Activation of Apoptosis by a Simian Virus 5 Mutant

猿猴病毒 5 突变体激活细胞凋亡

• 批准号: 6877082
• 负责人: Griffith D. Parks
• 金额: $ 7.18万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6877082
• 关键词: Paramyxoviridae; RNase protection assay; apoptosis; cell growth regulation; cysteine endopeptidases; enzyme activity; enzyme inhibitors; flow cytometry; gene mutation; host organism interaction; human tissue; immunofluorescence technique; lung; respiratory infections; simian virus; virus cytopathogenic effect; western blottings

DESCRIPTION (provided by applicant): The cellular anti-viral response to infection can be an important determinant of the outcome of an infection, viral tropism, and viral pathogenesis. Infection of most cells with the paramyxovirus Simian Virus 5 (SV5) results in minimal cytopathic effect, cells remain persistently infected and high titers of virus are produced for long periods. Thus, SV5 has very potent mechanisms to avoid activation of host anti-viral pathways that can lead to cell death. By contrast to rSV5-WT, our published data have shown that host apoptotic pathways are activated by infection with a recombinant SV5 (rSV5-P/V-CPI-) containing naturally occurring substitutions in the P/V gene. The basic features of the apoptotic pathways activated by this rSV5- P/V-CPI- have not been determined. The long-term goal of this research project is to determine the mechanisms by which SV5-WT prevents activation of cellular death pathways. In this short term R03 pilot project we will determine the pathways activated by the cytopathic SV5 P/V mutant and test the hypothesis that infection with WT rSV5 leads to inactivation of caspase-3. In aim 1, we will identify the apoptotic pathways that are activated by infection with the highly cytopathic SV5 variant rSV5-P/V-CPI-. Time course studies of apoptotic events in human lung cells infected with the rSV5-P/V mutant will determine if this cytopathic mutant induces apoptosis primarily through the extrinsic caspase-8 pathway or the intrinsic caspase-9 pathway. Remarkably, our preliminary results have demonstrated that human lung cells infected with WT SV5 show a time-dependent loss of immuno-staining for active caspase-3 enzyme. We hypothesize that WT SV5 infection results in either a loss of caspase-3 polypeptide, a change in subcellular location of caspase-3 or an increase in expression of cellular inhibitors of apoptosis (IAP). In aim 2, we will distinguish between these hypotheses through immunofluorescence, western blotting, and RNAse protection assays. There is intense interest in defining mechanisms employed by viruses in their battle against host antiviral responses. Our preliminary data have shown that SV5 has potent mechanisms to counteract activation of host apoptotic pathways, and we have in hand a set of defined mutants that are defective in blocking these steps. At the completion of this project, we will have established a firm baseline from which we can pursue detailed mechanistic questions concerning interactions of paramyxoviruses with host antiviral pathways.

描述（由申请人提供）：细胞抗病毒对感染的反应可能是感染，病毒疗法和病毒发病机理结果的重要决定因素。大多数细胞感染拟氧化病毒邻苯二醇病毒5（SV5）导致细胞病变效应最少，细胞仍然持续感染，并且长期产生了高滴度。因此，SV5具有非常有效的机制，可以避免激活可能导致细胞死亡的宿主抗病毒途径。与RSV5-WT相反，我们已发布的数据表明，宿主凋亡途径通过含有P/V基因中天然发生的自然替代的重组SV5（RSV5-P/V-CPI）感染而激活。该RSV5- P/V-CPI激活的凋亡途径的基本特征尚未确定。该研究项目的长期目标是确定SV5-WT阻止细胞死亡途径激活的机制。在这个短期R03试点项目中，我们将确定由细胞病SV5 P/V突变体激活的途径，并检验以WT RSV5感染导致CASPase-3失活的假设。在AIM 1中，我们将确定通过高度细胞病SV5变体RSV5-P/V-CPI激活的凋亡途径。感染RSV5-P/V突变体的人肺细胞中凋亡事件的时间过程将确定该细胞疗法突变体是否主要通过外在的Caspase-8途径或内部caspase-9途径诱导凋亡。值得注意的是，我们的初步结果表明，感染WT SV5的人肺细胞显示为活性caspase-3酶的免疫染色的时间依赖性丧失。我们假设WT SV5感染会导致caspase-3多肽的损失，caspase-3的亚细胞位置的变化或细胞凋亡（IAP）细胞抑制剂表达的增加。在AIM 2中，我们将通过免疫荧光，蛋白质印迹和RNase保护分析区分这些假设。定义病毒在与宿主抗病毒反应的斗争中使用的机制有强烈的兴趣。我们的初步数据表明，SV5具有抵消宿主凋亡途径激活的有效机制，并且我们手头有一组定义的突变体，这些突变体在阻止这些步骤时有缺陷。该项目完成后，我们将建立一个牢固的基线，我们可以从中提出有关帕马班病毒与宿主抗病毒途径相互作用的详细机械问题。