MECHANISM OF RNA REPLICATION OF NEGATIVE-STRAND VIRUSES

这项提议的目标有两个方面。第一，通过什么机制 负链RNA病毒水泡口炎病毒(VSV)复制 它的基因组RNA将被调查。第二，企业内部控制机制。 对野生型VSV RNA基因组复制的干扰 将对VSV的缺陷干扰(DI)粒子进行研究。这两个都是 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。这个系统是 来源于受感染的细胞，并已被证明支持复制 VSV基因组RNA，以及将其封装到核衣壳中。一个 两种VSV蛋白(N和NS)的复合体先前已被鉴定为 在支持基因组RNA的体外复制方面发挥积极作用。尝试将 来提纯这种活性蛋白质复合体，并研究这种复合体是如何 参与控制从转录到复制的转换 病毒核糖核酸。还将进行实验，以确定这是否 在体外分离的蛋白质之间可以形成复合体，如果是这样的话， 如果这个复合体能够支持RNA复制。这个建筑群将是 检查宿主细胞组件的存在，如果检测到， 将进行实验以确定这些细胞蛋白是否 在建筑群中发挥作用。我还计划生产单抗。 针对病毒的NS和L蛋白，然后用这些来确定 这些蛋白质在病毒RNA体外复制中的作用。因为这些 蛋白质可能是多功能的，是一种单一局部抗体的集合 针对每种蛋白质的特定位点可能有助于确定它们在 复制。缺陷干扰(DI)粒子的机制 将对VSV干扰VSV RNA复制的情况进行调查 将纯化的DI颗粒添加到标准VSV RNA复制反应中 正链和负链42S RNA相对敏感度的测定 被DI抑制。还将评估对合成的影响，如果 VSV在这些反应中处于领先地位。两个温度敏感(Ts)突变体 在体外RNA复制系统中将采用VSV。这些是 携带N蛋白损伤的IV组突变体tsG41和 II突变，NS蛋白受损。这两个突变体都有 非温度敏感型病毒粒子相关转录酶和产生 病毒mRNA在不允许的温度下，但每个都无法复制 病毒基因组RNA在不允许的温度下。这些T的影响 损伤将在体外进行评估。

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.