Hepatitis C virus entry: Role of host and viral factors

Infection, Genetics and Evolution 12 (2012) 1699–1709

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Review

Hepatitis C virus entry: Role of host and viral factors Baila Samreen b,1, Saba Khaliq a, Usman Ali Ashfaq c, Mahwish Khan b, Nadeem Afzal a, Muhammad Aiman Shahzad d, Sabeen Riaz d, Shah Jahan a,1,⇑ a

Department of Immunology, University of Health Sciences Lahore, Pakistan National Center of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan c Department of Bioinformatics and Biotechnology, Government College University Faisalabad (GCUF), Pakistan d Department of Microbiology and Molecular Genetics, University of the Punjab, Lahore, Pakistan b

a r t i c l e

i n f o

Article history: Received 17 February 2012 Received in revised form 13 July 2012 Accepted 16 July 2012 Available online 2 August 2012 Keywords: HCV HCV receptor HCV infection HCV therapeutic targets

a b s t r a c t Hepatitis C virus (HCV) has been considered to be a significant risk factor in developing liver associated diseases including hepatocellular carcinoma all over the world. HCV is an enveloped positive strand virus comprising a complex between genomic RNA and viral envelope glycoproteins (E1 and E2), which are anchored within host derived double-layered lipid membrane surrounding the nucleocapsid composed of several copies of core protein. HCV cell entry is the first step in infection and viral replication into host cells mainly hepatocytes. HCV cell entry is a complex process involving both the viral (envelope glycoproteins E1/E2) and host factors (cellular receptors and associated factors i.e. CD81, SR-BI, LDL-R, CLDN1, Occludin, DC-SIGN, L-SIGN and Glycosaminoglycans). Besides these the expression of certain other conditions such as polarization and EWI-2 expression inhibits the viral cell entry. Exploring the mechanism of HCV entry will help to better understand the viral life cycle and possible therapeutic targets against HCV infection including viral and host factors involved in this process. New strategies such as RNAi represents a new option for targeting the host or viral factors for prevention and therapeutic against HCV infection. In the current review we try to summarize the current knowledge about mechanism and interaction of cellular and viral factors involved in HCV cell entry and its implication as therapeutic target to inhibit HCV infection. Ó 2012 Elsevier B.V. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Model systems for the study of hepatitis C virus infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Viral proteins E1 and E2 mediating hepatitis C virus cell entry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cellular receptors and factors facilitating hepatitis C virus entry and target of siRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Cluster of differentiation 81 (CD81) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Low-density lipoprotein receptor (LDL-R) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Scavenger receptor class B type I (SR-BI). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Claudin 1 (CLDN 1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Occludin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6. DC-SIGN and L-SIGN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6.1. Other candidate receptors (NPC1L1 and CD5) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7. Glycosaminoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Association of hepatitis C virus with cells other than hepatocytes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. EWI-2wint – a host cell factor inhibiting hepatitis C virus entry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Polarization restricts hepatitis C virus entry into hepatocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: E1, E2, Envelop proteins 1, 2; HCC, Hepatocellular carcinoma; HCV, Hepatitis C virus; CD81, Cluster of differentiation 81; SR-BI, Scavenger receptor class B Type I; LDL-R, Low-density lipoprotein receptor; CLDN 1, Claudin 1; PEG-INF-a, pegylated interferon alpha; RNAi, RNA interference; shRNA, short hairpin RNA; siRNAs, small interfering RNAs. ⇑ Corresponding author. Address: Department of Immunology, In Charge Resource Lab, Department of Physiology, University of Health Sciences Lahore, Khayaban-e-Jamia Punjab, Lahore 54600, Pakistan. Tel.: +92 300 5081072. E-mail address: [email protected] (S. Jahan). 1 These authors contributed equally to this work. 1567-1348/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.meegid.2012.07.010

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6. 7. 8. 9.

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Potential mechanism of hepatitis C virus entry . . . . . . . . . . . . . . . . . . . . . . . . . Correlation of serum lipoprotein composition and hepatitis C virus infection Hepatitis C virus infection; gene silencing as therapy . . . . . . . . . . . . . . . . . . . . Conclusion and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Hepatitis C virus (HCV) infection is a global health problem, almost 2–3% of the world population an estimated 170 million people are infected with HCV worldwide (Machlin et al., 2011) and in Pakistan the prevalence of HCV is 6% (Memon et al., 2012). HCV infection leads to chronic hepatitis up to 60–80% and is associated with liver steatosis, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) (Bartenschlager et al., 2011). The variable effects of chronic infection among the individuals are due to the difference of their age, gender, immunity level and environmental healthcare but large proportions of patients develop severe form of liver disease like cirrhosis and hepatocellular carcinoma (Alter et al., 1992). In account of its adverse threat to life, existing therapies are inadequate and improvements of appropriate therapeutic and prophylactic vaccines are still considered to be a significant challenge in this era. Genome of HCV was identified 24 years ago; proficient information has been accumulated even though the difficulties in propagation of virus in cell culture. As for the other members of Flaviviridae family HCV is blood borne RNA-enveloped virus having genome of 9.6 kb, an overlapping +1 reading frame that encodes a single polyprotein of 3010 amino acids (Bartenschlager et al., 1993). Actually, two HCV ORFs have been identified; depending on genotypes, they include a polyprotein of 3000 aa encoded from an IRES in the 50 -UTR of the viral genome, and an ARFP/ F/core +1 protein resulting from a +1 frame shift overlapping the core-coding region. The open reading frame is flanked by two nontranslated regions (NTRs). The NTR is 341 nt in length and has at least two different functions: first, an internal ribosome entry site (IRES) directing cap-independent translation of the viral RNA; second, a promoter for initiation of (+) RNA synthesis (Friebe and Bartenschlager, 2009). On infection, this single polypeptide is processed by cellular as well as viral proteases to generate 10 proteins (Penin et al., 2004). The structural proteins Core, E1, E2/p7 are released after cleavage of polyprotein by host endoplasmic reticulum (ER) signal peptidase(s), whereas the non-structural proteins NS2, NS3, NS4A, NS4B, NS5A, NS5B are released after the enzymatic cleavage of HCV proteases NS2-3 and NS3-4A. HCV displays a high level of genetic heterogeneity which is associated mainly with natural error of RNA-dependent-RNA-polymerase due to lack of proof reading activity, facilitating high HCV replication rate in vivo. During replication, each progeny RNA genome contains mutations that lead to a continuous diversification of the viral population. Consequently, HCV circulates in vivo as a quasispecies, which divergent but closely related genomes subjected to a continuous process of genetic variation. This results in diverse population of viral variants known as quasispecies (Neumann et al., 1998). The large pool of variants provided by the quasispecies shows a great challenge for the control of HCV infection and has important biologic implications for viral persistence, host cell tropism, antiviral drug resistance, and development of an HCV vaccine. As the HCV infection progresses it may lead to insulin resistant (IR), liver cirrhosis, fibrosis and steatosis in substantial number of patients (Pekow et al., 2007).

