MECHANISM OF RNA REPLICATION OF NEGATIVE-STRAND VIRUSES

这项建议有两个目的。 第一， 负链RNA病毒水泡性口炎病毒（VSV）复制 将对其基因组RNA进行研究。 第二， 干扰野生型VSV RNA基因组的复制， 将研究VSV的缺陷干扰（DI）颗粒。 这两 目标将使用我开发的体外系统进行研究， 它非常适合研究这些问题。 该系统 来源于受感染的细胞，并已被证明支持复制 VSV基因组RNA，以及其进入核衣壳。 一 两种VSV蛋白（N和NS）的复合物先前已被鉴定为 在体外支持基因组RNA复制中具有活性。 尝试将 纯化这种活性蛋白质复合物，并研究这种复合物 参与控制从转录到复制的转换 病毒RNA。 还将进行实验以确定这是否 分离的蛋白质之间可以在体外形成复合物，如果是这样， 如果这个复合体能够支持RNA复制的话。 该综合体将是 检查宿主细胞成分的存在，如果检测到， 将进行实验以确定这些细胞蛋白是否 在复杂的功能。 我还计划生产单克隆抗体 针对病毒的NS和L蛋白，然后用这些来确定 这些蛋白质在体外病毒RNA复制中的作用。 由于这些 蛋白质可能是多功能的，是单域抗体的集合 针对每种蛋白质的特定位点可能有助于确定它们在 复制的 缺陷干扰（DI）颗粒 VSV干扰VSV RNA复制的研究将通过 将纯化的DI颗粒加入到标准VSV RNA复制反应中， 确定正链和负链42S RNA对 抑制DI。 还将评估对合成的影响， VSV在这些反应中的领导者。 两个温度敏感（TS）突变体 将VSV用于体外RNA复制系统。 这些是 IV组突变体tsG41，其携带N蛋白中的损伤，以及 II型突变体，NS蛋白有病变。 这两个变种人 非温度敏感性病毒体相关转录酶和产生 病毒mRNA在非允许的温度下，但每种都不能复制 病毒基因组RNA在非允许温度下。 这些影响 将在体外评估损伤。

The objective of this proposal is two fold. First, the mechanism by which the negative-stranded RNA virus vesicular stomatis virus (VSV) replicates its genome RNA will be investigated. Secondly, the mechanism of interference of the replication of wild-type VSV RNA genomes by defective-interfering (DI) particles of VSV will be studied. Both of these objectives will be investigated using an in vitro system which I developed, and which is ideally suited to study these problems. This system is derived from infected cells and has been shown to support replication of VSV genomic RNA, as well as its encapsidation into nucleocapsids. A complex of two VSV proteins (N and NS) has been previously identified as active in supporting replication of the genome RNA in vitro. Attempts will be made to purify this active protein complex, and study how this complex is involved in the control of the switch from transcription to replication of viral RNA. Experiments will also be performed to determine if this complex can be formed in vitro between the separated proteins, and if so, if this complex is able to support RNA replication. The complex will be examined for the presence of host cell components, and if detected, experiments will be performed to determine if these cellular proteins are functional in the complex. I also plan to produce monoclonal antibodies against the NS and L proteins of the virus, and then use these to determine the role of these proteins in viral RNA replication in vitro. Since these proteins may be multifunctional, a collection of monoclocal antibodies against specific sites of each protein may help define their role in replication. The mechanism by which defective-interfering (DI) particles of VSV interfere with the replication of VSV RNA will be investigated by adding purified DI particles to standard VSV RNA replicating reactions and determining the relative sensitivity of plus and minus stranded 42S RNA to inhibition by the DI. Effects will also be assessed on the synthesis if the VSV leader in these reactions. Two temperature-sensitive (ts) mutants of VSV will be employed in the in vitro RNA replicating system. These are a group IV mutant, tsG41, carrying a lesion in the N protein, and a group II mutant, with a lesion in the NS protein. Both of these mutants have non-temperature-sensitive virion-associated transcriptases and produce viral mRNA at the nonpermissive temperature, but each fails to replicate the viral genomic RNA at nonpermissive temperature. Affects of these ts lesions will be assessed in vitro.