Membrane rearrangement by positive-sense RNA viruses: Molecular mechanisms and cellular responses

脂质膜介导细胞内区室化，并在细胞器结构和生理学中发挥关键作用。大约三分之一的蛋白质是膜嵌入的，它们的活性受到脂膜的理化性质的调节。因此，细胞结构和功能取决于膜生理学和稳态的适当调节，但其潜在机制仍知之甚少。病毒长期以来一直被认为是剖析基础细胞生物学的绝佳工具。正义RNA病毒（或（）ssRNA病毒）是一大类广泛重塑宿主细胞内膜的病毒。我们使用 ( )ssRNA 病毒作为工具来了解调节脂膜重塑和稳态的基本机制，这对于维持正常细胞功能至关重要。 我们研究的主题之一是了解 ()ssRNA 病毒如何利用宿主因子和细胞机制来重新排列细胞膜。使用丙型肝炎病毒（HCV）作为模型（）ssRNA病毒，我们确定宿主蛋白亲环蛋白A（CypA）是HCV诱导的细胞内膜重排的关键参与者。我们现在寻求定义 CypA 如何调节膜重排。 CypA 与 HCV 蛋白 NS5A 相互作用，后者在膜重排中发挥核心作用。我们假设 CypA-NS5A 相互作用调节脂质运输以驱动细胞内膜重塑。 为了维持细胞功能，膜扰动必须由细胞“缓冲”。我们研究的第二个主题是表征细胞如何响应膜重排。我们假设细胞将膜扰动（例如由 ( )ssRNA 病毒引起的扰动）感知为应激信号，并激活恢复膜稳态的细胞反应。我们最近的研究结果表明 CypA 在调节蛋白激酶 R (PKR) 依赖性应激反应中发挥作用。引人注目的是，CypA 抑制似乎激活了 PKR 依赖性抗病毒程序，该程序对 ( )ssRNA 病毒（即重排膜的病毒）特别有效。我们现在将确定 CypA 如何调节影响膜重排的 PKR 依赖性反应。此外，我们将探讨其他应激相关蛋白在感知细胞内膜扰动中的作用。 意义：我们预计我们的发现将为调节脂膜稳态和细胞内膜重塑的机制提供新的理解。通过加深我们对这些基本细胞过程的理解，我们预计我们的发现对于研究细胞生物学许多方面的研究人员来说非常重要，从脂质代谢到膜蛋白再到细胞内在的抗病毒反应。更广泛地说，我们的工作将通过在细胞和分子生物学领域最先进的技术方面培训多名总部专家，使加拿大受益，为我们未来的科学领导者提供在自然科学职业中脱颖而出所需的专业知识。

Lipid membranes mediate intracellular compartmentalization and play a key role in organelle architecture and physiology. Approximately one third of proteins are membrane-embedded, and their activities are regulated by the physiochemical properties of the lipid membrane. Cellular structure and function therefore depend on proper regulation of membrane physiology and homeostasis, yet the underlying mechanisms are poorly understood. Viruses have long been considered excellent tools to dissect fundamental cellular biology. Positive-sense RNA viruses (or (+)ssRNA viruses) are a large group of viruses that extensively remodel host intracellular membranes. We use (+)ssRNA viruses as tools to understand fundamental mechanisms regulating lipid membrane remodeling and homeostasis, which are crucial for the maintenance of normal cellular function. One theme of our research is to understand how (+)ssRNA viruses co-opt host factors and cellular mechanisms to rearrange membranes. Using hepatitis C virus (HCV) as a model (+)ssRNA virus, we identified the host protein cyclophilin A (CypA) as a key player in HCV-induced intracellular membrane rearrangement. We now seek to define how CypA regulates membrane rearrangements. CypA interacts with the HCV protein NS5A, which has a central role in membrane rearrangement. We hypothesize that the CypA-NS5A interaction regulates lipid trafficking to drive intracellular membrane remodelling. In order to maintain cellular functionality, membrane perturbation must be “buffered” by the cell. A second theme of our research is to characterize how cells respond to membrane rearrangements. We hypothesize that cells sense membrane perturbation (such as that induced by (+)ssRNA viruses) as a stress signal and activate cellular responses that restore membrane homeostasis. Our recent findings suggest a role for CypA in regulating protein kinase R (PKR)-dependent stress responses. Strikingly, CypA inhibition appears to activate a PKR-dependent antiviral program that is particularly potent against (+)ssRNA viruses (i.e., viruses that rearrange membranes). We will now determine how CypA regulates PKR-dependent responses that impact membrane rearrangement. Furthermore, we will probe the role of other stress-related proteins in sensing intracellular membrane perturbation. Significance: We anticipate that our findings will provide new understanding of the mechanisms regulating lipid membrane homeostasis and remodelling of intracellular membranes. By advancing our understanding of these fundamental cellular processes, we expect our findings will be important for researchers studying many aspects of cell biology, ranging from lipid metabolism to membrane proteins to cell intrinsic antiviral responses. More broadly, our work will benefit Canada by training several HQP in state-of-the-art techniques in cellular and molecular biology, providing our future scientific leaders with the required expertise to excel in natural science careers.