Network-layer Tools

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transmembrane proteins; claudins, occludins, junction associated molecules(JAMs) and the Coxsackie virus receptors(Greber and Gastaldelli,2007).

Claudin-1 is a new protein belonging to claudin gene family associated with HCV entry. The hepatocytes tight junction plays a major role in several liver functions including bile formation and secretion. They also regulate paracellular transport of solutes, water and ions. With the discovery of tight junction proteins as entry factors, the role of cell differentiation and polarization in the HCV cell entry process has become important.The dependence of HCV assembly on an active VLDL assembly suggests indirectly that cell differentiation is important for HCV production because VLDL assembly is a metabolic function that characterized differentiated hepatocytes.HCV infection has been shown to provoke downregulation of claudin-1 and occludins expression and to induce the polarization of infected hepatocytes with potential pathological consequences(Liu et al, 2009)

The actual role of CLDN-1 and OCLN in HCV entry remains unclear but interestingly direct interactions between HCV envelop glycoprotein and OCLN has been shown in the work of Benedicto et al, (2008).Furthermore, the knock down of OCLN in a cell to cell fusion assay where fusion activity depends on cell surface expression of the HCV glycoprotein complex diminishes fusion activity, suggesting that OCLN may be implied in the HCV fusion process(Cukierman et al,2009).

Two other members of claudin family, CLDN 6 and CLDN9 also mediate HCV entry(Meertens et al,2008), these molecules are expressed in the liver and also present in peripheral blood monocellular cells, hence providing another replication site in addition to the human hepatocytes (Zheng et al, 2007).

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Glycosaminoglycans exist in different forms but only highly sulphated GAGS in particular appears to be involved in the interaction process with several virus triggering their subsequent uptake and docking between HCV and GAGs.A high sulphated GAG,heparin and heparinase inhibits attachments to cells and treating cell with glycosidases reduces HCV infectivity (Basu et al, 2007).

However,the high sulphate GAGs binds sE2 with high affinity but studies with HCVpp which carries E1-E2 heterodimer on their surface fails to confirm these findings. These observations suggests that either the heparin binding site is not accessible on the functional E1-E2 heterodimer or the attachment of authentic HCV particle to cellular GAGs is mediated by lipoproteins associated with virus particle.

HCV can bind and enter the cells by GAGs dependent pathway due to the interaction of the virus lipoproteins with its lipase mediating cell entry(Andreo et al,2007).

2.3.5 Lectins: (DC-SIGN and L-SIGN)

Lectins are another class of molecules involves in cell binding and entry of several viruses.DSIGN and L-SIGN are homotetrameric type II proteins from the C-typelectin family. They are involved in the binding, internalization and elimination of a variety of pathogens(Cambi et al, 2005).

2.3.6 Mechanism of Viral Cell Entry

From recent studies, it is clear that the interaction between virus and the host cell during the first step of virus-host encounter is not just limited to the hospitality of the host cells to the virus resulting in cellular binding and entry. Virus-host interaction is a two way dialogue in which the virus takes advantage of the host cells owned signal transduction system to transmit signals to the cells (Smit and Helenius, 2004).These signals usually generated at the cell surface induces change to facilitate entry, prepares the cells for invasion and neutralizes the host defense mechanism.

HCV entry is first engage by the initial capture of the viral particles by attachment factors and receptors on the cell surface in a spatiotemporally regulated manner (Lefevre et al,2014). The initial attachment is mediated by heparin sulfate

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proteoglycans(HSPG) syndecan-1 or SR-B1 depending on the virion density. It was initially thought that HCV glycoprotein are responsible for the virion binding to HSPG or SR-B1,however more recent research data suggest that apoE could be involve in this contact(Dao Thi et al,2012 and Jiang et al,2013).

Figure 2.5: Hepatitis C virus entry into hepatocytes (Mirjam et al, 2011)

To infect a target cell, the viral particles needs to proceed through multistep entry process during which each step is tightly regulated in time and space as illustrated in figure 2.5. The steps involved in entry are viral attachment to cells, internalization and fusion of the viral particles with cellular membranes, the release of the viral genome into the host cell cytosol and the initiation of viral replication.

Upon infection, the HCV particles are transported via the blood stream and enter in contact with the hepatocytes after crossing the fenestrated endothelium of the liver sinusoids. Once present in the space where basolateral membranes of hepatocytes are exposed, it becomes significant.

As earlier discussed, the HCV entry is initiated by anchoring of the envelop glycoprotein to the cell surface thereby offering multiple novel targets for antiviral therapy. Multiple strategies evolved by the virus in order to escape from the host

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immune system have to be taken into account for the design of efficient novel antiviral strategies. Current approaches for HCV antiviral drugsdesigns should target viral enzyme interfering with HCV entry process. Also as the understanding of molecular mechanism underlying HCV interaction with the host cell is growing, it holds promise for future drug design.

Viral entry may be inhibited by blocking interaction between the virus and the target cell, interfering with post binding events, interfering with viral fusion. Viral proteins are recognized as foreign particles by the host immune system and induced the production of antibodies, small proportion of this antibodies exhibits antiviral potentials in vitro and are referred to as virus-neutralizing antibodies. These antibodies render thevirion non infectious by interfering with receptor binding and entry.Many successful vaccines are based on the induction of neutralizing antibodies.

Isolation and characterization of antibodies targeting distinct steps of HCV entry is an important strategy for protection against this virus and provides a rationale basis for the development of HCV vaccines.Antibody mediated neutralization occurs during HCV infection in vivo but the role of antibody in the control of HCV infection is not clear (Walker, 1999).Antibodies with HCV neutralizing properties was first described in experimental infection of chimpanzee, these antibodies were directed against epitome in the Hypervariable region (HVR) of the HCV glycoprotein E2.The presence of antibodies directed against HVR has also been associated with viral clearance in HCV infected humans(Zibert et al,1997).

Chapel et al,(2001) with his colleague carried out a study on HCV infected patients with primary antibody deficiency, the result showed that the antibody deficient patients have an accelerated rate of disease progression, establishing HCV infection despite the induction of the humoral immune response that targets various epitomes of the envelop glycoprotein.

Recently, a functional study analyzing the neutralizing antibody response during acute to chronic infection using HCV model system demonstrated a lack of neutralizing antibody in the majority of the patients with acute HCV infection.Other studies using

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HCVpp models systems demonstrated that neutralizing antibodies are induced in the early phase of infection by patients who subsequently clears the virus or controlled viral infection (Lavillette et al,2005), the result suggested that a strong early neutralizing antibody response may play a role in the outcome. HCV infected patients who do not clear the virus develop high viral titre and even cross neutralization antibodies during the chronic phase of the infection(Pestka et al,2007).