Activation of Apoptosis by a Simian Virus 5 Mutant

描述（由申请方提供）：对感染的细胞抗病毒应答可能是感染结局、病毒嗜性和病毒发病机制的重要决定因素。用副粘病毒猿猴病毒5（SV 5）感染大多数细胞导致最小的细胞病变效应，细胞保持持续感染并且长时间产生高滴度的病毒。因此，SV 5具有非常有效的机制来避免可导致细胞死亡的宿主抗病毒途径的激活。与rSV 5-WT相比，我们发表的数据表明，宿主凋亡途径被感染重组SV 5（rSV 5-P/V-CPI-）激活，该重组SV 5在P/V基因中含有天然存在的取代。由该rSV 5- 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SV 5-WT阻止细胞死亡途径激活的机制。在这个短期R 03试点项目中，我们将确定细胞病变SV 5 P/V突变体激活的途径，并测试WT rSV 5感染导致caspase-3失活的假设。在目标1中，我们将鉴定由高度致细胞病变的SV 5变体rSV 5-P/V-CPI-感染激活的凋亡途径。对感染rSV 5-P/V突变体的人肺细胞中凋亡事件的时间进程研究将确定该致细胞病变突变体是否主要通过外源性半胱天冬酶-8途径或内源性半胱天冬酶-9途径诱导凋亡。值得注意的是，我们的初步结果已经证明，感染WT SV 5的人肺细胞显示出对活性半胱天冬酶-3酶的免疫染色的时间依赖性损失。我们假设WT SV 5感染导致caspase-3多肽的丢失、caspase-3亚细胞位置的改变或细胞凋亡抑制因子（IAP）表达的增加。在目标2中，我们将通过免疫荧光、蛋白质印迹和RNA酶保护测定来区分这些假设。有强烈的兴趣，在确定机制所采用的病毒在他们的战斗对宿主的抗病毒反应。我们的初步数据表明，SV 5具有有效的机制，以抵消激活宿主凋亡途径，我们手头上有一组定义的突变体，是有缺陷的，在阻止这些步骤。在这个项目完成后，我们将建立一个坚实的基线，我们可以追求详细的机制问题，副粘病毒与宿主抗病毒途径的相互作用。

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.