Molecular Characterization of HCV NS5A & E2 in Patients Receiving Interferon Therapy

Abstract

Hepatitis C virus is one of important pathogens that has a special predilection for liver and cause the disease commonly known as Hepatitis C. In the decades of 70’s and 80’s Hepatitis A and Hepatitis B viruses were known and their screening was mandatory for blood donors. During that screening procedure another pathogen was brought into notice. Initially it was named as NANB (Non-A Non-B). Later on in 1989 it was given the name of Hepatitis C virus. Globally 200 million individuals are infected with HCV, out of which around 130 million reported to became chronic HCV carrier. In our country Pakistan, despite intensive awareness, treatment and prevention programs by public sector and NGO’s, rapidly increasing rate of HCV infection has evolved as epidemic over last decade. Genotype 3a predominantly found in Pakistan. Therapeutic use of antiviral agents like IFN, PEG-IFN and DAAs has significantly declined morbidity and mortality rate. However, still HCV rendered itself resistant against these therapeutic agents through interaction with certain host factors. The objectives of this study was to analyze structural changes in two regions of HCV genome, known as NS5A and E2 and their possible outcome. The common property of these two regions is binding to protein kinase R (PKR) with subsequent inhibition of its activity. Concurrent occurrence of mutations in these two regions might be the consequence of this common property. We included five hundred patients in our study. The gender selection was random, ratio of male to female patients was nearly equal. The study was performed in Department of Biotechnology, University of Karachi where nested PCR of HCV seropositive isolates was performed. Other lab parameters were carried out in Rahila Diagnostic Research and Reference lab (pvt) Ltd; including serum Alanine transaminase level, ELISA for HCV antibodies, qualitative & quantitative RT PCR and genotyping. We analyzed NS5A region (2213-2352) including the ISDR, PKRBD & short sequence outside PKRBD (2281-2335). Notable substitutions found in this region were: glycine (Gly) replaced by alanine (Ala), proline (Pro) and serine (Ser); alanine (Ala) replaced by XVII serine (Ser) and threonine (Thr); valine (Val) replaced by is isoleucine (Ile); proline (Pro) replaced by leucine (Leu), serine (Ser), threonine (Thr); lysine replaced by arginine (Arg). In E2 we found mutations in PePHD region and among residues just before and after its span. These were histidine (His) to glutamine (Glu), asparagine (asp); glutamine (Glu) to leucine (Leu); leucine (leu) to arginine (Arg); leucine (Leu) to phenylalanine (Phe). Most important mutations found in the E2 region showed hydrophobic mutations within the hydrophilic area of PePHD at codon 668 and 669 of HCV-3a, that might affect interaction with other proteins including neutralizing antibodies. The similar changes in NS5A and E2 has been studied abroad in the context of vaccine development against HCV, that appears to be a big challenge because of quasispecies nature of HCV. Therefore effective vaccine development is the major demand for the prevention of HCV. Our study on HCV genotype 3a along with other peer research work in our country and other parts of world would provide new parameters and better understanding of structural changes in NS5A & E2 region of HCV genome, that would help to modulate therapeutic approach and vaccine preparation against Hepatitis C virus. In addition, it demonstrates the importance of application of bioinformatics tools for the study of proteins that are difficult to be investigated by other experimental procedures.