The Study On Template Selection In Genomic Replication Of Hepatitis C Virus

Both DNA and RNA can act as genetic materials. The molecular mechanism of DNA replication has been lucidly illuminated, however, the RNA replication has not been fully understood. The RNA replication is peculiar to viruses. Using positive-strand RNA viruses to research RNA replication is significant for the basic theoretics of molecular biology and genetics, as well as for the understanding of positive-strand RNA viruses, the latter may lead to the development of new anti-viral strategies.Hepatitis C virus (HCV) is an enveloped positive-strand RNA virus possessing a single positive-strand RNA genome of- 9.6 kb. The genomic RNA consists of 3 regions: a 5'-untranslated region (5'-UTR), a single ORF and a 3'-untranslated region (3'-UTR). The ORF encodes a polyprotein of ~3010 amino acids, which is cleaved by cellular and viral proteases into at least 10 structural and non-structural proteins. As the non-structural protein NS5B provides RNA-dependent RNA polymerase (RdRp) activity, it is a key enzyme for viral genome replication. The replication of HCV genome, similar to other positive-strand RNA virus, comprises two steps: synthesis of complementary negative-strand RNA using the genomic positive-strand RNA as template and subsequent synthesis of the positive-strand viral RNA using the newly synthesized negative-strand RNA as template. Therefore both 3' terminal regions of HCV positive- and negative-strand RNA are assumed to be important for the initiation of the replication of HCV genome. Asymmetric replication has been observed when the infecting HCV genomic RNA being replicated: the quantities of positive-strand RNA are 10- to 100-fold surpassed over those of negative-strand RNA, demonstrating that there is a strong template selection in HCV genomic replication. The molecular events involved in this process remain unclear. To address this issue, we investigated the RNA synthesis in vitro from RNA templates corresponding to the 3' terminuses of HCV positive- and negative-strand RNA by a purified HCV NS5B.The HCV NS5B gene was cloned to a prokaryotic vector pET-His, then the recombinant plasmid was transformed into Escherichia coli BL21(DE3). To obtain thesoluble protein, the hydrophobic C-terminal 21 amino acids were deleted. The soluble NS5B protein was expressed after IPTG induction, and purified by the Ni-NTA affinity chromatography. The purified NS5B protein was confirmed by Western blot analysis.The DNA sequences corresponding to the 3' terminuses of HCV positive- and negative-strand RNA were cloned to the transcription plasmid pGEM3Zf(+). The 3' terminuses of positive- and negative-strand RNA were prepared via in vitro transcription, and used as RNA templates for RdRp assay respectively. Northern blot and RT-PCR were employed to detect the products from these two templates. No products were detected from the positive-strand RNA; however, a full-length product was generated from negative-strand RNA, demonstrating 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. Therefore, the NS5B exhibited template selection on negative-strand RNA from these two templates. The template selection on negative-strand RNA suggests that in vivo the first step of HCV genomic replication (synthesis negative-strand RNA from positive-strand RNA) is under control, whereas the second step (synthesis positive-strand RNA from negative-strand RNA) can process on by NS5B independently. Thus, it provides a reasonable explanation for the asymmetric replication of HCV genome. In addition, these findings suggest that there must be other viral or cellular factors involve in the initiation of RNA synthesis from positive-strand RNA. Which protein acts this role and its characters remain to be determined.The product from negative-strand RNA was a full-length positive-strand RNA, demonstrating the initiation of RNA synthesis belongs to de novo fashion. De novo initiation demands RNA template harbors cw-acting elements at its 3' terminus for RNA synthesis. To determine the properties of the negative-strand RNA, different mutagenesis analyses were performed to evaluate the importance of the 3'-proximal stem-loop and the first 3'-cytidylate (3'-C) of the negative-strand RNA in the synthesis of the positive-strand RNA. Deletion of the 3'-proximal stem-loop resulted in -90% decrease in RNA synthesis. Disruption of the 3'-proximal stem-loop structure by nucleotide substitutions led to 70-80% decrease in RNA synthesis. However, the restoration of thestem-loop by compensatory mutations in the stem region restored also the RNA synthesis. These results show that it is the secondary structure, not the sequence of the 3'-proximal stem-loop of negative-strand RNA that paly an important role in the synthesis of positive-strand RNA. Likewise, the deletion of the first 3'C or substitution by guanylate (G) led to 90% decrease in the RNA synthesis; while the substitution by adenylate (A) or uridylate (U) resulted in 60-80% decrease in the RNA synthesis. These findings demonstrate that the 3'-proximal stem-loop and the first 3'C of the negative-strand RNA of HCV are two cw-acting elements involved in the synthesis of the positive-strand RNA. The 3'-proximal sequence/structure of positive-strand RNA, however, does not possess these two elements (the 3' nucleotide is U), which may lead to a weak template activity in vitro.