Characterization of monoclonal antibodies that specifically recognize the palm subdomain of hepatitis C virus nonstructural protein 5B polymerase

The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is an RNA-de pendent RNA polymerase (RdRp) which plays an essential role in viral RNA re plication. Antibodies that specifically recognize NS5B will have utilities in monitoring NS5B production and subcellular localization, as well as in s tructure-function studies. In this report, three mouse monoclonal antibodie s (mAbs), 16A(9)C(9), 16D(9)A(4) and 20A(12)C(7), against a recombinant NS5 B protein (genotype 1, H-77 strain) were produced. These mAbs specifically recognize HCV NS5B, but not RdRps of polivirus (PV), bovine viral diarrhea virus (BVDV) or CB virus B (CBV-B). The mAbs can readily detect NS5B in cel lular lysates of human osteosarcoma Saos2 cells constitutively expressing t he nonstructural region of HCV (NS3-NS4A-NS4B-NS5A-NS5B). NS5B proteins of different HCV genotypes/subtypes (1a, 1b, 2a, 2c, 5a) showed varied affinit y for these mAbs. Interestingly, the epitopes for the mAbs were mapped to t he palm subdomain (amino acid 188-370) of the HCV RdRp as determined by imm unoblotting analysis of a panel of HCV/GBV-B chimeric NS5B proteins. The bi nding site was mapped between amino acid 231 and 267 of NS5B for 16A(9)C(9) , and between 282 and 372 for 16D(9)A(4) and 20A(12)C(7). Furthermore, thes e mAbs showed no inhibitory effect on the NS5B polymerase activity in vitro . (C) 2001 Elsevier Science B.V. All rights reserved.