the formation of several disulfide bonds (Vieyres 2010). Furthermore, at this stage interaction of HCV particles with lipoproteins is suggested to occur.

Finally, infectious HCV virions are secreted from the plasma membrane.

Model systems for HCV research

For a long time HCV research was limited due to a lack of small animal models and efficient cell culture systems. The development of the first HCV replicon system (HCV RNA molecule, or region of HCV RNA, that replicates autonomously from a single origin of replication) 10 years after the identification of HCV offered the opportunity to investigate the molecular biology of HCV infection in a standardised manner (Lohmann 1999).

HCV replicon systems. Using total RNA derived from the explanted liver of an individual chronically infected with HCV genotype 1b, the entire HCV ORF sequence was amplified and cloned in two overlapping fragments. The flanking NTRs were amplified and cloned separately and all fragments were assembled into 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, 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, were generated.

In vitro transcripts derived from these constructs without the genome region coding for the structural HCV proteins were used to transfect the hepatoma cell line Huh7 (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 Huh7 clones can replicate replicon-specific RNA in titres of approximately 108 positive-strand RNA copies per microgram total RNA. Moreover, all encoded HCV proteins are detected predominantly in the cytoplasm of the transfected Huh7 cells. The development of this replicon was a milestone in HCV research with regard to the investigation of HCV RNA replication and HCV protein analyses.

More recently, 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 to investigate the influence of the structural proteins on HCV replication. Thus it became possible to analyse the intracellular localisation of these proteins although viral assembly and release has not been achieved.

Another important milestone was reached 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 Huh7, such as HepG2 or HeLa were transfected efficiently with transcripts derived from the JFH-1 replicon (Date 2004, Kato 2005).

HCV pseudotype virus particles (HCVpp). The generation of retroviral pseudotypes bearing HCV E1 and E2 glycoproteins (HCVpp) offers the opportunity to investigate E1-E2-dependent HCVpp entry into Huh7 cells and primary human hepatocytes (Bartosch 2003a, Hsu 2003, Zhang 2004). In contrast to the HCV replicons where cells were transfected with HCV-specific synthetic RNA molecules, this method allows a detailed analysis of the early steps in the HCV life cycle, e.g., adsorption and viral entry.

Infectious HCV particles in cell culture (HCVcc). Transfection of Huh7 and ‘cured’ Huh7.5 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. Importantly, the secreted viral particles are infectious in cell culture (HCVcc) (Wakita 2005, Zhong 2005, Lindenbach 2005) as well as in chimeric mice with human liver grafts as well as in chimpanzees (Lindenbach 2006).

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. Huh7 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 and it is now possible to investigate the whole viral life cycle from viral adsorption to virion release. These studies will help to better understand the mechanisms of HCV pathogenesis and they significantly accelerate the development of HCV-specific antiviral compounds. Nevertheless, hepatoma cell lines do not represent primary human hepatocytes, the host cells of HCV in the liver. The most relevant biological differences between hepatoma cells and hepatocytes are the ongoing proliferation of hepatoma cells and some differences in cellular morphology. In contrast to hepatoma cells, primary hepatocytes are highly polarised cells that play an important role, e.g., in viral adsorption, entry, and release. Further efforts must be made to develop HCV replication systems reflecting in vivo conditions as realistically as possible.

Small animal models. Substantial progress was also achieved in establishing two mouse models for HCV infection via genetically humanised mice (Dorner 2011). In this experiment, immunocompetent mice were transduced using viral vectors containing the genetic information of four human proteins involved in adsorption and entry of HCV into hepatocytes (CD81, SR-BI, CLDN1, OCLN). This humanisation procedure enabled the authors to infect the transduced mice with HCV. Although this mouse model does not enable complete HCV replication in murine hepatocytes it will be useful to investigate the early steps of HCV infection in vivo. Moreover, the approach should be suitable for the evaluation of HCV entry inhibitors and vaccine candidates.

A second group of investigators have chosen another promising strategy for HCV-specific humanisation of mice. After depleting murine hepatocytes human CD34+ hematopoietic stem cells and hepatocyte progenitors were co-transplanted into transgenic mice leading to efficient engraftment of human leukocytes and hepatocytes, respectively (Washburn 2011). A portion of the humanised mice became infected with primary HCV isolates resulting in low-level HCV RNA in the murine liver. As a consequence HCV infection induced liver inflammation, hepatitis, and fibrosis. Furthermore, due to the co-transplantation of CD34+ human hematopoietic stem cells, an HCV-specific T cell immune response could be detected.

Both strategies are promising and have already delivered new insights into viral replication and the pathogenesis of HCV. However, the methods lack some important aspects and need to be improved. As soon as genetically humanised mice that are able to replicate HCV completely are created, they can be used for the investigation of HCV pathogenesis and HCV-specific immune responses. The Washburn method should be improved in order to achieve higher HCV replication rates. A reconstitution of functional human B cells would make this mouse model suitable to study the important HCV-specific antibody response.

Finally, a humanised mouse model that is able to produce infectious HCV accompanied by human-like HCV pathogenesis would be an ideal tool for preclinical monitoring of putative HCV-specific therapeutics and vaccines.

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