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HCV virion an entire virus particle, consisting of an outer protein shell viral proteins, envelope glycoproteins (E1 and E2) are anchored within host derived double-layered lipid membrane surrounding the nucleocapsid as essential components of the virion for viral entry (Bartosch et al., 2003). A hallmark of HCV particles is their connection with cellular lipoproteins and lipids that determine both morphology and biophysical properties of the virion. Because in vivo liver cells and cultured human hepatoma cells can differ in their capability to produce lipoproteins, HCV particles vary in their properties, depending on the host cell in which they are produced (Bartenschlager et al., 2011) Besides viral glycoproteins cell surface receptors are also described as the potential HCV receptors having affinity with HCV envelope proteins i.e. CD81, SR-BI, LD-R, Claudins, occludins, L-Sign, D-SIGN and glycosaminoglycans (Agnello et al., 1999; Yang et al., 2008). Recent advances in cell culture models have significantly contributed to our understanding of the molecular virology of HCV infection, in particular the entry steps. In the present review we try to summarize the current information about HCV cell entry during infection, viral and cellular genes involved in this interaction and the possible mechanism involved.

2. Model systems for the study of hepatitis C virus infection  HCV infection system The study of the HCV cell entry mechanism has been quite complicated for a long time due to the absence of suitable animal model and competent in vitro cell culture system sustaining the complete HCV life cycle and enabling the production of infectious virus particles. So, several substitute models were considered for the study of viral entry mechanism before the development of in vitro cell culturing system that allows the reproduction of all steps of HCV replication cycle as well as viral entry pathway (Ashfaq et al., 2011a; Wakita et al., 2005).  Hepatitis C virus sub-genomic replicon The HCV replicon system replicates a modified HCV genome to high level Huh-7 Proteins encoded by the HCV genome. Replicon is either subgenomic (containing only the non-structural proteins for RNA replication) or genomic in length (contains the entire HCV genome). Both types of replicon contain the neomycin phosphotransferase gene for selection. Sub genomic replicon; have been extremely useful for the screening of chemical libraries for novel molecules with antiviral actions against HCV (Ashfaq et al., 2011a).  Infectious HCV virion Recent studies have led to the development of infectious HCV culture systems. Wakita and his colleague developed genotype 2a full length replicon (JFH-1) which was isolated from a Japanese patient with fulminant hepatitis. This HCV full length genome replicates efficiently and produce virus particle (HCVcc) in Huh-7 (Ashfaq et al., 2011a; Wakita et al., 2005). Chimaeric constructs of JFH-1 with the structural region of the J6 genotype 2a clone improved the infectivity (Ashfaq et al., 2011a; Wakita et al., 2005).

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 HCV producing pseudo particle (HCVpp) Recombinant E2 glycoprotein candidate receptor CD81 and human scavenger receptor class B (SR-BI/Cla1) were explored to be involved in HCV entry (Yang et al., 2008), also giving the evidence of interaction of E2 with heparin sulphate (HS) proteoglycans but these systems have limitations as naturally occurring E1 and E2 glycoproteins form hetrodimer on viral envelope and isolated E2 may behave differently (Barth et al., 2003). Further advancement introduced HCV pseudoparticles (HCVpp), which closely imitate the reliable HCV cell entry. This system is based on the production of lentiviral particles that incorporate unmodified HCV glycoproteins into the lipid envelope, but unfortunately it mimics only the early steps from particle binding to the liberation of capsid (Bartosch et al., 2003). Most importantly, unlike the natural cells, HCVpp are not associated with lipoproteins and do not synthesize them. Another model system of cell culture produced HCV (HCVcc) is based on particular genotype 2a virus strain, JFH-1 that is cloned from the serum of a patient with fulminant HCV. Subclones of human hepatoma cell line Huh-7 transfected with JFH-1 genome efficiently replicate the virus and secrete infectious particles (Wakita et al., 2005) but these system posses’ limitations as it is restricted to two cell lines (i.e. Huh7 and LH86), which have impaired lipoprotein metabolism. 3. Viral proteins E1 and E2 mediating hepatitis C virus cell entry E1 and E2 with molecular weights of 33–35 and 70–72 kDa, respectively are HCV envelop glycoproteins, which are type I membrane proteins necessary for viral entry and cell fusion (Nielsen et al., 2006). They contain a large N-terminal ectodomain of 160 and 334 amino acids and short C-terminal membrane domain. E1 and E2 form non-covalent heterodimers, which are the building blocks of the viral envelope (Wakita et al., 2005). As HCVpp show that these proteins form noncovalent heterodimer to be fully functional whereas E1 or E2 alone are non-infectious (Bartosch et al., 2003). The ectodomains of E1 and E2 translocate to the ER lumen by their transmembrane domains (TMDs) where they are modified by extensive N-linked glycosylation possessing up to well conserved 6 and 11 potential glycosylation sites, respectively (Goffard et al., 2005). These glycans have functional role in HCV entry and in defence against neutralization by envelop-specific antibodies (Fig. 1), as deletion or mutation of some glycosylation sites reduces the infectivity and at the same time mask the CD81 binding site, which inhibit HCV entry (Goffard et al., 2005; Helle et al., 2007). In addition to serve as membrane anchor, TMD posses a signal sequence in Cterminal that plays a major role in the localization of E1 and E2 to the ER and potentially involved in the assembly of the envelope proteins (Lavillette et al., 2007). Furthermore, the C-terminal

