MECHANISM OF RNA REPLICATION OF NEGATIVE-STRAND VIRUSES

负链病毒RNA复制机制

• 批准号: 3132820
• 负责人: RICHARD W PELUSO
• 金额: $ 10.03万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-06-01 至 1987-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3132820
• 关键词: Vesiculovirus; genetic transcription; interfering virus; messenger RNA; microorganism immunology; monoclonal antibody; temperature sensitive mutant; tissue /cell culture; virus genetics

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.

该提案的目标有两个。 首先，其机制 负链RNA病毒水泡性口病毒（VSV）复制 其基因组RNA将被研究。 其次，机制 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。 这两个 将使用我开发的体外系统来研究目标， 它非常适合研究这些问题。 这个系统是 源自受感染的细胞，并已被证明支持复制 VSV 基因组 RNA 及其衣壳化为核衣壳。 一个 两种 VSV 蛋白（N 和 NS）的复合物先前已被鉴定为 积极支持基因组 RNA 的体外复制。 尝试将 纯化这种活性蛋白复合物，并研究这种复合物如何 参与从转录到复制的转换的控制 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， 如果该复合物能够支持 RNA 复制。 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 在综合体中发挥作用。 我也计划生产单克隆抗体 针对病毒的 NS 和 L 蛋白，然后用它们来确定 这些蛋白质在病毒 RNA 体外复制中的作用。 由于这些 蛋白质可能是多功能的，是单克隆抗体的集合 针对每种蛋白质的特定位点可能有助于确定它们的作用 复制。 缺陷干扰 (DI) 粒子的机制 VSV 干扰 VSV RNA 复制的研究将通过 将纯化的 DI 颗粒添加到标准 VSV RNA 复制反应中， 确定正链和负链 42S RNA 的相对敏感性 DI 的抑制。 还将评估对合成的影响，如果 这些反应中 VSV 的领导者。 两个温度敏感 (ts) 突变体 VSV 将用于体外 RNA 复制系统。 这些都是 IV 组突变体 tsG41，在 N 蛋白中携带损伤，以及一组 II 突变体，NS 蛋白出现损伤。 这两个突变体都具有 非温度敏感病毒颗粒相关转录酶并产生 病毒 mRNA 在不允许的温度下，但均无法复制 病毒基因组 RNA 在不允许的温度下。 这些 ts 的影响 病变将在体外进行评估。