Role of autophagy during picornavirus replication.

自噬是蛋白质降解的重要途径，其中在细胞质中形成的膜囊泡将细胞质液和细胞器递送到溶酶体进行降解。自噬是由雷帕霉素（TOR）激酶和Akt/PKB信号通路的目标，响应营养剥夺控制。在静息条件下，TOR激酶是活性的并抑制自噬。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。自噬也代表了一种重要的先天细胞防御感染，可以消除细胞内的细菌。最近，人们已经认识到，自噬有可能通过破坏溶酶体中的病毒，并通过将降解产物呈递给MHC II类蛋白和Toll受体，在先天性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产生抑制自噬的蛋白质。相比之下，新的证据表明，自噬可能促进而不是抑制（+）链RNA病毒（如小核糖核酸病毒和冠状病毒）的复制。这项工作的主要重点是确定能够调节自噬和改变感染结果的病毒蛋白。口蹄疫病毒（FMDV;一种小核糖核酸病毒）感染诱导细胞内膜重排，产生被认为是病毒复制平台的囊泡结构。对于该提议，重要的是，我们已经表明FMDV感染或病毒2C蛋白的表达诱导自噬。这些观察结果为FMDV提出了一个悖论。看起来好像在复制过程中需要细胞膜迫使FMDV激活一个破坏性的途径，有可能在病毒离开细胞之前破坏病毒。在这个提议中，我们试图确定自噬的激活是否代表了对FMDV感染的细胞防御，或者操纵自噬体以提供复制平台。我们将利用我们观察到的FMDV的2C蛋白激活自噬，并将其用作探针来了解FMDV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FMDV或2C激活后进展多远，并观察这是否导致自噬体/溶酶体融合。可能与有风险使用自噬体作为复制位点的细菌一样，FMDV具有抑制自噬体成熟和与溶酶体融合的机制，作为对自噬的抗病毒臂的防御。这项工作代表了一个新的和令人兴奋的领域，研究宿主-病原体相互作用的基础细胞生物学。重要的是，这些研究将定义FMDV在复制开始时的“宿主-病原体界面”，此时细胞中病毒蛋白的水平非常低，并且病毒特别容易受到特异性阻断病毒复制的抗病毒试剂的影响。此外，了解FMDV调节自噬的方式将有助于深入了解病毒如何调节这种新发现的先天细胞对感染的反应。

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.