【作者】 叶力；

【导师】 叶林柏；

【作者基本信息】 武汉大学 ， 微生物学， 2005， 博士

【摘要】 DNA和RNA都是遗传物质,人们对于DNA的复制机制已经有了比较透彻的了解,但对RNA的复制还不清楚。RNA的复制为病毒所特有,利用正链RNA病毒开展有关RNA复制的研究在分子生物学和遗传学基本理论方面具有重要意义,同时也可以加强对RNA病毒的认识,并为抗病毒治疗提供新的策略。 丙型肝炎病毒(hepatitis C virus,HCV)是一种具囊膜的正链RNA病毒,基因组长约9.6kb,由5’编码区、3’编码区和一个大的开放阅读框架(ORF)组成。ORF编码一个约3000氨基酸残基组成的多聚蛋白前体,经宿主蛋白酶和HCV编码的蛋白酶切割后产生病毒的结构蛋白和非结构蛋白,其中非结构蛋白NS5B具有依赖于RNA的RNA聚合酶(RNA-dependent RNA polymerase,RdRp)活性,是HCV基因组复制的关键酶。HCV的基因组复制和其它正链RNA病毒类似,先以正链RNA为模板合成出互补的负链RNA,然后再以新合成的负链RNA为模板合成子代正链RNA,因此一致认为HCV正链RNA和负链RNA的3’端序列在起始病毒基因组复制的RNA合成中起重要作用。HCV在基因组复制时出现不对称复制:产生的正链RNA数量比负链RNA多得多,前者是后者的10—100倍,这一现象表明HCV基因组复制时对HCV正、负链RNA模板的选择性是非常强的。目前尚不清楚模板选择性的机制。本论文利用纯化的HCV NS5B蛋白以及HCV正链、负链3’末端RNA,在体外条件下研究NS5B利用两种模板进行RNA合成的情况,以探讨HCV基因组模板选择性的机制。 将HCV NS5B基因克隆至原核表达载体pET-His,转化至大肠杆菌BL21(DE3)中进行表达。为提高蛋白的可溶性表达,将NS5B C末端疏水性的21个氨基酸缺失,在一定的培养条件下,经过IPTG诱导获得可溶性蛋白。进一步通过组氨酸亲合层析获得纯化的蛋白,经Western blot检测确定是HCV NS5B蛋白。 把对应于HCV正、负链RNA3’末端序列的DNA序列克隆至体外转录质粒pGEM3Zf(+),通过体外转录制备HCV正链、负链3’末端RNA,分别作为RNA模板进行RdRp反应,用Northern blot和RT-PCR检测产物合成情况,结果没有检测到以正链RNA为模板的负链RNA产物,而以负链RNA为模板,可以合成一条全长的正链RNA产物;进一步将两种模板混合,进行模板竞争性实验,正链RNA模更多还原

【Abstract】 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.更多还原