IN VITRO CHARACTERIZATION OF HEPATITIS C VIRUS RNA DEPENDENT RNA POLYMERASE

Abstract

Hepatitis C virus (HCV) is a major health problem throughout the world with high morbidity and mortality. HCV has high mutation rate and is classified into six genotypes (GT). The major prevalent genotype in Pakistan is 3a. While inhibitors of the HCV protease are now available to treat patients infected with GT1 HCV, treatment options are limited for other HCV genotypes. The aim of the study was to analyze the effect of different inhibitors / HCV structural genes (Core, E1, E2 & P7) on the activity of HCV polymerase in 5BR assay and to develop a global consensus sequence of HCV NS5B. The 5BR assay was used to screen non-nucleoside inhibitors (NNI) against the GT3a HCV RdRp and determine the EC50 of HCV-796. Binding of hits to recombinant GT3a RdRp was determined using a differential fluorimetry assay. RNA synthesis by the recombinant protein was used to determine the IC50 of HCV-796. A mutation in the 3a RdRp that conferred resistance to inhibition by HCV-796 was identified using the 5BR assay and by RNA synthesis in vitro. Pan genotypic effect of HCV-796 was analyzed by using HCV polymerase from all the six HCV genotypes in 5BR assay. Effect of HCV structural genes on the activity of HCV polymerase was analyzed by using wild type polymerase, ∆21 polymerase and GAA mutant polymerase in 5BR assay. To develop a global consensus sequence of HCV NS5B; 236 HCV NS5B sequences belonging from all over the world were aligned and a representing phylogenetic tree was drawn. xix Inhibitors screening showed that HCV-796 decreased the activity of GT3a RdRp with an EC50 of 90 nM in the 5BR assay. In biochemical assays, HCV-796 had an IC50 of 88 nM for de novo initiation and 229 nM for primer extension. In the differential scanning fluorimetry assay, binding to HCV-796 significantly altered the denaturation profile of the 3a RdRp. A C316Y mutant that conferred resistance to HCV-796 in GT1 RdRp was found to render the 3a RdRp resistant to HCV-796 by more than one log in both the 5BR and the biochemical assays. When the effect of HCV structural genes was analyzed on the activity of HCV polymerase in cell bases assay, HCV core gene showed maximum increase of 10 fold by using wild type polymerase. Consensus sequence analysis showed that the active site residues D220, D225, D318 and D319, which bind the divalent cations, are highly conserved among all the HCV genotypes. The HCV NS5B phylogenetic tree showed the clusters of different genotypes and their evolutionary relationship. Given the high mutation rate of HCV, the residues which are present in the catalytic pocket, sugar selection and template/primer interaction are highly conserved, although we observed, at many places where change in nucleotide sequences did not affect the amino acid sequences of HCV NS5B. The NNI HCV-796, a known inhibitor for GT1 HCV RdRp, could also inhibit the 3a polymerase. HCV-796 should serve as a useful scaffold for further development of effective non-nucleoside inhibitor for GT3a HCV. HCV core gene increased the activity of HCV polymerase in cell based assay. The phylogenetic analysis suggested that different HCV genotypes evolved from genotype 1a.