Stress granules in virus infections

应激颗粒（stress granule，SG）是由停滞的mRNA、翻译因子、转录因子等组成的非膜性、相分离的复合体 以及响应于不同的应激信号在细胞质中瞬时形成的RNA结合蛋白， 包括病毒感染。在病毒感染期间形成的SG，抗病毒应激颗粒（avSG），不同于 典型的SG，因为它们招募抗病毒蛋白和信号传导途径的成分，作为一种抗病毒蛋白， 建立一个有效的抗病毒反应的平台。然而，其组成、时间变化和功能 病毒感染期间的avSG仍然知之甚少。SG的动态性质表明，宿主细胞使用 avSG作为抗病毒防御的信号中枢。另一方面，病毒已经进化出机制， 拮抗SG的形成和改变SG的用途以促进复制，突出了在病毒中的重要作用。 发病机制SG组成、组装和动力学的应力特异性差异如何影响 病毒感染的结果仍然不清楚，代表了我们对病毒感染的理解的一个关键差距。 信号事件和宿主-病毒相互作用。我们最近的工作突出了avSG作为平台的作用 用于通过dsRNA病原体相关分子模式激活模式识别受体（PRR） （PAMP）和随后的干扰素（IFN）应答。病毒感染期间产生的DsRNA 感染作为PAMP通过结合胞质Rig-like解旋酶来刺激干扰素（IFN）的产生 （RLH）、内体Toll样受体（TLR）、dsRNA依赖性蛋白激酶（PKR）或2 '-5'寡腺苷酸 合成酶（OAS）。此外，病毒感染诱导病毒和细胞RNA降解的活性， 干扰素诱导核糖核酸内切酶我们最近发现RNase L的dsRNA产物 激活PKR，磷酸化真核生物翻译起始因子-2（eIF 2）， 以抑制翻译并诱导avSG形成。我们的结果表明，avSG组件提供了一个 RNA配体与PRR有效相互作用的平台，以增强干扰素产生和抗病毒作用。 我们对这一提议的总体假设是，avSG的组成、组装和拆卸 在病毒感染期间改变，以允许宿主细胞通过限制损伤来响应和清除感染。在 我们的初步研究表明，RNase L的激活诱导了由SG蛋白G3 BP 1标记的avSG的形成 含有抗病毒蛋白Rig-I、PKR、OAS和RNase L。我们开发了一种新的方法来纯化avSG 和生物化学表征的蛋白质-蛋白质相互作用的avSG，将在本提案中使用。我们将 用以下具体目标来检验我们的中心假设。目的1：确定压力特异性差异， 标准SG和avSG中的SG组成，目的2：确定在正常情况下SG的动态变化的生理作用。 病毒感染中的avSG组成。实现这些目标将使我们能够在以下方面取得进展的基础上再接再厉： 纯化avSG以通过遵循组装或拆卸的动力学来剖析SG的抗病毒作用， 分析所述avSG组成以提供应激反应途径和先天信号传导的相关性。

Stress granules (SG) are non-membranous, phase-separated complexes of stalled mRNA, translation factors and RNA binding proteins that are formed transiently in the cytoplasm in response to diverse stress signals, including viral infections. SG formed during viral infection, antiviral stress granules (avSG), are distinct from canonical SG in that they recruit antiviral proteins and components of signaling pathways to function as a platform to mount an effective antiviral response. However, the composition, temporal changes and function of avSG during viral infections are still poorly understood. The dynamic nature of SG suggest that host cells use avSG as a signaling hub for antiviral defense. On the other hand, viruses have evolved mechanisms to antagonize SG formation and repurpose SG to facilitate replication highlighting an important role in viral pathogenesis. How the stress-specific differences in SG composition, assembly and dynamics impact the outcome of viral infections remains unclear and represents a critical gap in our understanding of signaling events and host-virus interactions. Our recent work has highlighted a role for avSG as a platform for the activation of pattern recognition receptors (PRRs) by dsRNA pathogen associated molecular patterns (PAMPs) and subsequent interferon (IFN) response during viral infections. DsRNAs produced during viral infections serve as PAMPs to stimulate interferon (IFN) production by binding the cytosolic Rig-like helicases (RLHs), endosomal Toll-like receptors (TLRs), dsRNA-dependent protein kinase (PKR) or 2’-5’oligoadenylate synthetase (OAS). Furthermore, virus infection induces degradation of viral and cellular RNAs by the activity of interferon-induced endoribonuclease, RNase L. We have recently shown that dsRNA products of RNase L activity function as PAMPs and activate PKR, phosphorylate eukaryotic translation initiation factor-2 (eIF2) to suppress translation and induce avSG formation. Our results demonstrate that avSG assembly provides a platform for efficient interaction of RNA ligands with PRRs to enhance interferon production and antiviral effect. Our overarching hypothesis for this proposal is that composition, assembly and disassembly of avSG change during viral infection to allow host cells to respond and clear infection by limiting damage. In preliminary studies we show that activation of RNase L induces avSG formation marked by SG protein, G3BP1 and contain antiviral proteins Rig-I, PKR, OAS and RNase L. We developed a novel method to purify avSG and biochemically characterized protein-protein interactions in avSG that will be used in this proposal. We will test our central hypothesis with the following specific aims. Aim 1: To determine stress-specific differences in SG composition in canonical SG and avSG, Aim 2: To determine the physiological role of dynamic change in avSG composition in virus infections. Completion of these aims will allow us to build on the advances in purifying avSG to dissect the antiviral role of SG by following the dynamics of assembly or disassembly and analyzing the avSG composition to provide correlation of stress response pathways and innate signaling.