Hepatitis c virus

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INTRODUCTION

Hepatitis C virus (HCV) infection affects a large number of patients worldwide. HCV is a major cause of chronic liver disease and affects over 270 million individuals worldwide (Wilson JA et al., 2003). HCV is believed to be more prevalent than hepatitis B virus infection (HBV) (Cooreman et al., 1996). It is estimated that 40-60% of infected individuals progress to liver disease, including cirrhosis and hepatocellular carcinoma (Alter, 1997; Bradley, 2000). Currently, the available treatment for hepatitis C patients is alpha interferon (IFN-a) alone or in combination with ribavirin. However, there is still no cure for a large proportion of patients, because there is a significant variation among different HCV genotypes as well as sub types (McHutchison et al., 2003). Thus, alternative therapeutic approaches for hepatitis C are needed. Some have reported that RNA interference represents a promising treatment option for HCV infections (Kapadia et al., 2003; Randall et al., 2003; Seo et al., 2003; Wilson et al., 2003).

Small interfering RNAs (siRNAs) can be used as potential therapeutic agent, because HCV replicates in the cytoplasm of liver cells without integration into the host genome, and because the HCV genome is a single-stranded RNA that functions as both an mRNA and as a replication template, destruction of HCV RNA could eliminate not only virally directed protein synthesis, but also viral replication. Furthermore, because replication depends on a (-)-strand template as a replication intermediate, both (+)- and (-)-strands are potential targets' for RNA interference (RNAi). In tissue culture, siRNA directed against the viral genome, effectively blocked the replication of viral replicons (Kronke et al., 2004; Kapadia et al., 2003; Yokota et al., 2003; Randall et al., 2003). Naturally occurring siRNAs are small RNA duplexes approximately 21 nucleotides long (Elbashir et al., 2001a) with characteristic termini involving nucleotide 3'-overhangs and 5'-phosphate groups. These termini allow for incorporation of siRNA into protein complexes that regulate gene expression in a sequence-specific manner (Elbashir et al., 2001a; Elbashir et al., 2001b). In its natural form, RNAi is a cellular defense process that protects against genome-invading transposable genetic elements and viruses (Fire et al., 1998). The siRNAs are short enough to bypass the Interferon (IFN) system in mammalian cells, and are incorporated directly into the RISC complex that targets sequence-specific mRNA for destruction (Elbashir et al., 2001c; Caplen et al., 2001; Hamilton et al., 2002). Simeoni et al demonstrated the successful delivery of siRNAs into the cytoplasm, that promoted the down regulation of target mRNA by RNAi (Simeoni et al., 2003; Simeoni et al., 2005). The HCV life-cycle is entirely cytoplasmic. Replication occurs through a minus (-)-strand intermediate in a membrane-bound compartment (Moradpour et al., 2004), yielding dsRNA intermediates. As an RNA virus, HCV is a prime candidate for RNAi.

In present study, gene expression will be blocked from HCV replicons that will inhibit its replication. Several sequences will be discovered that can be effectively targeted by different siRNAs, in mammalian cells, achieving the better inhibition rates. Specific inhibitory effects of siRNAs will be observed, in vitro and their dose responsiveness and effective duration. Ten expression plasmids will be constructed expressing all individual HCV genes. Each will be targeted by different siRNAs against each genes separately and also in different combinations. Double-stranded siRNA Molecules, designed to target all individual HCV genes will be cotransfected along with the plasmid having HCV gene into a human hepatoma cell line (Huh-7). Effective siRNAs will be screened by measuring the expression of all genes by RT-PCR and Western Blot analysis.

Our research may suggest that siRNA will represent a new approach for the treatment of persistent HCV infection.

