Hepatitis C virus infections afflict more than 170 million people worldwide with the great majority of patients with acute hepatitis C developing chronic HCV infection. Infection ultimately leads to liver cirrhosis, hepatic failure or hepatocellular carcinoma, all of which are responsible for hundreds of thousands of deaths each year. HCV was identified in 1989 by immunoscreening an expression library with serum from a patient with post-transfusion non-A, non-B hepatitis. (Chevaliez et al., 2006) However, the virus was not visualized conclusively, the low viral titers in serum and liver tissue precluded biochemical characterization of native viral products and most importantly, it was not possible to grow the virus in cell culture, hence impeding elucidation of the viral lifecycle and the development of specific antiviral drugs and preventive vaccines.
Great progress has been made in the study of HCV over the past 18 years using heterologous expression systems, functional cDNA clones that are infectious in vivo in chimpanzees, a replicon system, pseudoparticles (engineered retroviral particles bearing functional HCV envelope proteins) that enable the study of viral entry under reproducible and conveniently measurable conditions and most importantly complete cell culture systems. (Chevaliez et al., 2006).
HCV Genome Classification
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The Flaviviridae family is divided into three genera: flavivirus, pestivirus and hepacivirus. HCV, with at least 6 genotypes and numerous subtypes is a member of the hepacivirus genus. The members of the Flaviviridae family share a number of basic structural and virological characteristics. They are all enveloped in a lipid bilayer in which two or more envelope proteins are anchored. The envelope surrounds the nucleocapsid, which is composed of multiple copies of a small basic protein, and contains the RNA genome. HCV harbors a positive stranded RNA genome composed of a 5' UnTranslated Region (UTR) which includes an internal ribosome entry site (IRES), an open reading frame that encodes structural and non-structural proteins and a 3' UTR. The HCV genome encodes a polyprotein with a length of over 3000 amino acids. (Thurner et al., 2004) This polyprotein is proteolytically cleaved to at least 10 viral gene products of which the structural proteins are encoded by the N-terminal part of the Open Reading Frame (ORF), whereas the remaining portion of the ORF codes for the non-structural proteins. Sequence motif-conserved RNA protease-helicase and RNA dependant-RNA polymerase (RdRp) are found at similar locations in the polyproteins of all of the Falviviridae. An eleventh protein, F, is also encoded by HCV through ribosomal frame shifting; the ORF for F resides in the Core region.
The structural proteins which form HCV particle include core protein and envelope glycoproteins E1 and E2. The non-structural proteins include the p7 ion channel, the NS2 protease, NS3 serine protease and RNA helicase, NS4A polypeptide, NS4B and NS5A proteins and NS5B RdRp. (Penin et al., 2004)
Figure1 The 9.7-kb positive-strand RNA genome is schematically depicted at the top. Simplified RNA secondary structures in the 5'- and 3' UTR and the core gene, as well as the NS5B stem-loop 3 cis-acting replication element (5B-SL3) are shown. Internal ribosome entry site (IRES)-mediated translation yields a polyprotein precursor that is processed into the mature structural and non-structural proteins. Solid diamonds denote cleavage sites of the HCV polyprotein precursor by the endoplasmic reticulum signal peptidase. The open diamond indicates further C-terminal processing of the core protein by signal peptide peptidase. Arrows indicate cleavages by the HCV NS2-3 and NS3-4A proteases. The dots in E1 and E2 indicate the glycosylation of the envelope. (Moradpour et al., 2007)
Replication of hepatitis C virus
General mode of entry of Flaviviridae
Members of Flaviviridae family like yellow fever virus, classical swine fever virus, dengue fever virus, upon entry onto host bind to one or more cellular receptors organized as a receptor complex trigger receptor mediated endocytosis. Fusion of the virion envelope with cellular membranes results in internalization of nucleocapsid in the cytoplasm of host cell. After, decapsidation, translation of viral genome occurs in cytoplasm, thereby leading to production of a precursor polyprotein, which is then cleaved by both cellular and viral proteases into structural and non-structural proteins (Penin et al., 2004). Replication of HCV genome is carried out by viral replication complex which is associated with cellular membranes. Viral replication occurs in cytoplasm. For this a full length negative stranded RNA is produced which is employed as a template to produce multiple copies of the positive stranded HCV genome. Progeny virions are assembled from cytoplasmic vesicles formed by budding through intracellular membranes. Finally mature virions are released into the extracellular environment by exocytosis.
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HCV particles are 40-70 nm in diameter. It is thought that core protein and the envelope glycoproteins E1 and E2 are key protein components of virion. E1 and E2 are anchored to a host cell derived double-layer lipid envelope which surrounds a nucleocapsid containing the genomic RNA and multiple copies of core protein. HCV circulates in various forms in infected host and can be associated with low density lipoproteins (LDL) and very low density lipoproteins (VLDL), both of which are thought to be the infectious fraction, HCV also circulates as virions bound to immunoglobulins as well as free virions (Moradpour et al., 2007).
Genome Organization and Function
HCV contains a 9.7kb positive strand RNA genome composed of a 5' UTR, a long open reading frame encoding a polyprotein precursor and a 3' UTR. Mechanisms regulating translation, replication and packaging of the viral genome have been studied but remain somewhat elusive. The 5' UTR is highly conserved and contains the IRES essential for cap-independent translation of viral RNA.HCV genome also contains four highly structured domains, which are composed of numerous stem-loops. Domain I is not required for IRES activity, but domains I and II are both essential for HCV RNA replication (Friebe et al., 2001).The 3' UTR is approximately 225 nt. It is organized in three regions: a variable region of approximately 30-40 nt, a long poly (U)-poly (U/UC) tract, and a highly conserved 3' terminal stretch of 98 nt (the X-tail).
