Comparison Of Protease Of NS3/4A Of Hepatitis C Virus Between Genotypes And Identification Of Its Cellular Partners

Hepatitis C virus (HCV) infection is a major worldwide health problem. HCV usually results in a chronic infection in 80% of infected individuals with up to 20% progressing to liver failure, cirrhosis and hepatocellular carcinoma. It has been found that viral genotype influences disease severity and treatment response (some HCV types are more pathogenic than others, i.e., HCV type 1 over other types, and subtype 1b over subtype 1a). Considering the genetic variation of NS3 encoded gene among different isolates, the indispensability of NS3 protease in viral maturation and its association with carcinogenesis, it's hypothesized that the variation in enzymatic activity of NS3 protease might contribute to an altered prognosis of hepatitis C between genotypes. However, exactly how HCV genetic variation leads to these clinically important viral phenotypes is still poor understood. This study was therefore initiated to define the impact of genetic variation on the activity of the best characterized HCV NS3 protease and its molecular mechanism.With the full-length cDNA clones of 1b and 1a as templates for PCR amplification, an efficient, sensitive, cell-based assay system for the study of NS3 serine protease activity was set up first. NS3/4A-SEAP chimeric gene was constructed, in which the SEAP (secreted alkaline phosphatase) was fused in-frame to the downstream of NS4A/4B cleavage site. The protease activity of NS3 was reflected by the activity of SEAP in the culture media of transiently transfected or stable expression cells. Stable expression cell lines were obtained by G418 selection. Pefabloc SC, a potent irreversible serine protease inhibitor, was used to treat the stable cell lines to assess the system for screening NS3 inhibitors. To compare the activity of serine proteases from 1b and 1a, two chimeric clones were constructed and introduced into both transient and stable expression systems. Results showed that the SEAP activity in the culture media could be detected in both transient and stable expressionsystems, and was apparently decreased after Pefabloc SC treatment. In both transient and stable systems, NS3/4A-SEAP chimeric gene from HCV genotype 1b produced higher SEAP activity in the culture media than that from 1a. It could be concluded that this cell-based system is efficient, sensitive enough for detection, comparison of NS3 protease activity and screening of anti-NS3 inhibitors. The functional differneces between NS3/4A from 1a and 1b subtypes revealed by this system provides a clue for further investigation.To understand the molecular mechanism of variation in enzymatic properties from 1a and 1b geontypes, sequence alignment, space conformational analysis, site-directed mutagenesis and functional fragments substitution test were performed. Alignment of NS3 protease amino acid from 1a and 1b genotypes revealed that the 60 different amino acid residues between two subtypes were dispersive along the full-length NS3 protein, suggesting the differences in enzymatic activity may be the accumulative results of variation in multiple residues. Gene fragment substitution experiments supported the above hypothesis. Substitution of NS3P, NS4A, NS3H, or NS3P+NS4A from 1b to 1a could increased the NS3/4A protease activity from 2.44+0.37 (mu/ml,1a type), to 6.75+0.69mu/ml, 8.02+1.37mu/ml, 10.99+0.43mu/ml and 12.63+0.87mu/ml respectively, but still lower compared to NS3/4A from 1b type (29.44+3.69 mu/ml). The above results implied that the natural genetic variation along the full-length NS3 protease affects protease activity and possible viral replication, which may partly explain the molecular mechanism of variation in enzymatic properties from 1a and 1b genotypes and its association with different clinical outcomes.To further investigate the impact of NS3 protease on host cells and its role in viral pathogenesis, the effect of the expression of different NS3/4A on cell cycles and transcriptional activity of p21 promoter were analyzed. Functional analysis showed that the NS3/4A of 1b inhibited the tr...