Monday, September 12, 2011

HCV Viral Structure

HCV is a member of the Flaviviridae family with marked structural and sequence similarities to members of the genera Pestivirus and Flavivirus and belongs to Hepacivirus, its own new genus. It is an enveloped virus of 45-65 nm observed by electron microscopy in human plasma, chimpanzee liver and experimentally infected and transfected cell lines (Moradpour et al., 2002 and Penin et al., 2004).

The virion is composed of a viral genome and a capsid-phosphorylated protein that is encased in a lipid bilayer (a low-density beta lipoprotein) coated with the envelope proteins (E1 and E2). The capsid is icosahedral formed by polymerization of the HCV core protein and influences the HCV gene expression and regulation while the envelope is responsible for HCV antibody reaction (Gowans, 2000 and Dubuisson et al., 2002).
HCV circulates in various forms in the serum of an infected host including (i) virions bound to very-low and low density lipoproteins which appear to represent the infectious fraction; (ii) virions bound to immunoglobulin and (iii) free virions (Wünschmann et al., 2000 and Maillard et al., 2001).
Genome (Figure I):
The viral genome is a positive-sense, single-stranded RNA genome approximately 10 kilo base pair long. The sequence comprises a 5' and 3' untranslated regions (5' UTR and 3' UTR), flanking a single open reading frame (ORF) encoding for a polyprotein of 3,010-3,033 amino acids (a.a.). The differences in length of the polyprotein are originated in variable regions in the E2 and non structural 5 A (NS5A) genes (Xu et al., 2003).

Consistent with the known functions of most Flavivirus proteins, the polyprotein undergoes post-translational cleavages to form functional viral proteins as follows: the N-terminal proteins, comprises three structural proteins (C, E1, and E2/NS1) that form the viral particle and the C-terminal proteins comprises six non structural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) required for HCV replication and maintenance of infection (Macdonald and Harris, 2004).

A second translation product, the F protein, is produced by ribosomal frame shift at codon 11 of the core sequence, thus F and core share the N-terminal 10 a.a. Little is known about the function of this protein, although it has been shown to localize to the endoplasmic reticulum (ER) (Roussel et al., 2003).

Parts of the genome

**The 5' untranslated region (5' UTR)
It is a non coding region of at least 341 bases which precedes the long ORF. It contains an internal ribosomal entry site (IRES) that has a unique structure consisting of four stem loop structures (I–IV). Functionally, it is required for efficient RNA replication (as RNA packing) and translation of the HCV ORF (Spahn et al., 2001 and Kikuchi et al., 2005).

The IRES resides approximately between nucleotides 40 and 355 and comprises most of the 5'UTR sequence that extends and includes 30-40 nucleotides (nt) of the core coding sequence. It mediates the translation of the ORF and the stem-loops I and II constitute a part of the minimal promoter for initiation of minus-strand RNA synthesis and replication (Friebe et al., 2001 and Fletcher et al., 2002).

5'UTR is the most highly conserved portion of the genome and thus has been used in most laboratories to develop sensitive detection assays for HCV RNA (Zein, 2000).
Figure (I) : Organization of the HCV genome.
A) It shows the 5' UTR and 3'UTR regions, the single ORF and its cleavage sites.
B) The relative sizes and functions of the resulting proteins (Simmonds, 2001).

**Structural Proteins
A)-Core region
It is the first 573 nt of HCV polyprotein with more than 90% homology conserved between different genotypes. It is involved in virus replication and in guiding protein translation (Simmonds, 2001).

As it is highly antigenic and containing several linear B cell epitopes near the N terminus, it induces specific cellular and humoral responses and could be used for antibody detection against HCV (Lai and Ware, 2000).

It regulates apoptosis of infected cells and might be directly implicated in the pathogenesis of liver disease, cell proliferation and liver cancer development (Ruggieri et al., 1997 and Alberti et al., 2004).

The core proteins, as well as NS5A, have also been reported to interfere with intra-cellular metabolism of lipids and of lipoproteins with a direct effect on the development of steatosis which is a characteristic feature of chronic hepatitis C infection (CHC) (Perlemuter et al., 2002).