Besides the 5' and 3' UTR an essential cis acting replication element (CRE) has been identified in the sequence that encodes the C-terminal region of the NS5B. The 5B-SL3.2 stem loop was identified within a larger cruciform RNA element, designated NS5B-SL3. The upper loop of 5B-SL3.2 was found to interact with the stem loop of the X-tail in the 3'UTR (Moradpour et al., 2007), hence suggesting that a pseudoknot is formed at the 3' end of the HCV genome which is essential for RNA replication (Figure1).
HCV core is a highly basic RNA-binding protein, which forms the viral capsid. Core contains three distinct domains which are as follows: the N-terminal hydrophilic domain, the C-terminal hydrophobic domain and a signal peptide which serves as a signal for downstream envelope protein E1 (Grakoui et al., 1993; Harada et al., 1991; Santolini et al., 1994). The Core protein can also be involved in regulating activity of cellular genes; is thought to induce hepatocellular carcinoma when expressed in transgenic mice (Moriya et al., 1998; Moriya et al., 1997). Core can also stimulate the formation of lipid droplets and play a role in steatosis (Barba et al., 1997).
The two envelope glycoproteins, E1 and E2 are essential components of the HCV viral envelope and are necessary for viral entry and fusion (Bartosch et al., 2003; Nielsen et al., 2004). E1 and E2 are transmembrane glycoproteins and their transmembrane domains are composed of two stretches of hydrophobic amino acids separated by a short polar region containing fully conserved charged residues. E1 and E2 have numerous functions like membrane anchoring, ER localization and heterodimer assembly (Cocquerel et al., 1998; Cocquerel et al., 2000). E2 plays a vital role in the initial stages of infection. Viral attachment is thought to be initiated by the interaction of E2 with one or several components of the receptor complex. Less is currently known about E1, but it is considered to be involved in intra-cytoplasmic virus-membrane fusion (Flint and McKeating, 2000; Rosa et al., 1996).
The F protein or ARFP (alternate reading frame protein) is generated as a result of a -2/+1 ribosomal frameshift in the N-terminal core ending region of the HCV polyprotein. However the exact translational mechanisms of this protein during HCV infection remain poorly understood. Although its role remains enigmatic it is thought to be involved in viral persistence (Baril and Brakier-Gingras, 2005).
p7 is a small polyprotein that has been shown to be an integral membrane protein (Carrere-Kremer et al., 2002). The p7 protein appears to be essential as mutations or deletions in its cytoplasmic loop suppresses infectivity of intra-liver transfection of HCV cDNA in chimpanzees (Sakai et al., 2003). In vitro studies suggested that p7 belongs to the viroporin family and likely acts as a calcium ion channel (Gonzalez and Carrasco, 2003).
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NS2 is a non-glycosylated transmembrane protein. It contains two internal signal sequences that are responsible for ER membrane association (Santolini et al., 1995; Yamaga and Ou, 2002). NS2 together with the amino terminal domain of the NS3 protein forms the NS2-3 protease. NS2 is a short lived protein that loses its protease activity after self cleavage from NS3. In addition to its protease activity, NS2 also interacts with various host cell proteins.
NS3 is a multifunctional viral protein which contains a serine protease domain and a helicase domain. NS4 serves as a co-factor of NS3 for its protease activity. NS3-4A has additional properties in which they interact with the host cell pathways and proteins that are important in the lifecycle and of infection (Pawlotsky., 2006).
NS4B is an integral membrane protein, and is predicted to harbor at least four transmembrane domains and an N-terminal amphipathic helix responsible for membrane association. Another function of NS4B is that it serves as a membrane anchor for formation of replisome (Egger et al., 2002; Elazer et al., 2004; Gretton et al., 2005).
NS5A is a phosphorylated zinc metalloprotein that plays an important role in virus replication and regulation of cellular pathways, it contains three domains I to III. Mutations in NS5A inhibit HCV replication (Elazer et al., 2003; Penin et al., 2004) and were abolished by alterations in the zinc binding site (Tellinghuisen et al., 2004).
The mechanism by which NS5A regulates HCV replication remain unclear. NS5A associates with lipid rafts derived from intracellular membranes. This interaction appears to be crucial for HCV replication complex formation (Gao et al., 2004). Level of phosphorylation also plays a vital role in the viral lifecycle by regulating a switch from replication to assembly. Furthermore NS5A can interact directly with NS5B but the mechanism by which the NS5A protein transforms the RdRp activity is poorly understood (Shimakami et al., 2004).
NS5B belongs to a class of membrane proteins termed tail anchored proteins (Ivashkina et al., 2002; Schmidt-Mende et al., 2001). Interactions between NS5B and cellular components have also been reported to play an important role in the formation of HCV replication complex (Gao et al., 2004; Schmidt-Mende et al., 2001).
The development of novel anti-HCV therapeutic agents has been hindered by the lack of an efficient in vitro viral infection system. Although significant progress has been made through genetic and biochemical approaches to better understand HCV replication but much more needs to be learned. HCV RdRp is an error prone enzyme, resulting in production of quasi-species (Pawlotsky., 2006).
The objective of this research is to better understand the function of Core and E1 proteins. Towards that end we have cloned their ORF's in an inducible expression system which will permit us to study the impact of their effect on signaling pathways. Overall, we aim to study the influence of their expression on phenotypes of cultured hepatocytes.