Virus-specific cofactor requirement and chimeric hepatitis C virus/GB virus B nonstructural protein 3

GB virus B (GBV-B) is closely related to hepatitis C virus (HCV) and causes acute hepatitis in tamarins (Saguinus species), making it an attractive su rrogate virus for in vivo testing of anti-HCV inhibitors in a small monkey model. It has been reported that the nonstructural protein 3 (NS3) serine p rotease of GBV-B shares similar substrate specificity with its counterpart in HCV. Authentic proteolytic processing of the HCV polyprotein junctions ( NS4A/4B, NS4B/5A, and NS5A/5B) can be accomplished by the GBV-B NS3 proteas e in an HCV NS4A cofactor-independent fashion. We further characterized the protease activity of a full-length GBV-B NS3 protein and its cofactor requ irement using in vitro-translated GBV-B substrates. Cleavages at the NS4A/4 B and NS5A/5B junctions were readily detectable only in the presence of a c ofactor peptide derived from the central region of GBV-B NS4A. Interestingl y, the GBV-B substrates could also be cleaved by the HCV NS3 protease in an HCV NS4A cofactor dependent manner, supporting the notion that HCV and GBV -B share similar NS3 protease specificity while retaining a virus-specific cofactor requirement. This finding of a strict virus-specific cofactor requ irement is consistent with the lack of sequence homology in the NS4A cofact or regions of HCV and GBV-B. The minimum cofactor region that supported GBV -B protease activity was mapped to a central region of GBV-B NS4A (between amino acids Phe22 and Val36) which overlapped with the cofactor region of H CV. Alanine substitution analysis demonstrated that two amino acids, Val27 and Trp31, were essential for the cofactor activity, a finding reminiscent of the two critical residues in the HCV NS4A cofactor, Ile25 and Ile29. A m odel for the GBV-B NS3 protease domain and NS4A cofactor complex revealed t hat GBV-B might have developed a similar structural strategy in the activat ion and regulation of its NS3 protease activity. Finally, a chimeric HCV/GB V-B bifunctional NS3, consisting of an N-terminal HCV protease domain and a C-terminal GBV-B RNA helicase domain, was engineered. Both enzymatic activ ities were retained by the chimeric protein, which could lead to the develo pment of a chimeric GBV-B virus that depends on HCV protease function.