LITERATURE REVIEW

HCV infection has reached epidemic proportions. HCV has infected an estimated 270 million people worldwide (Wilson JA et al., 2003), and HCV is believed to be more prevalent than hepatitis B virus infection (HBV) (Cooreman et al., 1996). HCV is the leading reason for liver transplantation. The development of vaccine has been hampered, by the great heterogeneity of the HCV genome, so the main emphasis should be on treatment. HCV was the first virus discovered by molecular cloning without the direct use of biologic or biophysical methods. This was accomplished by extracting, copying into cDNA, and cloning all the nucleic acid from the plasma of a chimpanzee infected with non-A, non-B hepatitis by contaminated factor XIII concentrate (Choo et al., 1989).

HCV has been classified as a member of the genus Hepacivirus within the family Flaviviridae (Van Regenmortel et al., 2000). Based on nucleotide sequence comparisons, HCV genomes can be grouped into at least six genotypes, or clades, that differ from each other by 31 to 34%. Furthermore, several subtypes have been defined, with a nucleotide sequence diversity of about 20%. The HCV has a positive polarity genome with the length of about 9,600 nucleotides, and it carries a single long open reading frame (ORF) that is flanked at both termini by nontranslated regions (NTRs). Viral proteins are generated as a polyprotein precursor that is translated via the internal ribosome entry site (IRES) located in the 59 NTR (Tsukiyama et al., 1992; Wang et al., 1993).

The encoded polyprotein is co- and posttranslationally cleaved by viral and host cell enzymes into at least 10 viral proteins (core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Recently, the production of an additional viral protein by a ribosomal frame shift has been reported (Walewski et al., 2001; Xu et al., 2001).

The structural proteins that are located in the amino-terminal region of the polyprotein are the core protein and the envelope glycoproteins E1 and E2 (Hijikata et al., 1991). The nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B are separated from the structural proteins by the short hydrophobic polypeptide p7, which has an unknown function (Lin et al., 1994; Mizushima et al., 1994). NS2 and the aminoterminal region of NS3 constitute the NS2-3 proteinase responsible for cleavage at the NS2/3 site (Grakoui et al., 1993; Hijikata et al., 1993). Three enzymatic activities reside in NS3: a serine-type proteinase in the; 180 amino-terminal residues and nucleoside triphosphatasehelicase activities that are located in the remainder of the protein (Bartenschlager et al., 1993; Grakoui et al.,1993; Kim et al., 1995; Suzich et al., 1993; Tomei et al., 1993). NS4A is an essential cofactor of the NS3 proteinase that forms a stable heterodimeric NS3-NS4A complex that mediates cleavages at the NS3/4A, NS4A/B, NS4B/5A, and NS5A/B sites (Bartenschlager et al., 1994; Failla et al., 1994; Lin et al., 1994; Tanji et al., 1995). The function of NS4B is not known. NS5A is a highly phosphorylated polypeptide that may be involved in resistance to the antiviral activity of alpha interferon (Enomoto et al., 1996; Gale et al., 1997; Gale et al., 1998). Phosphorylation is mediated by an as yet unknown cellular kinase (Ide et al., 1997; Reed et al., 1997; Tanji et al., 1995). A major phosphoacceptor site has been mapped for a genotype 1a isolate, but this site is not conserved with NS5A proteins of other HCV genotypes (Reed et al., 1999). Most HCV isolates contain two phosphoprotein variants with apparent molecular masses of 56 and 58 kDa corresponding to the basal and the hyperphosphorylated forms, respectively (Kaneko et al., 1994; Reed et al., 1997; Tanji et al., 1995). It is not known whether NS5A has a direct role in RNA replication and whether phosphorylation is important for its function(s). NS5B is the RNAdependent RNA polymerase (RdRp) (Behrens et al., 1996; Lohmann et al., 1997, Yamashita et al., 1998).

In most cases, the virus has established a persistent infection, frequently associated with chronic hepatitis and liver fibrosis. Chronic hepatitis C often progresses to cirrhosis and eventually to hepatocellular carcinoma (Hoofnagle et al., 1997; Theodore et al., 2000).