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sequences of E1 and E2 contain signals, which lead to a reinitiation of translocation in the ER lumen (Cocquerel et al., 2002). Interestingly, mutation of specific residues in the transmembrane domains of E1 and E2 involved in E1E2 heterodimerization play a major role in the fusion properties of these envelope glycoproteins, suggesting that these domains also play a major role in the viral entry into cell (Ciczora et al., 2007). The N terminus of E2 contains a hypervariable region called HVR1 (residues 384–410 aa) whose high variability may contribute to HCV escape from the immune response (von Hahn et al., 2007). Indeed, this region contains several basic amino acids, which modulate HCVpp infectivity (Callens et al., 2005), suggesting that this region may be involved in HCV entry. Despite variability in the sequences of HCV envelope glycoproteins, the glycosylation sites are highly conserved, and have been reported to play specific roles in HCV glycoprotein folding or in virus entry (Goffard et al., 2005). HCV E2 protein residues 384–661, a receptor binding domain, binds with high affinity to the large extracellular loop of CD81, a tetraspanin found on the surface of different cell types including hepatocytes and epithelial cells to play an important role in the early steps of viral entry (Cocquerel et al., 2003). Furthermore, it has been shown that E1 and E2 heterodimers have stronger CD81 interactions than E2, suggesting that E1 can modulate the binding of E2 to CD81 [64]. HCV envelope glycoproteins are potential target for the development of antiviral molecules that could block HCV entry. E1 and E2 play essential roles at different steps of the HCV life cycle such as virus entry and assembly of infectious particles. The function of E1 in HCV infection remains poorly understood, but it appears to be involved in intracytoplasmic virus-membrane fusion (Cocquerel et al., 2003; Lavillette et al., 2007). E2 glycoprotein takes part as a key component in the interaction between the virus and its major cellular receptors CD81and SR-BI/Cla1. E2 glycoprotein having HVR1 is involved in virus interaction with human SR-BI (SR-BI/Cla1) (Yang et al., 2008). This region contributes to virus escape from host immune response because of its high variability (von Hahn et al., 2007). Whereas antibodies directed against HVR-1 inhibit cell entry of HCVpp and cellular binding of HCV-LPs regardless of the high sequence variability, the conformation and highly conserved structural properties of HVR-1 suggesting that HVR1 is indeed functional (Bartosch et al., 2003; Penin et al., 2004). 4. Cellular receptors and factors facilitating hepatitis C virus entry and target of siRNA Besides envelope glycoproteins, host factors including Cellular receptors and its co-factors are involved in HCV entry into the cell making it a complex process and these receptors can be potential

Fig. 1. Schematic representation of HCV envelope glycoproteins E1 and E2. Glycosylation sites are indicated by N followed by the number of the site position in the sequence. Glycans involved in protein folding, virus entry or protections against neutralization are highlighted. Residues 420, 437, 438, 441, 442, 527, 530 and 535 are involved in the E2–CD81 interaction are indicated by arrows. TMD, transmembrane domain; HVR1; hyperariable region 1.

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therapeutic targets to inhibit HCV infection. As discussed above, entry and binding of HCV is believed to be a multi-step process involving interaction of several host cell surface molecules such as CD81, SRBI, LDL-R, CLDN (CLDN1, 6 and 9) with envelope glycoproteins to play key role in HCV entry. All these co-entry factors have been shown to play an important role for viral entry, several type of cell lines expressing all known entry factors are non permissive for HCV entry, signifying the existence of other cellular factors modulating viral entry. In recent years, several host restriction factors targeting various steps in viral life cycle including entry, replication and viral gene expression protecting cells from viral infection have been identified. 4.1. Cluster of differentiation 81 (CD81) CD81 is transmembrane protein member of tetraspanin family identified as a potential entry factor for HCV. Tetraspanins are type III membrane glycoproteins, producing 2 extracellular loops and a short intracellular loop. One of the 2 extracellular loops, the long extracellular loop (LEL) contains the marked structural feature of the tetraspanin family of proteins. There are disulfide bonds between the 4 cysteine residues in the LEL which form a sub-loop structure containing a region that is hypervariable between family members. They form structures on the plasma membrane called tetraspanin enriched microdomains (TEMS) having no intrinsic enzymatic activity. CD81 is first identified and best characterized HCV entry factor of tetraspanins, initially shown to interact with HCV envelop glycoproteins E2. Early studies utilized soluble E2 (sE2, lacking the transmembrane region) to identify CD81 as a HCV receptor due to the lack of an in vitro infectious system (Bartosch et al., 2003). Studies with sE2 have verified that the two disulphide bonds, which stabilize the loop domain, are required for the Lipoproteins and HCV cell entry interaction between sE2 and CD81 (Drummer et al., 2005). Furthermore E1E2 heterodimers have stronger CD81 interactions than sE2, suggesting that E1 can modulate the binding of E2 to CD81 (Cocquerel et al., 2003), whereas access to the CD81binding site on E2 is reduced by the presence of glycans at positions E2N1, E2N6 and E2N11 (Helle et al., 2007), suggesting that these glycans surround the CD81-binding site. An HCV E2 binding region maps to the LEL of CD81 (Bertaux and Dragic, 2006). Moreover, involvement of CD81 in HCV entry has been confirmed using the HCVpp and HCVcc systems (Burlone and Budkowska, 2009). Non-permissive human hepatoma cell lines such as HepG2 and HH29, which do not express CD81, become susceptible to HCVcc and HCVpp infection upon ectotopic expression of CD81 after transduction (Lavillette et al., 2007). Residues of CD81 involved in the interaction with E2 have been mapped in the LEL (Bertaux and Dragic, 2006; Drummer et al., 2005). Current studies elucidated the cellular pathways triggered by HCV binding to CD81 that plays a fundamental role in HCV infectivity. CD81 engagement activates the Raf/MEK/ERK signalling cascade, which affects post-entry steps of the virus life cycle (Brazzoli et al., 2008). CD81 may also play a role in the modulation of the adaptive immune response through virus interaction with CD81 on T and B cells that leads to viral persistence, liver pathogenesis and, polyclonal activation of B cells, to extra hepatic manifestations frequently observed in chronic hepatitis C patients (Machida et al., 2005). 4.2. Low-density lipoprotein receptor (LDL-R) LDL-R is potentially involved in the uptake of lipoproteinassociated HCV into hepatocytes as serum fraction composed of HCV with LDL, or very low-density lipoprotein (VLDL), was involved in binding to the LDL receptor (Agnello et al., 1999).

