ARBOVIRUS REPLICATION IN MOSQUITO AND VERTEBRATE CELLS

近400种节肢动物传播的病毒(虫媒病毒)，其中许多 导致严重的人类疾病，已经被确认。这些病毒落在 分成五个不同的家庭。辛比斯病毒，这是一种蚊子- 传播的虫媒病毒，以及Togaviridae家族的原型病毒， 甲型病毒属，是这项提议的主题。 很多工作都是为了理解四个非结构的 辛德比斯病毒编码的(NS)蛋白在合成 病毒RNA。利用我国分离的两株辛德比斯病毒突变体的研究 实验室(SVLM21，能够在缺乏蛋氨酸的蚊子中生长 细胞和对霉酚酸具有抗性的SVMPA)。 强有力的证据表明，RNA鸟苷酸转移酶和 甲基转移酶活性(覆盖和甲基化 病毒mRNAs的5‘端)与ns蛋白nsP1结合。 尽管所有其mRNAs有5‘帽的细胞质RNA病毒都必须在 通过某种方式对修改其5‘末端所需活动进行编码 在任何情况下，mRNAs的催化位点都不参与甲基化或 确定了甲基供体S-腺苷蛋氨酸的结合部位。 这项建议的主旨是本地化和确定催化剂 NsP1上与甲基化(和鸟苷酸)相关的位点 病毒mRNAs。为此，我们将表示删除形式的nsP1并进行测试 以检测甲基转移酶的活性。我们将使用站点定向 突变以确定MTR活性所需的氨基酸残基，以及 通过光亲和标记的方法，我们将确定结合部位 ADOMet。此外，我们还将开发其他活动的分析方法。 参与病毒mRNAs(即RNA)的封端和甲基化 三磷酸酶和鸟苷酸转移酶)，并将确定是否表达 NsP1有这些活动。因为SVLM21和SVMPA突变映射到 NsP1编码序列，因为我们已经表达了 在大肠杆菌中具有酶活性的nsP1我们很好地进行了 这些实验。NsP1上催化位置的定义是 参与病毒mRNAs甲基化将促进理性 可能有效抑制NOT的抗病毒化合物的设计 不仅是甲型病毒，还有辛德比斯病毒中的其他病毒 超级大家庭。

Close to four hundred arthropod-borne viruses (arboviruses), many of which cause serious human disease, have been identified. These viruses fall into five different families. Sindbis virus, which is a mosquito- transmitted arbovirus, and the prototype virus of the family Togaviridae, genus alphavirus, is the subject of this proposal. Much effort is being directed at understanding how the four nonstructural (ns) proteins encoded by Sindbis virus function in the synthesis of the viral RNAs. Making use of two mutants of Sindbis virus isolated in my laboratory (SVLM21, which is able to grow in methionine-deprived mosquito cells and SVMPA, which is resistant to mycophenolic acid) we have obtained strong evidence which associates the RNA guanylyltransferase and methyltransferase activities (the activities which cap and methylate the 5' end of the viral mRNAs) with the ns protein, nsP1. Although all cytoplasmic RNA viruses whose mRNAs have a 5' cap, must in some way encode the activities needed to modify the 5' terminus of their mRNAs, in no case has the catalytic site involved in methylation or the binding site for S-adenosylmethonine, the methyl donor, been identified. The thrust of this proposal is to localize and identify the catalytic sites on nsP1 associated with the methylation (and the guanylylation) of viral mRNAs. To that end we will express deleted forms of nsP1 and test them for methyltransferase activity. We will use site-directed mutagenesis to identify amino acid residues required for MTr activity, and by means of photoaffinity labeling we shall identify the binding site for AdoMet. In addition, we shall develop assays for the other activities involved in capping and methylation of viral mRNAs (i.e. RNA triphosphatase and guanylyltransferase) and will determine if expressed nsP1 has these activities. Because the SVLM21 and SVMPA mutations map to the nsP1 coding sequence and because we have already expressed enzymatically active nsP1 in E. coli we are well positioned to carry out these experiments. Definition of the catalytic site on nsP1 which is involved in the methylation of viral mRNAs will facilitate the rational design of antiviral compounds which might be effective in inhibiting not only alphaviruses, but also other viruses in the Sindbis virus superfamily.