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Mutational analysis of the structure and functions of hepatitis C virus RNA–dependent RNA polymerase
Author(s) -
Qin Weiping,
Yamashita Tatsuya,
Shirota Yukihiro,
Lin Yong,
Wei Wenxiang,
Murakami Seishi
Publication year - 2001
Publication title -
hepatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.488
H-Index - 361
eISSN - 1527-3350
pISSN - 0270-9139
DOI - 10.1053/jhep.2001.22765
Subject(s) - ns5b , rna dependent rna polymerase , rna polymerase , biology , rna , ns5a , microbiology and biotechnology , point mutation , mutant , polymerase , alanine scanning , hepatitis c virus , biochemistry , enzyme , virology , mutagenesis , virus , gene , hepacivirus
The hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is an RNA‐dependent RNA polymerase (RdRP), a central catalytic enzyme for HCV replication. To further understand the structure and functions of NS5B, we introduced a series of 27 clustered and 19 point substitution mutations within and outside the well‐known motifs conserved among RdRP by alanine scanning and investigated effects of these mutants on enzymatic activity of NS5B. Surprisingly, most of the mutations (22 of 27 clustered mutants) do not affect RdRP activity at all, indicating that the side chains of the corresponding amino acid residues are dispensable for the catalytic activity. On the other hand, 4 mutants, cm20t, cm194t, cm2t, and cm3t, are defective in RdRP activity. By further analysis with point mutations within these regions, E18, Y191, C274, Y276, and H502 were determined to be critical for the RdRP activity. Y276 was also shown to be critical for RNA template/primer association, although 3 amino acid sequences were identified to be important for RNA template binding by RNA‐filter binding assays. Finally, 4 discontinuous sequences of NS5B (aa139‐145, aa149‐155, aa 365‐371, and aa 382‐388) were found to be essential for binding to NS5A as determined by glutathione S ‐transferase (GST)‐pull down assays using GST‐NS5A and FLAG‐NS5B expressed in cotransfected cells, and GST‐pull down assay in vitro . In light of the crystal structure models of NS5B recently reported, our results indicate that the RdRP activity of NS5B requires the longer loop and the helix located at the distal of the thumb, which are unique among RdRPs as well as reverse transcriptases.