Several studies demonstrated that other members of the Flavivirus family also employ the LDL-R for viral entry. LDL-R is a modular protein of 839 amino acids consisting of seven adjacent LDL receptor type-A modules at N-terminal end, an YWTD domain, a serine and threonine rich region, a transmembrane region, and a 50 residue cytoplasmic tail (Beglova and Blacklow, 2005). Confirmation of LDL-R importance in HCV infection was gathered by studies done with primary human hepatocytes. LDL is most important ligand for LDL-R, which is dependable for the transportation of most of the plasma cholesterol. Treatment of hepatocytes with monoclonal antibodies against LDL-R or LDL also inhibits HCV infection with a strong correlation between the accumulation of HCV RNA into primary hepatocytes, expression of LDL-R mRNA (Molina et al., 2008). These conclusions and the relationship of HCV particles with lipoproteins suggest a role for LDL-R as a cellular receptor for HCV. In hepatoma cell lines, Lipo-viro-particles of HCV can enter through LDL-R as it recognizes apoB and apoE on the particles (Huang et al., 2007). The inhibition of HCVcc entry by anti-ApoE or -ApoB antibodies is another potential dispute in support of the role of LDL-R in HCV entry (Andreo et al., 2007). Moreover, a correlation of cell surface expression of LDL-R in patients with chronic HCV infection with a high viral load implies that LDL-R is actually involved in the viral replication (Andreo et al., 2007). From all information that has been accumulated, the LDL-R is probably an attachment factor for the lipoproteins associated with HCV. 4.3. Scavenger receptor class B type I (SR-BI) SR-BI is also called CLA-1, expressed in variety of mammalian cells but frequently expressed in liver and steroidogenic tissues. It’s a glycoprotein of 509 amino acids with 2 cytoplasmic domains, 2 transmembrane domains and a large extracellular loop with 9 potential N-glycosylation sites (Acton et al., 1994). SR-BI serves as a ‘‘multi-ligand’’ receptor for various classes of lipoprotein that is high, low and very low density lipoproteins (HDL, LDL and VLDL, respectively) as well as for lipoproteins, which are chemically modified like oxidized and acetylated LDL. SR-BI posses particular binding sites that can independently interact with their respective ligands as well as may play role of an endocytic receptor. SR-BI arbitrate an arrangement of the plasma membrane, which serve as a major cholesterol provider and modifying organization of CD81 at the plasma membrane, consequently ‘boosting’ the permissiveness of cell to HCV infection. SR-BI has been known to interact with HCV glycoprotein E2 as an additional potential entry factor for the virus (Yang et al., 2008). The involvement of SR-BI in HCV entry has been confirmed with the HCVpp and HCVcc systems as incubation of Huh-7 cells with anti-SR-BI antibodies significantly decreases HCV entry (Bartosch et al., 2003). On the basis of its reactivity with sE2, SR-BI has been projected to act as a putative entry molecule of HCV (Yang et al., 2008). SR-BI binding to soluble recombinant form of HCV E2 glycoprotein (sE2) found to be species specific because mouse SR-BI does not bind to sE2. Furthermore HVR-1 of E2 is responsible for binding to SR-BI and deletion of HVR-1 retard the interaction between SR-BI and sE2, which results in reduced HCVpp infectivity (Bartosch et al., 2003). Several studies suggest that the presence of HVR1 on E2 is important for its interaction with SR-BI as deletion of HVR1 inhibit the enhancing effect of HDL on HCVpp entry (Bartosch et al., 2003; Callens et al., 2005). Interestingly, kinetics of inhibition with anti-CD81 and anti-SRBI antibodies revealed that SR-BI may act concurrently with CD81 and referred as ‘post binding’ receptor (Zeisel et al., 2007). Finally, SR-BI is able to modify the lipid composition of the plasma membrane and possibly the enhanced activity on HCV entry is the consequence of such modification facilitating some early step in

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the HCV life cycle. In line with this hypothesis, it has been shown that HDL accelerates HCVpp endocytosis (Dreux et al., 2009). Consolidate SR-BI binds to serum amyloid A (SAA), an acute phase protein produced by hepatocytes during infection, tissue damage or inflammation (Dreux et al., 2009). In Huh 7.5 cells, human SAA inhibits HCV cell entry suggesting a beneficial role for this protein in host defense (Cai et al., 2007). Additionally, HDL reduces the antiviral effects of SAA, signifying a relationship between SAA, HDL and HCV infectivity, may be due to the competition between HDL and SAA. The decrease expression of SR-BI on the cell surface has been recently reported using interferon thus restricting viral attachment and entry into hepatocytes (Cormier et al., 2004). These data highlight the key role of SR-BI in cellular infection by HCV. 4.4. Claudin 1 (CLDN 1) Since the arrival of the HCVpp system, it had been noticed that expression of the known putative receptor CD81 and SR-BI were not enough to support HCV entry, which led to the hypothesis that there must be an additional factors required (Bartosch et al., 2003). The tight junction protein Claudin-1 (CLDN1) and Occludin have been recently identified as additional entry factors for HCV (Ploss et al., 2009). Claudin constitute the backbone of epithelial tight junction, which take a part apical and basolateral membrane compartments (Furuse and Tsukita, 2006). Predominantly CLDN1 expressed in liver and also in all epithelial tissues forming a network of tight junction (Furuse and Tsukita, 2006). This molecule is composed of 211 amino acid with 2 extracellular loops, 4 transmembrane segments and 3 intracellular domains, where the highly conserved domain in the first extracellular loop (EL1) seem to be implicated in HCV entry (Evans et al., 2007). However there is no sequence homology between tetraspanins and claudin. The region of CLDN1 involved in HCV entry corresponds to the first extracellular loop, particularly residues I32 and E48 (Evans et al., 2007). Functional studies revealed that CLDN1 plays important role in the post-binding of infection, following to HCV binding to CD81 and binding to SR-BI (Evans et al., 2007). Two other members of Claudin family are also known to mediate HCV entry (i.e. CLDN6 and CLDN9) (Zheng et al., 2007). Unlike CLDN1, these molecules expressed in liver cells as well as in peripheral blood mononuclear cells which provides another replication site to HCV in addition to hepatocytes. They contain highly conserved EL1 region that has a significant sequence homology with CLDN1 (Zheng et al., 2007). Current studies indicate that the distribution of CLDN1 in tight junctions correlates with the permissiveness to HCV infection (Liu et al., 2009; Yang et al., 2008), thus confirming that the localization of CLDN1 tight junctions is significant for viral entry and cellular tropism of HCV. In non-hepatic cell lines like 293T and SW13, expression of CLDN1 confers the susceptibility to HCVpp infection (Evans et al., 2007). However siRNA and shRNA interference targeting CLDN1 and occludin, confirmed the reduction of expression of both of these molecules along with inhibition of HCVpp and HCVcc cell entry (Lavillette et al., 2007). Likewise silencing of CLDN1 inhibits HCV infection in susceptible cells (Huh7.5) (Evans et al., 2007). While CLDN1 had never been reported to show a direct interaction with envelop glycoproteins of HCV particles (Evans et al., 2007; Zheng et al., 2007).