Currently, hepatitis C patients are treated with alpha interferon (IFN-a) alone or in combination with ribavirin. However, there is still no cure for a large proportion of patients, even with the most advanced therapy regimens (McHutchison et al., 2003).

Thus, alternative therapeutic approaches for chronic hepatitis C are needed. Some have reported that RNA interference represents a promising treatment option for HCV infections (Kapadia et al., 2003; Randall et al., 2003; Seo et al., 2003; Wilson et al., 2003). Since HCV cannot currently be grown in culture, RNA interference activity against HCV was analyzed using the HCV subgenomic replicon, a self-replicating HCV RNA that propagates in cell culture but does not yield infectious virus particles (Blight et al., 2000, Lohmann et al., 1999). RNA interference activity directed against multiple target sequences of the HCV genome has been found to effectively block the synthesis of replicon RNA (Randall et al., 2004).

A number of technologies have been used in an attempt to mediate the downregulation of gene expression. For example, anti-sense oligonucleotides and ribozymes have been used for more than a decade to target specific RNAs for degradation. Although these methods worked satisfactorily in some simple experimental models, they have generally not delivered effective gene silencing in complex mammalian systems. However, in the past 2 years, extraordinary developments in RNAi-based methodologies have seen siRNAs become the primary means by which most researchers attempt to target specific genes for silencing.

siRNA is a phenomenon in which small double-stranded RNA molecules induce sequence-specific degradation of homologous single-stranded RNA (Hannon, G. J., 2002) RNAi was first discovered in Caenorhabditis elegans, when it was noted that introducing a double-stranded RNA (dsRNA) that was homologous to a specific gene resulted in the post-transcriptional silencing of that gene.

It has been shown that the process is initiated via so-called siRNAs of approximately 21-23 bp, which are cleaved from double stranded precursor RNAs by the RnaseIII-like enzyme dicer. These siRNAs associate with various proteins to form the RNA-induced silencing complex (RISC), harbouring nuclease and helicase activity. The antisense strand of the siRNA guides the RISC to the complementary target RNA, and the nuclease component cleaves the target RNA in a sequence-specific manner (Elbashir et al., 2001; Hannon, 2002; Tijsterman et al., 2002). Inaddition, siRNAs can function as primers for synthesis of double-stranded (ds) RNA on the single-stranded (ss) RNA template by a host-encoded RNA-dependent RNA polymerase (RdRp) (Lipardi et al., 2001; Sijen et al., 2001). Only RNA molecules <30 bases in length can be used to exclusively induce RNAi in mammalian cells because longer molecules also activate the nonspecific double-stranded RNAdependent response (Ui-Tei et al., 2000; Elbashir et al., 2001). From a practical perspective, RNAi is proving to be a very powerful technique to ''knock down'' specific genes to evaluate their physiological roles in Caenorhabditis elegans (Hannon, G. J., 2002; Kim, S. K., 2001), Drosophila melanogaster (Clemens et al., 2000), and humans (McManus et al., 2002).

The inhibitory action of siRNAs has been documented for numerous viruses. Nevertheless, RNAi can be used to inhibit virus replication in mammalian cells. Most recently, it was shown that HCV RNA replication is also sensitive to siRNA (Kapadia et al., 2003; Randall et al., 2003; Wilson et al., 2003; Yokota et al., 2003).

OBJECTIVES

  • Constructing the expression vectors for each gene of HCV.
  • Designing and synthesis of siRNAs against all HCV genes.
  • Expression analysis to pick most effective siRNAs after co-transfection of siRNAs and each expression vector through RT-PCR and Western blotting.
  • Testing all effective siRNAs in alternative combinations against all genes.
  • Testing the dose response of siRNAs, by analyzing all HCV proteins inhibition.

