【作者】 伊光辉；

【导师】 张楚瑜；

【作者基本信息】 武汉大学 ， 微生物学， 2004， 博士

【摘要】 猪瘟病毒(Classical swine fever virus, CSFV)是一种具有囊膜的正链RNA病毒，属于黄病毒科瘟病毒属。其基因组由5’非编码区、3’非编码区和一个大的开放阅读框(ORF)组成。ORF编码一种由约3898个氨基酸残基组成的多聚蛋白，该多聚蛋白在病毒和细胞酶的作用下加工成4种结构蛋白和8种非结构蛋白，其中非结构蛋白NS5B，即RNA依赖的RNA聚合酶(RdRp)，是病毒复制酶最重要的组成部分。 一般认为猪瘟病毒基因组RNA的复制同其它正链RNA病毒RNA的复制类似：首先以基因组正链RNA为模板合成互补的负链RNA，然后再以合成的负链为模板起始子代病毒基因组正链RNA的合成，因此基因组正链和负链RNA模板的3’末端可能含有起始RNA复制所必需的顺式作用调控元件，如启动子、增强子等。目前研究表明正链RNA病毒RNA依赖的RNA聚合酶起始基因组RNA合成的分子机制主要有两种：引物依赖的RNA合成(Primer-dependent RNAsynthesis)和引物非依赖的RNA合成即从头合成(De novo RNA synthesis)。已有报道表明，表达猪瘟病毒NS5B蛋白的昆虫细胞粗提液起始RNA的合成主要是通过引物依赖的RNA合成机制(为模板引物)，然而昆虫细胞中存在着末端转移酶的活性，有可能在RNA模板3’末端添加额外的核苷酸并作为引物起始RNA合成，导致RNA合成方式发生改变。 为了深入了解猪瘟病毒起始基因组正链和负链RNA合成的分子机制，同时建立体外RdRp复制系统，我们利用RT-PCR的方法扩增NS5B基因，并在大肠杆菌BL21(DE3)中表达NS5B蛋白。为了提高蛋白的可溶性表达，将NS5B C末端24个氨基酸的疏水序列缺失，并构建表达载体pET-NS5BA 24，通过组氨酸亲和层析柱纯化到一定量的可溶性蛋白。RT-PCR和实时荧光定量PCR的结果证明从大肠杆菌中纯化的NS5BA94蛋白具有RNA依赖的RNA聚合酶活性。 分别以病毒正常的基因组RNA和3’-OH封闭的RNA为模板，通过Northernblot分析和RT-PCR的方法对猪瘟病毒RdRp起始病毒RNA合成的分子机制进行研究。结果表明，猪瘟病毒RdRp主要是通过引物非依赖的3’末端从头合成机制起始病毒正链和负链RNA的合成。在以负链RNA为模板的反应中发现，RdRp除了以从头合成机制起始RNA合成外，还可以通过模板转换的方式合成高分子量的RNA。随后以(-)IRES为模板，对RdRp合适的反应条件进行摸索，发现csFv RdRP既能在镁离子(MgZ+)也能在锰离子(N加2+)存在的反应体系中起始RNA合成，但在N伪2+存在的情况下其活性更高，如果没有MgZ+或N位2+离子Rdl切就不能合成新生的RNA。高浓度三磷酸核昔酸(N仰)预孵育能够增强Rdl切合成正链和负链RNA的能力。将正链和负链RNA模板3’末端的胞啥吮C (即模板起始核昔酸，T+:)突变为尿啼陡(U)、腺嚎吟(A)或鸟嗓吟(G)都将导致RNA合成能力明显下降，表明模板3’末端起始核昔酸对起始RNA合成具有重要作用。将正链RNA模板的3’末端C突变为U后，其活性要比突变为G或A时的活性相对要高，而负链RNA模板的3’末端C突变为G后，Rd助也能合成少量的新生RNA，表明3’末端的其它序列对起始RNA合成可能也有一定的影响。 在病毒Rdl切起始基因组RNA合成过程中，RNA依赖的RNA聚合酶通过与起始三磷酸核昔酸(NTPi)，模板起始核昔酸(T+:)以及模板RNA等形成起始复制复合体以后才能有效起始病毒基因组RNA的合成。本实验通过No川五一Westem blot的方法证明了复性后的CSFV Ns5B似4蛋白能够与正链和负链RNA模板相结合，模板竞争试验表明这种结合是特异性的，表明Ns5BC末端的疏水序列对RdRP的识别与结合活性是非必需的.体外RdRP复制系统的建立为深入研究CSFV基因组RNA复制的顺式、反式作用调控元件奠定了基础。 细胞转染试验发现CSFV全长RNA依赖的RNA聚合酶主要是定位在核膜周围，而C末端截短的RdRP则分布于细胞核内，并形成聚集状。表明虽然RdRPC末端24氨基酸的疏水序列对RdRP的识别与结合活性是非必需的，但它可能作为膜锚定序列起作用。将RdRPC末端24个氨基酸的疏水序列连接到绿色荧光蛋白(GFP)下游，瞬时表达发现24个氨基酸不能完全将外源GFP引导至细胞核膜周围。 对猪瘟病毒NSSA基因的功能了解甚少，一般认为非结构蛋白是病毒复制酶的组成成分，在病毒基因组RNA的复制中起调控作用。我们通过体外表达GsT.Ns5A的融合蛋白来探讨Ns5A在CSFv RdRP起始RNA合成中的作用。细胞转染试验发现NSSA分布于细胞核和细胞质内。更多还原