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transmembrane regions, 2 extracellular loops and N- and Cterminal region. The polypeptide of occludin is delivered to the plasma membrane in microtubule and temperature dependent manner, Occludin is known to take part in both cell–cell adhesion in the paracellular space and anchoring of the junctional complex to the cytoskeleton of polarized epithelial cells. Confocal microscopic analyses revealed that occludin accumulates in ER and co-localizes with the E2 protein of HCV (Benedicto et al., 2008). Further association of E2-occludin using JFH1 variant was confirmed by co-immuno precipitation and pull down assays (Benedicto et al., 2008; Liu et al., 2009). It has been experimentally proved recently that human occludin render murine cell infectable with HCVpp (Ploss et al., 2009). This information suggests that occludin directly interacts with E2 and facilitates viral entry through hepatocytes tight junctions, requiring a delicate molecular architecture of tight junction proteins. This is a remarkable observation that HCV infection may result in altered localization of tight junction proteins (Benedicto et al., 2008; Liu et al., 2009). These studies reveal that HCV infection leads to the remarkable reduction of tight junction proteins in HCV infected cells. Indeed, tight junction proteins are important in maintaining the polarity of hepatocytes so the altered expression of tight junction proteins lead to various reported symptoms including Cholestatic disorders. 4.6. DC-SIGN and L-SIGN DC-SIGN (dendritic cell specific intercellular adhesion molecule-3-grabbing nonintegrin; CD209) and L-SIGN (DC-SIGNR; liver and lymph node specific; CD209L), which are homo tetrameric type II membrane proteins belong to C type lectin family, are known to function as capture receptors for several viruses in cell entry pathway including HIV type I (HIV-I) (Geijtenbeek et al., 2000). DC-SIGN is expressed on Kuppfer cells, Denderitic Cells and lymphocytes while L-SIGN is expressed in liver sinusoidal endothelial cells. Both of them contain calcium dependent carbohydrate recognition domain (CRD) in their extracellular C-terminal region and membrane-proximal heptade-repeat region for oligomerization (Geijtenbeek et al., 2000). Through this CRD, DC-SIGN and L-SIGN are involved in capturing viral particles, binding, internalization and elimination of variety of pathogens (Engering et al., 2002). DC-SIGN and L-SIGN have been demonstrated to bind specifically with recombinant soluble sE2 of HCV, natural viruses from patient sera (Gardner et al., 2003), and retrovirus pseudo typed HCV particles with strong affinity through high mannose type oligosachrides. HCV captured by SIGN molecules depending on CRD, indicates that recognition of this mannose oligosaccharides in the viral envelop glycoproteins is crucial and studies shown that anti-L-SIGN and anti-DC-SIGN mAbs recognizing CRD of SIGN receptors as well as mannan, inhibit soluble E2 and HCV capture and thus confirming that DC-SIGN and L-SIGN serve as capture receptors capable of transmitting the virus to permissive cells and may play role in promotion of transinfection of human liver cells and tissue tropism (Gardner et al., 2003). Moreover, several studies revealed that primary human DC mediate transinfection of target cells by a DC-SIGN-dependent mechanism. while, L-SIGN+ liver sinusoidal epithelial cells (LSEC) may facilitate infection of hepatocytes, whereas DC-SIGN+ dedritic cells (DC) may transmit HCV to hepatocytes as well as subpopulations of B and/or T lymphocytes (Cormier et al., 2004).

4.5. Occludin Current studies have given an important clue about another transmembrane element that is Occludin, structurally related to Claudins involve in HCV cell entry and initiation of HCV infection (Lavillette et al., 2007). Occludin is a protein of 60 kDa with 4

4.6.1. Other candidate receptors (NPC1L1 and CD5) Sainz et al. (2012) has not only identified Niemann-Pick C1–like 1 (NPC1L1) as an HCV cell entry factor but also discovered it as a new antiviral target and potential therapeutic agent. Viral entry represents a potential multifaceted target for antiviral intervention;

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however, to date, FDA-approved inhibitors of HCV cell entry are unavailable. Now it has been showed that the cellular NPC1L1 receptor is an HCV entry factor and target for therapeutic intervention (Ray, 2012). Specifically, NPC1L1 expression is necessary for HCV infection, as silencing or antibody-mediated blocking of NPC1L1 impairs cell culture-derived HCV (HCVcc) infection initiation (Sainz et al., 2012). T cell susceptibility to HCV needs CD5, a lymphocyte-specific glycoprotein belong to the scavenger receptor cysteine-rich family. Inhibition of T cell CD5 with antibody or silencing with specific siRNA decreased cell susceptibility to HCV, while increasing CD5 expression by mitogen stimulation had the reverse effect (Sarhan et al., 2012). Moreover, transfection of naturally CD5-deficient HEK-293 fibroblasts with CD5 facilitated HCV infection. In contrast to T cells, hepatocytes do not express CD5 which shows that CD5 plays important role in HCV entry into human T cells (Sarhan et al., 2012). (Mizuochi et al., 2009) demonstrated that peripheral CD5 () B cells were more susceptible to apoptosis than CD5 (+) B cells in CHC it means that peripheral CD5 (+) are more susceptible to HCV infection which leads to CHC. The over expression of CD81 and the expansion of the population of CD5(+) peripheral B cells in HCV-infected patients shows involvement of CD 5 with CD 81 receptor in HCV infection (Zuckerman et al., 2002). 4.7. Glycosaminoglycans Glycosaminoglycans are present at the surface of cells and seem to be an initial harboring site for HCV attachment and other viruses of Flaviviridae (Barth et al., 2003;Yang et al., 2008). Virus initially attach to host cells, binds to these specific entry factors, which are responsible for initiation of series of events that in due course lead to the release of viral genome into the cytosol. There are several different types of GAG e.g. chrondroitin sulfate, dermatansulfate, keratan sulfate, heparan sulfate, heparinand hyaluronan. Several authors by using different model systems like sE2, HCVpps, HCVccs and virus isolated from plasma have shown that heparin (heparin sulphate homolog) and heprinase, enzyme able to degrade heparin sulphates at the surface of cell, inhibit HCV attachment to target cells (Barth et al., 2003). Though, other GAGs do not exhibit any inhibitory activity. Intracellular forms of sE2 are known to have strong affinity for heparin and HVR1 has been anticipated as essential region for this particular interaction (Barth et al., 2003). However, no interaction was observed between E2 and heparin in case of E1E2 heterodimers, isolated from HCVpps (Callens et al., 2005), and proposing that there is no heparin binding domain of E2 accessible on the functional heterodimer. Further experiments need to be done in order to comprehend the role of GAGs in HCV entry by using similar framework of envelop glycoproteins isolated from HCVccs. Although it is captivating that no heparin binding motif has been identified in the sequence of E2, while it is possible that such motif is formed at the surface of the folded E2 protein. Ultimately it could be put forward that HCV interacts indirectly with GAGs e.g. through lipoproteins associated with HCV particles. Recently it has been shown that lipoprotein lipase play an indirect role in the interaction between HCV and GAGs (Andreo et al., 2007). 5. Association of hepatitis C virus with cells other than hepatocytes HCV infects hepatocytes, B cells, T cells, and monocytes through CD81 and several receptor candidates, indicating that these types of cells are potential targets of HCV infection (Kondo et al., 2012). Replication of the hepatitis C virus (HCV) in peripheral blood mononuclear cells (PBMC), particularly B-cells, may impair immune functions and establish persistent infection (Ito et al., 2011; Panasiuk et al., 2009). HCV infects hepatocytes, B cells, T

