Viral interaction with host eIF2alpha kinases

总结 面对抗病毒反应，病毒使用许多策略来翻译它们的 mRNA，否则这些策略将导致抗病毒反应。 阻断病毒蛋白质的合成。影响蛋白质合成的最突出的抗病毒机制是 蛋白激酶 R (PKR) 使真核起始因子 2α (eIF2αα) 磷酸化。人类腺病毒 (hAds) 通过阻止 PKR 激活的病毒相关 (VA) RNA 克服了这一问题。然而，小鼠腺病毒 1 型 (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 eIF2α 激酶 GCN2 在抗病毒反应中的作用尚未得到充分认识，但它对 MAV-1 具有抗病毒作用。 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。长期目标是增加有关腺病毒与宿主相互作用的基础知识， 促成疾病。该应用的总体目标是确定 MAV-1 如何调节宿主 eIF2α 激酶并在这些宿主抗病毒防御存在的情况下成功复制。中央 假设 MAV-1 通过降低 PKR 水平来克服宿主抗病毒反应，而 MAV-1 激活但不逃避巨噬细胞（病毒复制的靶细胞）中的 GCN2。这个假设是基于 根据初步数据，PKR 在 MAV-1 感染中耗尽，GCN2 缺陷型巨噬细胞产生更多 病毒，而 GCN2 缺陷的小鼠更容易受到感染，并表达更多的促炎细胞因子。 MAV-1感染率高于对照小鼠。基本原理是了解 MAV-1 如何诱导和对抗宿主 抗病毒反应将能够操纵这些反应并在强大的体内测试其效果 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，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 发病机制和分子病毒学检测方法。 实验室。在目标 1 中，初步数据表明，在感染过程中 PKR 通过蛋白酶体降解，并且 PKR 翻译被抑制将得到验证和扩展，以确定其机制以及是否特定 病毒基因是罪魁祸首。方法将使用病毒突变体、PKR-/- 小鼠和细胞。目标 2 将解决如何 GCN2 的缺乏会导致更高的小鼠死亡率和促炎症反应，以及是否以及如何导致 MAV-1 使用病毒突变体、野生型和 GCN2-/- 小鼠和细胞诱导 GCN2 激活。方法 具有创新性，因为它将首次阐明 DNA 病毒如何诱导 PKR 耗竭，并且它将 扩大对 eIF2α 激酶 GCN2 作为宿主抗病毒反应的关注。这项研究意义重大，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.