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siRNAs (small interfering RNAs) are approximately 21 nucleotides (nt) long RNA duplexes. siRNA can silence gene expression specifically, when they are introduced into mammalian cells. After infection, HCV goes into the liver cells and starts its replicates in the cytoplasm without incorporation into the host genome. As HCV has a single stranded RNA genome, acting both as a template for replication and as a messenger RNA. So, destruction of its RNA will not only inhibits viral replication but also will diminish the viral protein synthesis. It has already been known that siRNAs can inhibit viral replication and its gene expression. Due to limitations in the available therapies of HCV infection, there is a need to develop some novel strategies that could target specifically to viral replication. The development in the study of siRNA as an influential tool for inhibiting gene expression has prompted a great interest in its therapeutic potential.
Introduction of Hepatitis C Virus (Structure, Prevalence, replication, protein functions)
Hepatitis C virus (HCV) was proposed as non-A non-B hepatitis in 1970s and proved decisively in 1989 (Choo et al., 1989). HCV is the member of family Flaviviridae and the genus is Hepacivirus (Van Regenmortel et al., 2000).
More than 350 million world's population and about 10 million people in Pakistan are infected with HCV (Giannini and Brechot, 2003; Hamid S et al., 2003). And out of which about 50% to 80% individuals leads to chronic hepatitis (Waheed Y et al., 2009). HCV is classified into six major genotypes (labeled 1 through 6), out of which 1a is the most prevalent genotype worldwide and 3rd most prevalent in Pakistan. (Idrees M et al., 2004)
Its genome is 9.6 kb long and is single stranded positive sense RNA having two un-translated regions (UTR) at 5´ and 3´ ends, and an open reading frame that encodes a single, about 3000 amino acids long, ampolyprotein precursor. This polyprotein precursor is then cleaved co and post translationally by its own viral proteases and host cell peptidases to get 10 different structural and non-structural proteins. The structural proteins consist of core (21 kDa), E1(27kDa), E2 (27kDa) and a membrane associated protein P7 (Penin F et al., 2004). The core and both envelope glycoproteins E1 and E2 are discharged from the precursor polyprotein by the signal peptidase of endoplasmic reticulum and P7 protein is also released by the host peptidases ( Griffin SD et al., 2004). There are six non-structural proteins comprising NS2 and NS3 (68kDa) which are proteases, NS4A (6 kDa), NS4B (26 kDa) NS5A (5658 kDa) polypeptides, and NS5B (65 kDa) protein which is RNA-dependent RNA polymerase (Penin F et al., 2004).
From the N-terminus, core is the first structural protein of the polyprotein precursor. Most probably this protein interact with the viral RNA, as it comprises the virion nucleocapid. Primary localization of core protein is the perinuclear regions and cytoplasm (Barba G et al., 1997).
The E1 and E2 (envelope proteins), contains a large N-terminal domain and a hydrophobic C-terminal connection to the membrane. These connections build a non covalent hetero-dimer that is opened on the surface of virion. A number of studies propose that E1 may have some involvement in virus encapsulation, while E2 has a role in intervening virus binding to the host cells (Deleersnyder V et al., 1997; Flint M et al., 2000). In the structural part of proteins there is also a small protein P7 which is integrated into the membrane and seems to act as an ion channel (Griffin SD et al., 2003; Pavlovic D et al., 2003).
Out of non-structural protein both NS2 and NS3 are the viral proteases that directs the cleavage of the left over part of non-structural proteins. During RNA replication NS3 also functions as a NTPase and helicase (Tai CL et al., 1996). NS4A act as a cofactor of NS3, and due to the NS3/NS4A heterodimer HCV becomes able to escape the immune response (Li K et al., 2005).
NS4B is a membrane integrated protein. It induces web formation in membrane has a role in fixing the replication complex to the cell membrane, similar to what has seen during the replication of other RNA viruses (Hugle T et al., 2001).
NS5A is a captivating protein. This serine phosphoprotein has a varying levels of phosphorylation (Kaneko T et al., 1994). This protein has a signal sequence that looks like a nuclear localization signal, but it is localized to the peri-plasmic membrane of the nucleus, where it seems to be involved in the viral replication (Blight KL et al., 2000).
The NS5B polypeptide has vital role in catalyzing the HCV replicase enzyme that is the RNA-dependent RNA polymerase. This protein has a hydrophobic domain on C-terminus that is responsible for the incorporation this protein into the plasma membrane and becomes capable to interact with other HCV proteins (Yamashita T et al., 1998).
All HCV products have a role in HCV life cycle that includes, binding and invading into the host cell, release and exposure of the viral genome, processing of polyprotein precursor by host and viral proteases, RNA replication, packaging and discharge from the host cell (Cocquerel L et al., 2006).
Figure 2 | HCV genes and gene products. a, The structure of the viral
genome, including the long open reading frame encoding structural and
nonstructural genes, and 5_ and 3_NCRs. The polyprotein processing
scheme is shown below. Closed circles refer to signal peptidase cleavage
sites; the open circle refers to the signal peptide peptidase cleavage site. All
other terms are defined in the text. b, The topology of HCV proteins with
respect to a cellular membrane.
HCV infection and replication is a dynamic process having a few hours viral half life and production and/or clearance of about 1012 virions per day in an infected individual (Neumann AU et al., 1998). After binding to liver cells, HCV enters into cells through clathrin-mediated endocytosis (Blanchard E et al., 2006). Till now several cellular co-receptors of HCV have been known, including glycosaminoglycans, the LDL receptor (LDLR), L-SIGN and DC-SIGN, SRBI, CD81 and claudin-1 (Sabahi A, 2009). Current studies proposed that the low pH environment in the endosome activates the fusion of virus with the endosomal membrane and the entry of the HCV genome into the cytoplasm (Lavillette D et al., 2006). Translation of the viral genome is initiated by the internal ribosomal entry site (IRES) located in the 5' UTR which is highly conserved. Translation starts after the IRES of HCV and the 40S subunit of ribosome complex is formed. This is followed by the attachment of the eIF2 Met-tRNA GTP complex and eIF3, and then 48S-like complex is formed at the initiation codon of the viral RNA. The final GTP-dependent step is the formation of 80S complex after the association of the 60S subunit (Otto GA and Puglisi JD, 2004). The translation followed by the processing of precursor polyprotein by the cellular and viral proteases results in the formation of structural and non-structural mature proteins. The structural proteins are processed by the signal peptidase of endoplasmic reticulum (ER) and the remaining non-structural proteins are subjected to be processed by the NS3-4A serine protease and NS2-3 protease (Moradpour D et al., 2007). After the HCV proteins expression, replication complexes are formed in the cytosol. The replication complexes are located close to the cell membrane which can be seen as a membrane alteration known as the membranous web (Gosert R et al., 2003). The HCV proteins expression and the viral replication is followed by the packaging of the viral particles and secretion. Most probably virions are made by budding of the ER and comes out via the secretory pathway.