cells, and monocytes through CD81 and several receptor candidates, indicating that these types of cells are potential targets of HCV infection (Kondo et al., 2012). Replication of the hepatitis C virus (HCV) in peripheral blood mononuclear cells (PBMC), particularly B-cells, may impair immune functions and establish persistent infection (Ito et al., 2011; Panasiuk et al., 2009). More than 175 million people worldwide are infected with hepatitis C virus (HCV), a positive-strand RNA virus that infects both hepatocytes and peripheral blood mononuclear cells (Kasama et al., 2010). 5.1. EWI-2wint – a host cell factor inhibiting hepatitis C virus entry Certainly, CD81 is a putative viral entry receptor having two major partners; EWI-F (also called CD9P-1, FPRP or CD315) and EWI-2 (also called PGRL, IgSF8 or CD316) (Kolesnikova et al., 2004). Rocha-Perugini et al., 2008 first identified a cleavage product of EWI-2, which associates with CD81 and inhibits its interaction with the HCV envelope glycoproteins. Most importantly, this molecule EWI-2wint (EWI-2 without its N-terminus) has an inhibitory effect on HCV entry, highlighting a potential new mechanism for the regulation of cellular invasion by this pathogen (RochaPerugini et al., 2008). EWI-2 wint is expressed in several cell lines but not in hepatocytes. Ectopic expression of EWI-2 wint in a hepatoma cell line susceptible to HCV infection blocked viral entry by inhibiting the interaction between the HCV envelope glycoproteins and CD81. By doing co-immunoprecipitation studies it has been concluded that EWI-2wint may significantly inhibit HCV entry by reducing E1E2–CD81 interactions in non-HCV-permissive cell lines but not in primary human hepatocytes, Huh7.5, as well as in Huh-7 cells for a number of possible reasons (Rocha-Perugini et al., 2008). Likewise, EWI-2wint may reduce CD81 accessibility to envelope glycoproteins by steric hindrance. Otherwise, the association of EWI-2wint with CD81 may induce conformational modifications in CD81, which blocks the binding of HCV heterodimers (Rocha-Perugini et al., 2008). CD81 may be potentially required for a post-binding step such as escorting the particle into the endocytic pathway or priming it for the pH triggered fusion mechanism. The association of EWI-2wint with CD81 could block such entry stages. EWI-2wint may exert its inhibitory effect on CD81 functionality in the endosomes. Furthermore, EWI-2wint may interfere with actin polymerization potentially required for HCV entry as HCV replication requires microtubule and actin polymerization and CD81 engagement leads to actin rearrangement (Rocha-Perugini et al., 2008). Although EWI-2wint expressed in different cell lines but absent in hepatocytes, it is not yet known either this expression profile is due to a differential expression of the protease responsible for EWI-2wint production or if the accessibility of EWI-2 to protease is cell type specific. In conclusion, absence of this natural inhibitor of CD81 in hepatic cells may thus facilitate virus entry and contribute to the hepatotropism of HCV (Burlone and Budkowska, 2009). 5.2. Polarization restricts hepatitis C virus entry into hepatocytes Polarity is characterized by a specific organization of plasma membrane proteins and defines the shape and architecture of a cell. Hepatocytes polarity requires the coordinated establishment and maintenance of TJs and apical membrane domains (Konopka et al., 2007). It has been demonstrated that disrupting epithelial barrier formation of simple polarity consisting cells increased HCV entry, suggesting that TJs impose a physical barrier and restrict viral access to receptors (Mee et al., 2009). The majority of immortalized hepatocyte-derived cell lines and primary hepatocytes dedifferentiate in culture and fail to demonstrate a complex polarized phenotype. However, several studies have reported that

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the human HepG2 hepatoblastoma line develops hepatic polarity in culture, forming apical cysts that are equivalent to as in the liver (Mee et al., 2009). As HepG2 cells polarize, discrete pools of CLDN1 at the TJ and basal/lateral membranes develop, consistent with the pattern of receptor in liver tissue. Mee et al., 2009 demonstrate that HepG2 polarization limits HCV entry by treating cells with PKA agonist, OSM, and Rho kinase inhibitor, which stimulate polarization via TJ and nonjunctional pools of CLDN1 with an altered association with CD81. Overall, the complex polarity limits HCV entry, suggesting that agents who disrupt hepatocytes polarity may promote HCV infection and transmission within the liver.

CD81 and may be after a lateral migration of the virus-receptor complex to the tight junctions. HCV entry thus shown to be controlled by the presence of various entry factors and probably also requires the absence of a specific inhibitory factor. The HCV entry process may be still more complex than already revealed. Further studies will also be necessary to understand the precise role of each entry factor in the HCV life cycle.

