Template Specificity of HCV NS5B in RNA Syntheses from 3′Terminal Sequences of HCV Positive and Negative Strand RNA
Abstract: A recombinant HCV RNA-dependent RNA polymerase (RdRp, NS5B protein) was expressed in E. coli strain BL21(DE3). The purified protein was used to investigate the RNA syntheses from the 3′ terminal sequences of HCV positive and negative strand RNA in vitro. The positive strand RNA did not direct the synthesis of negative strand RNA; however, the negative-strand RNA was able to generate a full-length positive strand RNA. These results demonstrate that NS5B has the template specificity on negative strand RNA. The template specificity was further confirmed by template competition assay. The competition of positive-strand RNA did not affect the RNA synthesis from the negative strand RNA. The template specificity of NS5B provides a reasonable explanation for the excesses production of positive over negative strand RNA during the HCV genome replication. In addition, these findings provide some clues to further work. The research on viral or cellular factors involved in the RNA synthesis from positive-strand RNA, as we
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