SIGNIFICANCE

        As Hepatitis C affects a large number of population worldwide, and limitations in the treatment of HCV infection have prompted the development of novel therapeutic strategies targeting specifically to viral replication. The rapid increase in the study of siRNA as a powerful tool for silencing gene expression has spurred considerable interest in its therapeutic potential.

For gene silencing the introduction of large dsRNA in most mammalian cells has disadvantage in that they act as interferon inducers. Interferon has non-specific and broad-spectrum action resulting in silencing and activation of multiple genes. But siRNA should have minimum adverse affects as compared to other antiviral drugs, as it selectively shut off the post-transcriptional expression of mRNA. So, Synthesizing siRNAs act as readymade products that circumvented this problem.

Rapid, inexpensive and selective silencing of a gene product in complex biological systems obviously may open new avenues in fields of virology, cancer research, genetic disorders, drugs designing, etc. The potential of using siRNA activity for treatment of viral diseases and cancer has aroused a great deal of interest in the scientific community. The inhibitory action of siRNAs has been documented for numerous viruses. Most recently, it has shown that HCV RNA replication is also sensitive to siRNA. siRNA silencing mechanism is believed to act as a natural defense against incoming viruses. Therefore siRNA may be a good tool to treat the Hepatitis C patients.

PLAN

First Year

  • Designing of primers for amplification of all HCV genes.
  • Sequence analysis & amplification of all genes from H/fl (HCV Clone) plasmid.
  • Cloning of Flag TAG oligoes into the vector pCR3.1 (pCR3.1 Flag TAG).
  • Cloning of 10 HCV genes into the vector pCR3.1 Flag TAG to construct expression plasmids.
  • Cloning confirmation through PCR amplification and sequencing.

Second Year

  • Analysing the expression of each expression vector in HUH7 cells (mammalian hepatoma cell line) with transient transfection through RT-PCR and Western Blotting using Flag TAG antibodies
  • Designing and synthesis of siRNAs against all HCV genes.
  • Expression analysis to pick most effective siRNAs after co-transfection of siRNAs and each expression vector through RT-PCR and Western blotting.

Third Year

  • Testing all effective siRNAs in alternative combinations against all genes.
  • Testing the dose response of siRNAs, by analyzing all HCV proteins inhibition.

METHODOLOGY

1. Constructing the expression vectors for each gene of HCV.

For this study, first primers will be designed to amplify all HCV genes individually from the H/fl plasmid, containing the whole genome of HCV 1a genotype. After PCR amplification of all genes, positional cloning will be done into a mammalian expression vector pCR3.1. In order to escape different antibodies for all 10 genes, a FLAG Tag expression system will be used, by cloning it in reading frame with HCV genes.

2. Designing and synthesis of siRNAs against all HCV genes.

        First, the target sequences for the siRNAs will be determined, followed by generation and synthesis of the HCV genes specific siRNAs. The homology to other coding sequences in humans will be checked by BLAST, found on the NCBI server at: http://www.ncbi.nlm.nih.gov/BLAST.

3. Expression analysis to pick most effective siRNAs.

        A mammalian liver cell line will be used for expression analysis of all constructed expression plasmids. All expression plasmids will be co-transfected individually along with their relevant gene specific siRNAs. Total RNA will be isolated from transfected cells for RT-PCR to check the expression of all HCV genes at RNA level. Western blot analysis for all HCV proteins will be performed using total protein lysate form transfected cells, to check the effect of each siRNA. The specific inhibitory effect of siRNAs against each HCV genes will be determined in transient transfection experiments. The effective siRNAs, showing the protein inhibition, will be screened, individually for all HCV genes.

4. Testing all siRNAs in alternative combinations against all genes.

        In order to minimize the development of an escape mutation and to increase the effect of siRNAs, multiple sites of the viral gene will be targeted by different combinations of most effective siRNAs.

5. Testing the dose response of siRNAs, by analyzing all HCV proteins inhibition.

        siRNAs will be co-transfected in different ratios, in order to check their dose response to inhibit specific protein expression.

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