6. Potential mechanism of hepatitis C virus entry

HCV RNA replication and virion assembly largely depend on cholesterol metabolism and fatty acid biosynthetic pathways in host cells inducing large changes in cellular lipid metabolism such as abnormal serum lipoproteins levels (reduced) in chronic HCV infection and accumulation of lipids (steatosis) (Andre et al., 2005; Huang et al., 2007). HCV particles circulate in patient sera as a very heterogeneous population with a density between 1.03 and 1.34 g/ml. Most of the circulating HCV particles are of low density due to association with b-lipoproteins (Agnello et al., 1999). Only a very low density population of serum-derived HCV (sHCV) was highly infectious in chimpanzees and tissue culture cells, and LDL-R appeared to play an important role in infection (Agnello et al., 1999). Low-density, highly infectious HCV in the serum primarily corresponds to LVPs, which are lipoprotein-like structures composed of triglyceride-rich lipoproteins containing apolipoproteins, ApoB and ApoE, viral nucleocapsids and envelope glycoproteins (Nielsen et al., 2006). The specific infectivity of HCVcc produced in vitro and recovered from experimentally infected chimpanzees or uPA-SCID mice with human liver grafts is higher and buoyant density is lower than those of the virus produced in cell culture (Lindenbach et al., 2006). High-density (immature) HCV particles produced in vitro are actively degraded in a proteasome independent manner, whereas low-density, i.e. VLDL associated, HCV particles are efficiently secreted from infected cells (Gastaminza et al., 2008). In accordance with these observations, HCVcc infection in vitro could be efficiently inhibited not only by antibodies directed against the HCV envelope but also by antibodies against ApoB-containing lipoproteins (Andreo et al., 2007), supporting the essential role of serum lipoproteins in virus cell entry and higher viral infectivity. These findings provide an explanation for the presence of very-low-density, infectious virus

The modern advancement of functional models to analyze the early steps of HCV life cycle has led to the identification of numerous cell surface proteins or receptors involved in HCV entry. The information, which has recently been discussed above suggests that HCV entry is a complex multistep and time-consuming process. The exact function of each molecule that takes part in HCV entry remains to be determined, but current knowledge let us to draw a model of HCV entry pathway (Fig. 2). Glycosaminoglycans (GAGs) and the LDL-R may facilitate initial attachment to the host cell. This interaction is may be mediated by the lipoproteins associated with HCV virions (represented by a big grey coloured sphere in Fig. 3. However, direct contact between HCV envelope proteins and these cellular proteins cannot be excluded. After the initial binding step, HCV particles are expected to interact with SR-BI and CD81. Although the sequence of HCV, which show the interaction with these two entry factors has not been clearly determined, current understanding suggests that HCV particles may necessarily first contact with SR-BI before to interact with CD81. The interaction with SR-BI can likely be direct or indirect, by means of HCV-associated lipoproteins (Yang et al., 2008). Importantly, these early steps of HCV entry may altered by different components of the serum, which can facilitate (HDL) or inhibit [oxidized LDL (LDLox), lipoprotein lipase (LPL) and SAA] HCV infectivity. Furthermore, the presence of EWI-2wint, which expressed in several cell types can block the interaction of the viral particles with CD81, thus prevents these cells from being infected but absent in hepatocytes. As mentioned earlier, CLDN1 acts at a late stage of the entry course, after interactions with SR-BI and

7. Correlation of serum lipoprotein composition and hepatitis C virus infection

Fig. 2. A model of HCV entry. HCV attachment to the host cells may involve glycosaminoglycans (GAGs) and LDL-R due to association of the LDL which potentially play a role in the initial step of entry. After initial binding to the cell, the particle appears to interact with four other entry factors: SR-BI, the tetraspanin CD81, the tight junction proteins Claudin-1 (CLDN1) and Occludin. Clathrin-mediated endocytosis helps to internalize, the HCV particle. Whereas several serum components may inhibit HCV entry like SAA, oxidized LDL (LDLox) and lipoprotein lipase (LPL) have also shown in the figure. In contrast, HDL has been reported to facilitate HCV entry.

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Fig. 3. Therapeutic strategies against HCV based on RNAi Technology. RNAi technology, inducing gene silencing at posttranscriptional level mediated by siRNA, can be applied to prevent HCV infection by targeting either viral entry surface receptors, LDL-R, SR-BI, CD81, CLDN1, Occludin, HCV structural and non structural genes including UTRs and intracellular host factors as well that are involved in HCV replication and therefore silencing of these targets could be considered as a potential therapeutic modality.

particles circulating in patient sera and the role of lipoprotein receptors for virus cell entry.

8. Hepatitis C virus infection; gene silencing as therapy Recently most common way of HCV treatment is the administration of IFN-a and ribavirin. Genotype 2 and 3 respond better to this current therapy up to 80% but there are evidences that genotype 2 respond better than genotype1, reason is not known. This entire strategy show only 50% efficacy, led the scientist to discover other ways of treatment. For the past decade, only 40–45% of HCV patients achieved a SVR when treated with PEG-IFN/ RBV but now the treatment has revolutionized with the availability of the two protease inhibitors bocepervir and Boceprevir and telaprevir are the first two protease inhibitors available for the treatment of patients infected with hepatitis C virus (HCV) genotype 1. Boceprevir is a linear peptidomimetic ketoamide serine protease inhibitor that binds reversibly to the HCV nonstructural 3 (NS3) active site (Venkatraman et al., 2006). Like other protease inhibitors, boceprevir must be given with peginterferon-ribavirin to minimize the emergence of viral resistance (Sarrazin et al., 2007). RNAi induces gene silencing at a post-transcription level by double-stranded small interference RNA (siRNA) and correspond to an exciting new technology that has uses in the treatment of viral diseases. As HCV is an attractive target for RNAi therapy because it is a positive single stranded RNA that functions both as the viral messenger RNA and a template for RNA replication through a negative strand intermediate. Combined siRNA targeting either viral or host factors might be consider effective tools to extensively block HCV infection and replication. This is an advance method which knockdown both viral and cellular factors that may add improvement in the therapeutic efficacy. Combined siRNA have been designed against HCV genes or cellular genes which are involved in entry, replication and assembly of HCV. The IRES, located at the 50 non coding region, plays an important role to bind eukaryotic ribosomal subunits and starts the assembly of the translationally active 80S complex. This sequence is known to be more conserved than any other part of the viral genome, at least among the six known HCV genotypes. Thus, IRES considered as an ideal target for RNAi mediated anti-HCV therapy and several groups have experimented efficient inhibition of HCV replication by designing siRNAs towards this region (Ray and Kanda, 2009). Furthermore, shRNA targeted to the Envelop inhibited virus replication and infectivity titters

