ARBOVIRUS REPLICATION IN MOSQUITO AND VERTEBRATE CELLS

有近400种节肢动物传播的病毒，其中许多导致严重的 人类疾病，分为5大家族。辛德毕斯病毒（SV） 披膜病毒科甲病毒属是本提议的主题。通过 研究Ae.纹伊蚊 蚊子细胞和脊椎动物细胞，我们已经能够分离出突变体 1）SVLM 21，其能够在蚊子中生长 已经被剥夺了甲硫氨酸并且具有低水平的ADO的细胞 met，2）SVmpa，其可以在已经处理过的蚊子细胞中生长 与霉酚酸，并有低水平的GTP，3）Svcp，这表明， 对蚊子细胞的细胞致病性增加，4）SVcl 35和SVc，158 5）SVAP 15/21， 限制在脊椎动物细胞中。利用重组DNA技术 和“感染性”Toto质粒，其含有一个cDNA拷贝的 辛德毕斯病毒RNA，我们正在绘制这些病毒的突变， 确定负责其表型的核苷酸变化;这 这种方法也使得将特定的生物化学物质 与各种SV非结构（NS）蛋白一起发挥功能。所以大多数 最近，我们已经能够使用SVLM，CDNA，将病毒RNA 甲基转移酶与非结构蛋白，nspl，并通过核酸 酸测序以鉴定SVLM中的两个氨基酸变化，nspl. 因为我们最终希望研究的是 nspl我们正在制定表达的构建策略 这些载体将使我们能够生产大量的 标准和突变型NSPL。以类似的方式，我们将使用SVmpa来 鉴定与RNA加帽活性相关的蛋白质（RNA 鸟苷酰转移酶）。这些研究的意义在于 到目前为止还没有严格的证据证明 可以将特定功能与四个SV ns中的任何一个相关联 proteins.由于SVLM 21和SVMPA是SV的仅有的突变体， 1）NS蛋白的变化和2）表明 改变特定的生化功能，它们将是无价的。 用于将至少两种功能与特定NS蛋白相关联。我们 从这些关于SV基因组及其复制的研究中学习 战略可能最终导致新的战略控制 医学上重要的病毒，由节肢动物传播。

There are almost 400 arthropod-borne viruses, many of which cause serious human disease, fall into 5 major families. Sindbis virus (SV) in the family Togaviridae, genus alphavirus, is the subject of this proposal. By studying the comparative aspects of viral replication in Ae. albopictus mosquito cells and vertebrate cells, we have been able to isolate mutants of SV with novel phenotypes: 1) SVLM21, which is able to grow in mosquito cells which have been deprived of methionine, and have low levels of ado met, 2) SVmpa, which can grow in mosquito cells which have been treated with mycophenolic acid, and have low levels of GTP, 3) Svcp which shows increased cytopathogenicity for mosquito cells, 4) SVcl35 and SVc,158 which are host-restricted in mosquito cells and 5) SVAP15/21 which is restricted in vertebrate cells. Making use of recombinant DNA technology and the "infectious" Toto plasmids which contain a CDNA copy of the Sindbis virus RNA, we are mapping the mutations in these viruses and determining the nucleotide changes responsible for their phenotypes; this approach is also making it possible to associate specific biochemical functions with various of the SV nonstructural (ns) proteins. Thus, most recently we have been able, using SVLM, CDNA, to associate the viral RNA methyltransferase with the nonstructural protein, nspl, and by nucleic acid sequencing to identify two amino acid changes in the SVLM, nspl. Since we wish eventually to study the structure-function relationships of nspl we are developing strategies for the construction of expression vectors which will enable us to produce large amounts of both the standard and mutant nspl. In a similar fashion we shall use SVmpa to identify the protein associated with the RNA capping activity (RNA guanylyltransferase). The significance of these studies lies in the fact that until now there has been little rigorous evidence which makes it possible to associate a specific function with any of the four SV ns proteins. Since SVLM2l and SVMPA, are the only mutants of SV which have both 1) a change in an ns protein and 2) a phenotype which points to an alteration in a specific biochemical function, they will be invaluable for associating at least two functions with specific ns proteins. What we learn from these studies concerning the SV genome and its replication strategy may ultimately lead to novel strategies for the control of medically important viruses, which are transmitted by arthropod vectors.