Hepatitis C virus (HCV) is a major cause of hepatitis which reduces the quality of life of some 170 million people worldwide [1]. HCV infection is frequently associated with chronic liver diseases and development of hepatocellular carcinoma. In Pakistan propensity of HCV infection in local population is ≥6% with prevalent genotype 3a [2]. The patients infected with genotype 3a respond efficiently to interferon therapy but unfortunately the rate of reoccurrence of infection is also very high and after relapse the patients show resistance to interferon therapy. Recent studies showed resistance to interferon in HCV infection has been partially ascribed to functional inhibition of PKR which is interferon induced anti-viral protein [3–7]. HCV is an enveloped sense, single stranded RNA virus belonging to family Flaviviridae. Its genome is 9.6 kb in size and encodes 10 proteins which are synthesized as a poly-protein precursor and subsequently processed by host as well as viral proteases to yield ten mature proteins. HCV also encodes an eleventh protein, ARFP or F that is produced by translational frame shifting from the core region [8]. Viral proteins are expressed in a cap-independent manner by means of an internal ribosome entry site (IRES) located in the 5' UTR [9–11].

The IFN system is the first line of defence against viral infection in mammals [12]. IFNs are glycoproteins commonly known as cytokines which are released by the cells during infections.Activation of type I IFN (IFN-α & β) genes at transcription level is mainly triggered by viral double-stranded RNA present in infected cells [13, 14]. Upon infection the virus particles replicate inside the host cell ultimately the cell dies and viral particles are released that can infect surrounding cells. However, the infected cell can warn neighbouring cells of a viral presence by releasing interferon. The neighbouring cells, in response to interferon signalling, produce large amounts of an enzyme known as (PKR). PKR is aserine/threonine kinase found in cells in dormant state.PKR is induced by interferon and activated upon autophosphorylation.It plays an important role in cellular antiviral defence as well as in apoptosis, signal transduction, and transformation [15]. Activation of PKR by autophosphorylation occur upon binding to its regulator, dsRNA molecules [16, 17].This permits the enzyme to phosphorylate its substrates. Best known of these is translational initiation factor eIF2, which is phosphorylated on serine 51 of its subunit. Phosphorylation of eIF2a controls a number of cellular processes, most important of which is the blockage of protein synthesis. Phosphorylation of many cellular and viral proteins, including the human immunodeficiency virus transactivator protein, Tat [18, 19], and 90-kDa proteins from rabbit reticulocytes [20] and human cells is mediated by PKR [21–24]. The roles of the other phosphorylation events are as yet unknown. Different genotypes of HCV exhibit different rates of response to IFN-alpha and these variants are characterized by mutations that may be accountable for IFN-alpha resistance. Two HCV proteins that have been involved in IFN resistance through inhibition of its downstream protein kinase (PKR) are NS5A and E2 [17, 25]. The E2 glycoprotein are supposed to be the first viral components that come in contact with the host cell surface receptors, and elicits production of neutralizing antibodies against the virus, and is involved in viral morphogenesis. It binds to external loop of CD81, a tetraspanin found on the surface of many cell types including hepatocytes [26]. This viral envelope glycoprotein is an obvious candidate for vaccine development as it is chosen target for humoral and cell-mediated immune responses [27, 28]. Taylor et al., 1999 reported that envelope protein 2 of HCV contains a 12 amino-acid sequence domain which is homologous to the autophosphorylation site of PKR and initiation factor eIF2a, which is growth they considered it a possible mechanism by which HCV circumvents the antiviral effect of IFN and cause chronic infections and hepatocellular carcinoma. They also reported that E2 proteins with a PePHD sequence identical to genotypes 2 and 3 of HCV, which are known IFN sensitive genotypes, showed only a weak inhibitory effect against PKR activity [28].

Potential phosphorylation sites in the cytoplasmic domain of the E2 protein was predicted, which may be possibly involved in mediating interferon resistance. Phosphorylation site predictor has the tendency to over-predict, therefore stringency was increased and only those motifs were selected that received a NetPhos score of 0.8 or higher or predicted by both NetPhos [29] (Figure 1) and Scansite [30]. The final yield was 31 putative sites (Ser- 11, Thr- 14 and Tyr- 6). Fine of them were predicted by both the servers.

The envelope protein 2 (E2) of hepatitis C virus (HCV) is recently reported to interact with double stranded RNA-dependent protein kinase (PKR) [9, 12, 15, 16, 23, 25]. HCV envelope protein E2 contains a domain homologous to phosphorylation sites of the interferon-inducible protein kinase PKR and the translation initiation factor eIF2 alpha, which is a target of PKR [23]. As a result E2 competes and inhibited the kinase activity of PKR and blocked its inhibitory effect on protein synthesis and cell growth [18, 21, 29, 32, 39]. We aim to find out the role of phosphorylation of envelope protein 2 on interferon & PKR response. Envelope protein coding genes were isolated from local HCV isolates and cloned in mammalian expression vector for further analysis. The cloned fragment was sequenced and the sequence was submitted to NCBI (EU 399722). The local envelope gene sequence was compared with other reported sequences for 3a genotypes from different regions of the world to find out the percentage nucleotide identity (PNI) as variations in the envelope genes accounts for different degrees of disease progression in HCV RNA positive individuals with different genotypes [3, 12, 25]. In silico N-linked phosphorylation sites were predicted (Figure 1) using the freely available online servers i.e. NetPhos 2.0 developed with neural network which predicts phosphorylation sites in sequences and structure [29] and Scanite which predicts target motifs for different kinases [30]. Scanite predictions were made on the "Low Stringency" to identify as many sites as possible. Further analysis was done using these predicted sites. The final yield was 31 putative sites (Ser- 11, Thr- 14 and Tyr- 6). Fine of them were predicted by both the servers (Table 2). The phosphorylation sites must be present on the surface of the protein. To find this local envelope protein sequence was analysed for finding the surface accessibility by using online server NetSurfP. Due to the unavailability of template model in Protein Data Bank (PDB) server [40], ab-initio model was developed using I-TASSER [32]. Sequences were uploaded and models were obtained. The phosphorylation sites predicted with two servers were shown in the 3D structure. Chimera [33] and SWISS PDB viewer [34] was used to infer whether potential phosphorylation sites were surface exposed or buried in which five residues were observed on the exposed surface of the model (aa: S66, S266, S267, S356, T311). The most reliable sites are visualized in the (Figure 2). From all this analysis two sites were found to be most reliable phosphorylation sites i.e. S266 and S267. Scansite was used for finding the phosphorylation interaction motifs and found that S66, S266 and S267 interact with the PKC, Casein Kinase, SRC Kinase and AKT Kinase which were then investigated in GeneCards and was confirmed from UniGene and UniProt. Protein sequences of local envelope gene sequence and other reported sequences for 3a genotypes from different countries of the world (Japan, China, USA, UK) were retrieved from NCBI. All sequences (EU399722, NC009824, D17763, AY958007, AY958005, AY958014, AY958012, AY958010, AY957994, DQ430819 and DQ437509) were then aligned by using CLUSTALW [35]. A neighbour joining tree was constructed using PHYLIP [41] (Figure 3). IFN normally acts by upregulating expression of many antiviral genes, including the double stranded RNA activated protein kinase (PKR), which in turn inhibits protein synthesis by phosphorylation of the translation initiation factor eLF2. HCV have evolved the strategies to surmount the antiviral effects of PKR [39] and phosphorylation of E2 protein may accounts for IFN resistance. This work will help to identify factors that favour a successful innate immune response against HCV infections.