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

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• 作者：Ingravallo ; Paul ; Lahser ; Frederick ; Xia ; Ellen ; Sodowich ; Bruce ; Lai ; Vicky C.H. ; Hong ; Zhi ; et. al.
• 关键词：Amino acids ; Antibodies ; Hepatitis C
• 刊名：Virus Research
• 出版年：2001
• 出版时间：June, 2001
• 年：2001
• 卷：75
• 期：2
• 页码：179-187
• 全文大小：398 K

文摘

The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is an RNA-dependent RNA polymerase (RdRp) which plays an essential role in viral RNA replication. Antibodies that specifically recognize NS5B will have utilities in monitoring NS5B production and subcellular localization, as well as in structure-function studies. In this report, three mouse monoclonal antibodies (mAbs), 16A₉C₉, 16D₉A₄ and 20A₁₂C₇, against a recombinant NS5B protein (genotype 1a, H-77 strain) were produced. These mAbs specifically recognize HCV NS5B, but not RdRps of polivirus (PV), bovine viral diarrhea virus (BVDV) or GB virus B (GBV-B). The mAbs can readily detect NS5B in cellular lysates of human osteosarcoma Saos2 cells constitutively expressing the nonstructural region of HCV (NS3-NS4A-NS4B-NS5A-NS5B). NS5B proteins of different HCV genotypes/subtypes (1a, 1b, 2a, 2c, 5a) showed varied affinity for these mAbs. Interestingly, the epitopes for the mAbs were mapped to the palm subdomain (amino acid 188-370) of the HCV RdRp as determined by immunoblotting analysis of a panel of HCV/GBV-B chimeric NS5B proteins. The binding site was mapped between amino acid 231 and 267 of NS5B for 16A₉C₉, and between 282 and 372 for 16D₉A₄ and 20A₁₂C₇. Furthermore, these mAbs showed no inhibitory effect on the NS5B polymerase activity in vitro.