Characterization of Pakistani HCV Genotype 3a NS3 Protease/Helicase and Development of Robust In-vitro Serine Protease Assay for Anti-Viral Drug Testin

Abstract

Hepatitis C Virus non-structural protein-3 (NS3) protease and helicase domains are essential for viral replication; they are highly conserved among various HCV strains. In the current study fifty six (56) HCV infected patients were enrolled from various regions of Pakistan to determine their genotypes, ALT level and virus titer by qRT-PCR. Next aim was to clone and sequence NS3/NS4A genes from selected HCV samples (n=5); two clones from each patient (n=10). Nucleotide sequence alignment of ten NS3/NS4A clones (NCVI 01-10)―showed high level of identities among 3a genotypes (94%). One of the samples (NCVI 01) showed unique amino acids substitutions, including R9Q, L332P, L354I, I605V, and S622C. To analyze the effect of substitutions on amino acids interactions, three dimensional (3D) structure was determined. To perform the functional analysis of NS3/NS4A, HCV-NCVI 01 clone was selected to express NS3 protease and helicase domains in E.Coli Rosetta DE3. Subsequently, these proteins were purified by His-Tags and found to be 95% purified by FPLC. Purifications steps yielded, 0.92 mg/Litre (>90%) of protease and 14.2 mg/Litre of helicase protein products. Further, Fluorescence Resonance Energy Transfer (FRET) based assays were established for detecting proteolytic activity of (NS3-4A) serine protease, using Anaspec peptide. Such an assay offers an attractive choice for high throughput screening (HTS) inhibitors against HCV in vitro. Since NS3 is necessary for subsequent viral replication, it is thought that development of a specific inhibitor of NS3 would be an attractive anti-HCV drug-lead. To test the anti-viral activity of organotins compounds, a pilot high-throughput screening assay was used to screen these 30 unknown compounds by infectious xix system, Gaussia luciferase (Gluc) using Jc1-FLAG2 (p7-nsGluc2A). The virus was infected to HuH7.5 cell lines (MOI 0.1) and luciferase secretion (Gluc) was monitored as an indicator of viral replication. Five compounds (FS155, FS174, FS169, FS102, FS167 and FS55) were screened, identified and presented very promising results as hits by Renilla luciferase reporter system whereas 12 inhibitors were identified as good only for the infectious system, stating that the assay is skillful for the identification of drugs. These Five compounds not only showed effectiveness at low concentration but the inhibition was steady with time and IC50 values were low as compared to the control. The qRT-PCR authenticates the decrease in viral replication by FS102 and FS167 which were very effective and the inhibitory concentrations were low especially for the FS102 versus the control. Furthermore, experiments were carried out to study the functional aspects of HCV-NS3 helicase protein. The NS3 helicase can unwind dsRNA as well as dsDNA and RNA-DNA heteroduplexes in the 3‘ to 5‘ polarity by using ATP as the energy source. NS3 helicase was cloned and purified protein was employed in in vitro helicase assay based on molecular beacons to test the enzymatic activity of this enzyme. In the present study a recombinant Adeno-associated virus vector pAD-NS3 was constructed, with the view to delineate the pathways of NS3 mode of action within liver of intact animals or humans and dendritic cells which have been proposed to be the vectors of choice for vaccination due to ex-vivo enhancement of antigen presenting functions. Two chimeric clones, JC1/NS3P-3a GT and JC1/NS3-3a GT developed in the current study could be further exploited to investigate viral replication/infection and anti-viral drug testing against Pakistani HCV. In summary, this work was carried for the first time to develop in vitro assay for the detection of xx anti-protease and anti-helicase activities (NS3) against the predominant Pakistani HCV genotype 3. The successful cloning, purification of NS3 coupled with the development of cell free assay and subsequent testing of various organotin compounds has shown the feasibility of drug testing. The hepatoma cell-based model of NS3 activity using the recombinant adenovirus encoding NS3 and HCV chimera has the impending to be exploited for high throughput screening of putative antiviral inhibitors against Pakistani HCV. The novelty of this work is that such functional molecular characterization at individual component level of HCV has not been established against genotype 3a.