against HCV genotypes 1a, 2a and 3a (Khaliq et al., 2011a). Additionally, RNAi directed against the viral core, NS3, NS4B, NS5A and NS5B regions can suppress HCV infection. McCaffrey et al. (2002) was the first who reveal the feasibility of siRNA targeting HCV NS5B in vivo. By co-expression of an NS5B-luciferase fusion gene with an anti-NS5B siRNA expression plasmid and found a significant reduction of luciferase expression in the mouse liver indicating selective degradation by the NS5B siRNA. Liu et al. (2006) has designed several siRNA against two structural genes core and E2 of HCV 1b genome to explore the silencing of these two genes. Recently Khaliq et al. (2011) designed and explore the potential of synthetic siRNAs against E1 and E2 genes of HCV 3a genotype in serum infected cell line showing up to 70–80% inhibition in viral RNA. Recently, siRNAs targeting against HCV genotype 1a structural and non-structural genes showed efficient inhibition of HCV replication in cell culture (Ashfaq et al., 2011b). Numerous host cellular factors, such as CD81, SR-BI, HSP90, p68 or USP18 are also considered as targets for potentiating RNAi antiviral therapy (Nakagawa et al., 2007) (Fig. 3). CD81 expressed in most human cells and known to bind to HCV E2 protein, therefore it is considered as a putative receptor for HCV entry. Further investigation shown that density of cell surface-exposed CD81is a key determinant for HCV entry into host cells by observing their ectopic expression in Huh7-Lunet cells (low expression of CD81) or modulation of its cell surface density in Huh-7.5 cells (high expression of CD81) by RNAi (Koutsoudakis et al., 2007). siRNA-CD81 silencing distinctly inhibit (>90%) the HCV serum infection irrespective of HCV genotype and viral load (Molina et al., 2008). SRBI is mainly expressed in the liver and steroidogenic tissues and identified as another potential HCV receptor based on co-precipitation with recombinant E2. A 90% down-regulation of SR-BI expression in Huh7 cells by RNAi caused a 30–90% inhibition of HCVpp infection, depending on the HCV genotype (Evans et al., 2007). SR-BI-specific siRNA are also known to markedly reduce the susceptibility of human hepatoma cells to HCV infection at an entry step (Zeisel et al., 2007). But, either CD81 or SR-BI alone is not enough for virus binding, showing that at least one additional host protein must be required for cell entry of enveloped virions via the CD81/SR-BI pathways, that is recently identified co-receptor, Claudin-1 (Evans et al., 2007). Silencing of CLDN1 inhibits HCV infection in susceptible cells (Huh7.5) (Evans et al., 2007; Liu et al., 2009). Targeting CLDN1 and occludin by siRNA and shRNA interference confirmed that reduction of the expression of both of these molecules inhibited HCVpp and HCVcc cell entry [114]. HCV circulating in patient sera contains ApoE and

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ApoB as a part of VLDL and/or LDL and use LDL-R for entry (Nielsen et al., 2006). siRNAs targeting ApoB or ApoE efficiently inhibit the release of HCV and HCV infectivity is positively correlated with levels of secreted ApoE (Huang et al., 2007). Recently scientists have targeted that both entry and replication simultaneously using shRNAs directed against two regions of the HCV RNA and one region of the host cell receptor, CD81. The triple shRNA expression vector was effective in concurrently reducing HCV replication, CD81 expression, and E2 binding (Henry et al., 2006). RNA interference is advance method which knockdown both viral and cellular factors that may add improvement in the therapeutic efficacy (Jahan et al., 2011b). siRNA has been designed against only HCV genes or cellular genes involved in entry, replication and assembly of HCV (Jahan et al., 2011b). As we have described in detail that by inhibiting HCV receptors and HCV E2 genes separately and in combination we inhibited HCV entry into the cell (Jahan et al., 2011a,b). Furthermore, it is presumed to be future perspective of siRNA therapy using against HCV plus cellular genes and/or addition of drugs. Recent insights make it clear that siRNA faces some major hurdles before it can be used as a drug such as stability of the RNA molecules, effects other than the targeted mRNA suppression called as off-target effects related to a wide variety of immune and toxicity effects intrinsic to the RNAi itself or its delivery vehicle and interference with the endogenous miRNA machinery.

9. Conclusion and perspectives In the recent years, due to the development of new HCV model systems, the mechanism of HCV cell entry as a multi-step process has been extensively explored with interaction of several viral and cellular host factors. HCV enveloped virus enters the cells through a complex interaction of viral envelope proteins with cellular receptors CD81 and SR-BI and tight junction proteins CLDN1 and occludin, as co-receptors although still the exact events of cell entry are remains to be revealed. HCV E2 glycoprotein is directly involved in HCV cell entry, glycosylation sites of E2 are essential HCV binding to cellular receptors. The exact role of E1 is less defined, mostly due to the lack of more advance HCV model. Several reports including siRNA inhibition and antibodies against CD81 blocking HCV infection leave no doubt that CD81 is indeed the most essential component of HCV viral-receptor entry complex. Like CD81, antibodies and siRNAs against SR-BI inhibit HCVpp infection as the association with sE2 is disturbed. Lipoproteins are also involved in HCV entry through virus–receptor interaction and activating signalling pathways which help in viral particle internalization in association with LDL particles and ApoB and ApoE containing lipoproteins. HCV infection is initiated by the interaction between lipoproteins and lipoprotein receptors SR-Bi and LDL-R. In addition to cellular receptors cell surface glycans also facilitate post-binding roles in a lipoprotein-dependent manner. Besides all these factors some human cells remain HCV free in the presence of CD81, SR-BI, lipoproteins and tight junction proteins, suggesting that either additional cellular entry factors remain to be discovered or some inhibitory factors may also be present in those cells. EWI-2wint a cellular partner and natural inhibitor of CD81 also regulate the viral entry into the host cell by inhibiting the CD81. Moreover, it is speculated that the expression of this particle restricts the infection into other host cells as hepatocytes does not express EWI-1wint that’s why only liver cells gets infected with HCV. A detailed understanding of the HCV entry mechanism is essential for the development of new therapeutic strategies against HCV infection. Targeted strategies like RNAi against envelope glycoproteins, cellular receptors and co-receptors have been proposed as potential antiviral targets to block HCV cell

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entry. Future advances in developing more robust HCV models will greatly enhance our current knowledge of HCV life cycle and the processes involved in early cell entry.

Acknowledgement We are highly acknowledged HEC, CEMB and UHS family and all those whom names are not there but they helped us in this work.

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