Viral interaction with host eIF2alpha kinases

摘要 病毒使用许多策略来翻译它们的mRNAs，以应对否则会产生的抗病毒反应 阻断病毒蛋白质合成。影响蛋白质合成的最突出的抗病毒机制是 蛋白激酶R对真核细胞启动因子2(eIF2)的磷酸化作用。人腺病毒 (HADS)通过阻止PKR激活的病毒相关(VA)RNA克服这一点。然而，鼠腺病毒 类型1(MAV-1)不编码VA RNA；MAV-1如何成功克服PKR尚不清楚。的作用 另一种eIF2MAV2激酶，GCN2，在抗病毒反应中的作用还不是很好，但它对-1是抗病毒的。 作为抗病毒药物的一部分，迫切需要了解PKR和GCN2如何影响病毒复制 回应。长期目标是增加对腺病毒-宿主相互作用的基础知识， 会导致疾病。本申请的总体目标是确定MAV-1如何调制宿主 在这些宿主抗病毒防御系统存在的情况下，eIF2激活并成功复制。中环 假设MAV-1通过降低PKR水平来克服宿主的抗病毒反应，而MAV-1 激活巨噬细胞中的GCN2，但不逃避GCN2，巨噬细胞是病毒复制的目标细胞。这一假设是有根据的 根据初步数据，在MAV-1感染中PKR被耗尽，GCN2缺陷的巨噬细胞产生更多 病毒和GCN2缺陷小鼠更容易感染，并在 MAV-1感染小鼠明显高于对照组小鼠。其基本原理是了解MAV-1是如何诱导和抵消宿主的 抗病毒反应将使操纵这些反应并在强大的体内测试它们的效果成为可能 模型，这将增加对疾病的了解。具体目标是确定1)MAV-1如何 对抗宿主PKR反应，以及2)宿主GCN2反应如何保护小鼠免受MAV-1感染。 这些目标将使用申请人建立的MAV-1致病机理和分子病毒学分析 实验室。在目标1中，初步数据表明，在感染期间，PKR通过蛋白酶体和 PKR翻译是被禁止的，将被验证和扩展以确定其机制以及是否特定 病毒基因对此负有责任。方法将使用病毒突变体、PKR-/-小鼠和细胞。Aim 2将解决如何 缺乏GCN2会导致更高的小鼠死亡率和促炎反应，以及是否和如何 MAV-1利用病毒突变体、野生型和GCN2-/-小鼠和细胞诱导GCN2激活。该方法 具有创新性，因为它将首次阐明DNA病毒是如何导致PKR耗尽的，而且它将 扩大对eIF2GCN2作为宿主抗病毒反应的研究重点。。这项研究具有重要意义，因为它 预计与病毒-宿主相互作用和人类疾病具有根本相关性。

Summary Viruses use many strategies to translate their mRNAs in the face of antiviral responses that would otherwise block viral protein synthesis. The most prominent antiviral mechanism affecting protein synthesis is phosphorylation of eukaryotic initation factor 2 (eIF2 by protein kinase R (PKR). Human adenoviruses (hAds) overcome this via virus-associated (VA) RNAs that block PKR activation. However, mouse adenovirus type 1 (MAV-1) does not encode VA RNAs; how MAV-1 successfully overcomes PKR is unknown. The role of another eIF2 kinase, GCN2, in antiviral responses is less well appreciated, but it is antiviral against MAV-1. There is a critical need to understand how PKR and GCN2 impact viral replication as part of the antiviral response. The long-term goal is to increase fundamental knowledge about adenovirus-host interactions that contribute to disease. The overall objective of this application is to determine how MAV-1 modulates host eIF2 kinases and successfully replicates in the presence of these host antiviral defenses. The central hypothesis is that MAV-1 overcomes the host antiviral response by reducing PKR levels, whereas MAV-1 activates but does not evade GCN2 in macrophages, target cells of viral replication. This hypothesis is based on preliminary data that PKR is depleted in MAV-1 infections, GCN2-deficient macrophages produce more virus, and GCN2-deficient mice are more susceptible and express more proinflammatory cytokines upon MAV-1 infection than control mice. The rationale is that knowing how MAV-1 induces and counteracts the host antiviral responses will enable manipulation of these responses and testing their effects in a powerful in vivo model, which will increase understanding of disease. The specific aims are to determine 1) how MAV-1 counters the host PKR response, and 2) how the host GCN2 response protects mice from MAV-1 infection. These aims will use assays of MAV-1 pathogenesis and molecular virology established in the applicant's laboratory. In Aim 1, preliminary data showing that during infection PKR is degraded via the proteasome and PKR translation is inhibited will be validated and extended to identify the mechanisms and whether specific viral genes are responsible. Approaches will use viral mutants, PKR-/- mice and cells. Aim 2 will address how the lack of GCN2 leads to higher mouse mortality and proinflammatory responses, and whether and how MAV-1 induces GCN2 activation, using viral mutants, and wild type and GCN2-/- mice and cells. The approach is innovative because it will elucidate for the first time how a DNA virus induces depletion of PKR, and it will broaden the focus on eIF2 kinase GCN2 as a host antiviral response. The research is significant because it is expected to have fundamental relevance to virus-host interactions and human disease.