ARBOVIRUS REPLICATION IN MOSQUITO AND VERTEBRATE CELLS

近四百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 五个不同的家庭。 辛德毕斯病毒，是一种蚊子- 传播的虫媒病毒，以及披膜病毒科的原型病毒， 甲病毒属是本提案的主题。 许多努力都是针对理解如何四个非结构性的 (ns)辛德毕斯病毒编码的蛋白质在合成 病毒RNA。 利用我所分离的两株辛德毕斯病毒突变株， 实验室（SVLM 21，其能够在缺乏甲硫氨酸蚊子中生长 细胞和SVMPA，这是耐霉酚酸），我们已经获得 强有力的证据表明，RNA鸟苷酰转移酶和 甲基转移酶活性（将甲基转移酶的活性加帽和甲基化） 病毒mRNA的5'末端）与ns蛋白nsP 1的连接。 虽然所有的细胞质RNA病毒，其mRNA有一个5'帽，必须在细胞质中， 以某种方式编码修饰其5'末端所需的活性， mRNA，在任何情况下都没有参与甲基化的催化位点， 结合位点的S-腺苷甲基，甲基供体，已确定。 这项建议的主旨是定位和确定催化剂， nsP 1上与甲基化（和鸟苷酰化）相关的位点 病毒mRNA。 为此，我们将表达nsP 1的缺失形式，并测试 甲基转移酶活性。 我们将使用网站导向 诱变以鉴定MTr活性所需的氨基酸残基，和 通过光亲和标记，我们将鉴定出 我见过。 此外，我们还将开发其他活动的测定方法， 参与病毒mRNA（即RNA）的加帽和甲基化 三磷酸酶和鸟苷酰转移酶），并将确定是否表达 nsP 1具有这些活动。 因为SVLM 21和SVMPA突变映射到 nsP 1编码序列，因为我们已经表达了 在E.我们有能力进行 这些实验。 nsP 1上催化位点的定义， 参与病毒mRNAs甲基化的基因将促进 设计可能有效抑制非病毒的抗病毒化合物 不仅是甲病毒，还有辛德毕斯病毒中的其他病毒， 超家族

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.