Role of autophagy during picornavirus replication.

自噬是蛋白质降解的一条重要途径，在细胞质中形成的膜泡将细胞质液体和细胞器输送到溶酶体进行降解。自噬由雷帕霉素(TOR)激酶和Akt/PKB信号通路的靶标控制，这些信号通路对营养缺乏做出反应。在静息条件下，TOR激酶是活跃的，并抑制自噬。自噬已被证明在发育和组织重塑、癌症以及与蛋白质聚集、衰老和细胞应激相关的疾病中发挥重要作用。自噬也是抵抗感染的一种重要的先天细胞防御，可以消灭细胞内的细菌。最近，人们认识到，自噬通过破坏溶酶体中的病毒，并通过向MHC II类蛋白和Toll受体递送降解产物，在先天抗病毒免疫中发挥重要作用。作为回应，辛德比斯病毒和疱疹病毒等病毒会产生抑制自噬的蛋白质。相反，新的证据表明，自噬可能促进而不是抑制(+)链RNA病毒的复制，如小核糖核酸病毒和冠状病毒。这项工作的主要重点是识别能够调节自噬和改变感染结果的病毒蛋白。口蹄疫病毒(FMDV；一种微小核糖核酸病毒)感染可诱导细胞内膜重排，产生泡状结构，被认为是病毒复制的平台。对于这一提议，我们已经证明了口蹄疫病毒的感染或病毒2C蛋白的表达诱导了自噬。这些观察为FMDV提出了一个悖论。看来，在复制过程中对细胞膜的需要迫使口蹄疫病毒激活一条破坏性途径，有可能在病毒离开细胞之前将其摧毁。在这项提议中，我们试图确定自噬的激活是否代表了对口蹄疫病毒感染的细胞防御，或者是操纵自噬小体为复制提供了平台。我们将利用我们的观察结果，即口蹄疫病毒的2C蛋白激活自噬，并将其作为探针来理解口蹄疫病毒如何激活自噬以产生用于复制的膜。我们还希望确定自噬途径一旦被口蹄疫病毒或2C激活后进展到什么程度，并看看这是否导致自噬小体/溶酶体融合。口蹄疫病毒可能与有可能将自噬小体用作复制地点的细菌一样，具有抑制自噬小体成熟并与溶酶体融合的机制，以防御自噬的抗病毒臂。这项工作代表了研究宿主-病原体相互作用的基础细胞生物学的一个新的和令人兴奋的领域。重要的是，这些研究将确定口蹄疫病毒在复制开始时的“宿主-病原体界面”，当时细胞中的病毒蛋白水平非常低，而且病毒特别容易受到专门阻止病毒复制的抗病毒试剂的攻击。此外，了解口蹄疫病毒调节自噬的方式将为病毒如何调节新发现的对感染的先天细胞反应提供有价值的见解。

Autophagy is an important pathway of protein degradation where membrane vesicles formed in the cytoplasm deliver cytoplasmic fluids and organelles to lysosomes for degradation. Autophagy is controlled by the target of rapamycin (TOR) kinase and Akt/PKB signalling pathways that respond to nutrient deprivation. Under resting conditions the TOR kinase is active and inhibits autophagy. Autophagy has been demonstrated to play important roles during development and tissue remodelling, cancer, and diseases associated with protein aggregation, ageing and cell stress. Autophagy also represents an important innate cellular defence against infection that can eliminate intracellular bacteria. Recently, it has been recognised that autophagy has the potential to play an important role in innate antiviral immunity by destroying viruses in lysosomes, and by presenting degradation products to MHC class II proteins, and Toll receptors. In response, viruses such as Sindbis virus and herpes viruses make proteins that inhibit autophagy. In contrast, new evidence suggests that autophagy may promote, rather than inhibit, the replication of (+) strand RNA viruses such as picornaviruses and coronaviruses. The main focus of this work is to identify viral proteins able to regulate autophagy and change the outcome of infection. Foot-and-mouth disease virus (FMDV; a picornavirus) infection induces rearrangements in intracellular membranes to produce vesicular structures that are believed to serve as platforms for virus replication. Significantly for this proposal we have shown that FMDV infection or expression of the viral 2C protein induces autophagy. These observations raise a paradox for FMDV. It looks as though a need for cellular membranes during replication forces FMDV to activate a destructive pathway with the potential to destroy virus before it can leave the cell. In this proposal we seek to determine if activation of autophagy represents a cellular defence against (FMDV) infection, or the manipulation of autophagosomes to provide platforms for replication. We will exploit our observation that the 2C protein of FMDV activates autophagy, and use this as a probe to understand how FMDV activates autophagy to generate membranes for replication. We also wish to determine how far the autophagy pathway progresses once it is activated by FMDV or 2C, and see if this results in autophagosome/lysosome fusion. It is possible that in common with bacteria that risk use of the autophagosome as a site for replication, FMDV has a mechanism to inhibit autophagosome maturation and fusion with lysosomes as a defence against the antiviral arm of autophagy. This work represents a new and exciting area for studying the basic cell biology of host-pathogen interactions. Importantly, these studies will define the 'host-pathogen interface' for FMDV at the start of replication, when the levels of viral proteins in cells are very low, and the virus is particularly vulnerable to antiviral reagents which specifically block virus replication. Furthermore, an understanding of the way in which FMDV regulates autophagy will give valuable insight into how the virus regulates this newly discovered innate cellular response to infection.