Activation of Apoptosis by a Simian Virus 5 Mutant

描述（由申请人提供）：细胞对感染的抗病毒反应可能是感染结果、病毒趋向性和病毒发病机制的重要决定因素。副黏液病毒类人猿病毒5 （SV5）感染大多数细胞后，其细胞病变作用最小，细胞持续感染，病毒的高滴度可长时间产生。因此，SV5具有非常有效的机制来避免宿主抗病毒途径的激活，从而导致细胞死亡。与rSV5-WT相比，我们发表的数据表明，宿主凋亡途径被含有P/V基因自然取代的重组SV5 （rSV5-P/V- cpi -）感染激活。该rSV5- P/V-CPI-激活的凋亡通路的基本特征尚未确定。本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。在这个短期的R03试点项目中，我们将确定细胞病变SV5 P/V突变激活的途径，并验证WT rSV5感染导致caspase-3失活的假设。在目的1中，我们将确定受高度细胞病变的SV5变异rSV5-P/V-CPI-感染激活的凋亡途径。对感染rSV5-P/V突变体的人肺细胞凋亡事件的时间进程研究将确定这种细胞病变突变体是主要通过外源性caspase-8途径还是内源性caspase-9途径诱导细胞凋亡。值得注意的是，我们的初步结果表明，感染WT SV5的人肺细胞表现出活性caspase-3酶免疫染色的时间依赖性丧失。我们假设WT SV5感染导致caspase-3多肽的缺失，caspase-3亚细胞位置的改变或细胞凋亡抑制剂（IAP）表达的增加。在目标2中，我们将通过免疫荧光、western blotting和RNAse保护试验来区分这些假设。人们对病毒在对抗宿主抗病毒反应中所采用的机制有着浓厚的兴趣。我们的初步数据表明，SV5具有有效的机制来抵消宿主凋亡途径的激活，并且我们已经掌握了一组确定的突变体，它们在阻断这些步骤方面存在缺陷。在这个项目完成后，我们将建立一个坚实的基线，从中我们可以追求有关副粘病毒与宿主抗病毒途径相互作用的详细机制问题。

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.