B)-Envelop (E1 and E2) regions
Envelope proteins (E1 and E2) are highly glycosylated type 1 transmembrane proteins. E1 (gp 35) and E2 (gp70) genes are highly variable, typically differing at over 50% of sites between different genotypes (Deleersnyder et al., 1997 and Simmonds, 2001).
Moreover, E1 and E2 proteins most likely play a pivotal role in HCV life cycle; in the assembly of infectious particle and in the initiation of viral infection by binding to the cellular receptor(s) in hepatocytes and lymphoid cells (Takikawa et al., 2000 and Quinn et al., 2001).
The E2 (or E2/NS1 as some authors report) contains two hyper variable regions; hypervariable region 1 (HVR1) located directly downstream of the putative cleavage site between E1 and E2 and consists of 27 amino acids and hypervariable region 2 (HVR2) is much smaller of about 6 amino acids (Ray et al., 2000).
It has been proposed that HVR1 is the major determinant for strain-specific neutralizing antibody responses. Through its continual evolution during chronic infection, the virus population is able to evade host immune surveillance to maintain a persistent infection (Zein, 2000).
E2 mediates viral binding to the cells, as shown by a decrease of infectivity by incubation of the virus with anti-E2 antibodies. Also, it was shown that E2 interact with the interferon-induced double-stranded RNA-activated protein kinase (PKR) enzyme. Upon induction by interferon (IFN) during the treatment course, this enzyme reduces protein synthesis via phosphorylation of translation initiation factor, but in cells containing E2, PKR is inhibited, allowing continuation of translation in the presence of IFN (Taylor et al., 1999 and Lauer and Walker, 2001).
Protein 7 (P7), a hydrophobic polypeptide, is located between E2 and NS2 with its N-terminus is at a.a. 747. Subgenomic replicon studies have shown that it is not necessary for genome replication and its role in infectious virus production remains unknown (Pietschmann et al., 2001). However, its main role may be to mediate cation permeability across membranes and virion release or maturation (Pavlovic et al., 2003).
**Non-Structural proteins
1) NS2
It comprises with the amino terminus of NS3, the NS2-3 protease responsible for cleavage between NS2 and NS3. This is required for NS2 protein translocation (Lohmann et al., 2003).

2) NS3
It is a bifunctional molecule carrying in the amino-terminal ~180 residues, a serine-type proteinase responsible for cleavage at the NS3/4A, NS4A/B, NS4B/5A and NS5A/B sites and in the carboxy-terminal remainder, nucleoside triphosphatase (NTPase)/helicase activities essential for translation and replication of the HCV genome. Also, it has other activities involved in interference with host cell functions (Kolykhalov et al., 2000 and Friebe et al., 2001).

3) NS4A
Being an essential cofactor that activates the NS3 protease function by forming a heterodimer, it is required for efficient cleavage at the NS4B/5A site and polyprotein processing (Bartenschlager et al., 1995).

4) NS4B
It is a hydrophobic protein that has been suggested to function as an anchor to secure part of the HCV replication apparatus to the ER membrane (Lundin et al., 2003).

5) NS5A (Figure II)
It is generally accepted that NS5A is a pleiotropic protein with key roles in both viral RNA replication and modulation of the physiology of the host cell. It is a 446 a.a. (codon 1973 - 2419), which exists, in two distinct species, termed phosphoproteins 56 and 58 (p56 and p58) (Sarrazin et al., 2002 & Macdonald and Harris, 2004).

NS5A has many effects to regulate the cellular response to IFN. The C-terminal region of NS5A includes protein kinase R (PKR)-binding domain, a region of 66 a.a. of which the first 40 a.a. constitutes an INF sensitivity determining region (ISDR), whose mutations may confer sensitivity to IFN therapy, and adjacent 26 a.a. that may be related to the resistance to response.
The cellular PKR is an IFN-inducible double stranded RNA activated protein kinas (PKR) whose activity causes inhibition of translation and the signals implicated in cell proliferation, transformation and apoptosis by phosphorylating eukaryotic initiation factor 2 α. The NS5A binds to and inhibits it. Also, NS5A was found to induce production of IL-8 which in turn, partially inhibits the IFN-induced antiviral response in vito (Francois et al., 2000, Podevin et al., 2001 & Pavio and Lai 2003).

6) NS5B
As it contains the characteristic sequence, Gly-Asp-Asp, it encodes the RNA dependent RNA polymerase (RdRp) that is one of the subunits expected to participate in HCV replication. It has distinct biochemical activities participating in RNA synthesis. Hence, it is a prime target for anti HCV viral treatment (Kao et al., 2001 and Ranjith-Kumar et al., 2004).

3' untranslated region (3'UTR)
The 3' UTR is 200-235 nt long and has a tripartite structure composed of a variable sequence following the stop codon of the ORF. Its three distinct stretches are: (i) short, nonconserved region (from 27-66nt) that is virus genotype specific, (ii) variable-length poly (U) and polypyrimidine (UC) tract of (30-90nt), and (iii) almost completely conserved stretch of 98 nt at the 3' terminus that is termed region X, (+) for genomic RNA and (–) for complementary RNA strand generated during virus replication. The X region has no homologous sequence in other Flaviviruses (Macejak et al., 2001, Friebe and Bartenschlager, 2002 and Tallet-Lopez et al., 2003).

It is required for virus in vivo infectivity and replication, and it probably contributes to genome stability and translation efficiency (Dutkiewicz and Ciesiolka, 2005 and Kikuchi et al., 2005).

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