【Abstract】 Classical swine fever virus (CSFV) is a small enveloped positive-strand RNA virus classified in the genus of Pestivirus, together with the genera Flavivirus and Hepacivirus, they form the family Flavivirdae. The genomic RNA contains a single long open reading frame (ORF) encoding a polyprotein of about 3898 amino acids which is flanked by 5’ and 3’ untranslated regions (UTR). This polyprotein is processed co- and/or posttranslationally into four structural proteins and eight nonstructural proteins (NS) by viral and cellular proteases. One of the nonstructural proteins, NS5B, which encodes an RNA-dependent RNA polymerase, is a key component of viral replicase responsible for the replication of viral RNA genome.The replication of CSFV genome is generally thought to be similar to other positive strand RNA viruses: synthesis of complementary minus-strand RNA with the plus-strand genomic RNA as template and subsequent synthesis of the progeny RNA with the minus-strand RNA as template. Thus, 3’ end of both plus- and minus-strand RNAs may contain the cw-acting elements, such as promoter or enhancer, involved in the initiation of viral RNA synthesis by RdRp. At present, it has been shown that positive strand RNA viruses initiate RNA synthesis by either of two major mechanisms: primer-dependent RNA synthesis and primer-independent de novo RNA synthesis. Previous reports have shown that recombinant CSFV NS5B (expressed in insect cells) catalyzed RNA synthesis was strictly primer-dependent and that intramolecular priming copy-back synthesis represented the preferred mechanism for initiation of RNA synthesis. However, the activity of cellular terminal nucleotidyle transferase (TNTase) was also demonstrated to present in the cytoplasmic extracts of insect cells. Since cellular TNTase could add extras nucleotide to the 3’terminus of the RNA template and might serve as primer for template-primed copy-back synthesis, it might not represent the original initiation of viral RNA synthesis in vitro.To better understand the initiating of CSFV RNA replication and to establish an in vitro RdRp replication system, we first cloned the NS5B gene by RT-PCR from the pig serum infected with CSFV Shimen strain, and then expressed this protein in Escherichia coli BL21 (DE3). To improve the solubility of the NS5B, 24 amino acids of the C-terminal was deleted, and expression was performed at low temperature. The truncated fusion protein (NS5BA24) was purified on a Ni-chelating HisTrap affinitycolumn and demonstrated to possess RNA-dependent RNA polymerase by RT-PCRand real-time quantitative PCR.Using the plus-strand RNA (+)3’UTR and minus-strand RNA (-)IRES as templates, we performed Northern blot and RT-PCR to study the mechanism of initiating CSFV RNA synthesis by RNA-dependent RNA polymerase. The results showed that CSFV RdRp preferred to initiate either plus- or minus-strand viral RNA synthesis by a primer-independent de novo manner but not primer-dependent RNA synthesis in vitro. Besides de novo RNA synthesis, high molecular RNA products, which were produced by template-switch mechanism were also observed with the minus-strand RNA as template. Using (-)IRES as template, the optimal reaction conditions were found. Both divalent cations Mg2+ and Mn2+ supported de novo initiation, however, RNA synthesis was more efficient in the presence of Mn2+ than in presence of Mg2+. De novo initiation of RNA synthesis was stimulated by preincubation with high concentration of NTP, and a 3’-terminal cytidylate on the viral plus- and minus-strand RNA templates was preferred for de novo initiation. Mutations of the 3’-terminal cytidylate on the viral RNA templates to guanidylate, adenylate or uridylate results in dramatically decreasing RNA synthesis. These results suggested that the presence of a 3’terminus cytidylate at viral RNA templates was necessary for efficient de novo RNA synthesis by CSFV RdRp.Initiation of RNA synthesis required the initiating recognition and specific binding between RNA-dependent RNA polymerase and template RNA. T更多还原