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Hepatitis C virus (HCV) infection afflicts a great deal of the worldwide population with majority of the patients diagnosed having acute hepatitis C, develop chronic HCV infection. Infection ultimately leads to liver cirrhosis, hepatic failure or hepatocellular carcinoma, all of which are accountable for thousands of deaths with each progressing year. HCV was recognized in 1989 by immunoscreening an expression library by extracting serum from a patient with post-transfusion non-A, non-B hepatitis. (Chevaliez et al., 2006) However, the virus was not visualized unquestionably, the low viral titers in serum and liver tissue barred biochemical classification of local viral products and most importantly, it was not possible to grow the virus in cell culture, hence hindering not only the exposition of the viral lifecycle as well as the development of explicit antiviral drugs and preventive vaccines.
Immense advancement has been made in the study of HCV over the past years by means of heterologous expression systems, practical cDNA clones that are infectious in vivo in chimpanzees, a replicon system, pseudoparticles (engineered retroviral particles bearing well-designed HCV envelope proteins) that facilitate the study of viral entry under reproducible and suitably measurable environment and most importantly complete cell culture systems. (Chevaliez et al., 2006).
1.1 HCV Genome Classification
The Flaviviridae family is categorized into three genera; flavivirus, pestivirus and hepacivirus. HCV, with six genotypes and several subtypes is a component of the hepacivirus genus. The constituents of the Flaviviridae family have many fundamental structural and virological characteristics in common. They are all sheathed in a lipid bilayer in which two or more envelope proteins are affixed. The envelope encircles the nucleocapsid, which is composed of numerous copies of a small basic protein, and comprises of the RNA genome. HCV harbors a positive stranded RNA genome comprising of a 5' UnTranslated Region (UTR) which consists of an internal ribosome entry site (IRES), an open reading frame that encodes both the structural and non-structural proteins and a 3' UTR. The HCV genome codes for 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 coded by the N-terminus of the Open Reading Frame (ORF), whereas the rest of the portion codes for non-structural proteins. The RNA protease-helicase and RNA dependant-RNA polymerase (RdRp) are found at similar positions in the polyprotein of all 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 whereas the non-structural proteins consist of 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)
Replication of hepatitis C virusFigure 1.1
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)
1.2 General mode of entry of Flaviviridae
Members of Flaviviridae family like yellow fever virus, classical swine fever virus, dengue fever virus, upon entrance into host bind to cellular receptors prearranged as a receptor complex and set off receptor arbitrated endocytosis. Union of virion envelope with cellular membranes leads to internalization of nucleocapsid in the cytoplasm of host cell. After, decapsidation, translation of viral genome takes place in the cytoplasm, hence leading to the development 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 done by viral replication complex which is linked with cellular membranes. Viral replication takes place 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 brought together from cytoplasmic vesicles shaped by budding through intracellular membranes. Finally mature virions are unconfined into the extracellular environment by the process of exocytosis.
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 affixed to a double-layer lipid envelope -derived from the host cell- which surrounds a nucleocapsid containing the genomic RNA and multiple copies of core protein. HCV circulates in a variety of forms in infected host and can be linked with low density lipoproteins (LDL) and very low density lipoproteins (VLDL), both of which are contemplated to be the infectious fraction. HCV also circulates as virions bound to immunoglobulins as well as free virions (Moradpour et al., 2007).
1.3 Genome Organization and Function
HCV contains a 9.7kb positive strand RNA genome consisting 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 about 225 nt in length. It is systematized into three regions: a variable region of approximately 30-40 nt, a long poly (U/UC) tract, and a extremely conserved 3' stretch at the end 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).
The core is an extremely 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 stimulate liver cancer 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 glycoproteins, E1 and E2 are vital components of the viral envelope and are required for viral admission and fusion (Bartosch et al., 2003; Nielsen et al., 2004). The transmembrane domains of the glycoproteins are composed of two stretches of hydrophobic amino acids which are partitioned by a small polar region consisting of fully conserved charged residues. E1 and E2 have various functions like membrane anchoring,localization in the ER and heterodimer assembly (Cocquerel et al., 1998; Cocquerel et al., 2000). E2 plays a crucial role in the initial phases of infection. The affixing of the viral particle is thought to be initiated by the interface of E2 with one or many constituents of the receptor complex (Rosa et al., 1996). Not much is at present known about E1, but it is thought-out to be involved in intra-cytoplasmic virus-membrane fusion (Flint and McKeating., 2000).
The F protein or alternate reading frame protein is generated as a result of a ribosomal frameshift in the N-terminus core terminating 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 considered to be involved in viral persistence (Baril., 2005).
p7 is a small protein 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 a deletion in its cytoplasmic loop represses (Sakai et al., 2003) infection of liver transfection of HCV cDNA in chimpanzees. In vitro studies propose that p7 belongs to the viroporin family and most likely operates as a calcium ion channel (Gonzalez., 2003).
NS2 is a non-glycosylated transmembrane protein. It consists of two internal signal sequences that play a role in ER membrane association (Yamaga., 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 proteolytic action as a result of 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 foreseen to comprise of at least four transmembrane domains and an N-terminus 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 zinc metalloprotein which is phosphorylated and 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 eliminated by changes in the zinc binding site (Tellinghuisen et al., 2004).
The mechanism by which NS5A regulates HCV replication remains unclear. NS5A connects with lipid rafts obtained from intracellular membranes. This interaction appears to be crucial for HCV replication complex formation (Gao et al., 2004). The degree of phosphorylation also plays
a crucial role in the viral lifecycle by controlling a switch from the replication process to assembly. Moreover, (Shimakami et al., 2004) NS5A can interrelate with NS5B but the means by which the NS5A protein transforms the RdRp action is poorly understood.
NS5B belongs to a class termed tail anchored proteins (Schmidt-Mende et al., 2001). Connections between NS5B and cellular constituents have been accounted to play a vital role in the development of HCV replication complex (Schmidt-Mende et al., 2001).
The production of fresh new anti-HCV agents has been stalled by the lack of an efficient in vitro viral infection model. Although momentous advancement has been made through various approaches in order 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.