Autophagy mechanism of coronaviral infection: Lessons from enteroviruses

我的研究计划的长期目标是了解阳性RNA病毒如何劫持宿主细胞机器以实现成功感染的分子基础。包括肠道病毒(EVS)和冠状病毒(CoVS)在内的正链RNA病毒覆盖了所有病毒属的三分之一以上，并感染包括植物、动物和人类在内的广泛宿主。之前的NSERC Discovery Grant使我们在了解柯萨奇病毒(EV)进化到利用宿主循环系统支持其生命周期的潜在机制方面取得了实质性进展。在这一更新应用中，我建议将我们的研究扩展到研究CoV和自噬之间的相互作用，自噬是细胞中的一种“自噬”过程，细胞通过这种过程回收受损的蛋白质和/或细胞器。冠状病毒是一组被冠状蛋白尖峰覆盖的单链RNA病毒，可感染人类和各种动物，并引起从无症状到致命性感染的一系列疾病。我的假设是，与EV类似，CoV通过促进病毒复制和防止病毒成分和/或颗粒的清除来颠覆宿主自噬机制，以确保成功感染。这一假说是基于我们新的观察结果，即(1)冠状病毒通过一条与传统自噬所需的酶无关的途径诱导自噬；(2)冠状病毒编码的蛋白酶针对参与自噬调节的许多宿主蛋白进行降解。尽管有这些发现，但CoV如何劫持宿主自噬途径的详细机制以及自噬蛋白裂解的功能后果仍不清楚。为了验证这一假设，我们将结合使用尖端的蛋白质组学和成像方法与传统的分子和细胞技术来研究冠状病毒如何操纵自噬途径，并确定调节冠状病毒感染的自噬途径中的关键酶和靶蛋白。我们在研究肠道病毒对自噬途径的颠覆方面的经验使我们处于一个独特的位置，可以理解宿主自噬和冠状病毒之间的相互作用。这项研究探索了一种新的基于自噬的病毒机制，将填补我们在理解宿主细胞有效感染冠状病毒的机制方面的一个关键知识空白。从这项研究中获得的知识也将对同一家族中其他病毒的宿主-病原体相互作用的研究产生更广泛的影响。拟议的研究计划还将通过培训3名自然科学博士研究生和5名本科生，为未来在学术研究和教学机构、医学实验室和生物技术公司的职业生涯，使加拿大和加拿大经济受益。

The long-term objective of my research program is to understand the molecular basis of how positive RNA viruses hijack the host cellular machinery to achieve successful infection. Positive-strand RNA viruses, including enteroviruses (EVs) and coronaviruses (CoVs), cover more than one-third of all virus genera and infect a wide range of hosts, including plants, animals, and humans. The previous NSERC Discovery Grant allowed us to make substantial progress towards understanding the underlying mechanisms by which Coxsackievirus (an EV) has evolved to exploit the host recycling system to support its lifecycle. In this renewal application, I propose to extend our research to study the interaction between CoVs and autophagy, a "self-eating" process in cells by which cells recycle damaged proteins and/or organelles. CoVs are a group of single-stranded RNA viruses covered by crown-like protein spikes, which can infect humans and a variety of animals, and cause a range of disorders from asymptomatic to lethal infection. My hypothesis is that similar to EVs, CoVs subvert the host autophagy machinery to ensure successful infection by enhancing viral replication and through preventing the clearance of viral components and/or particles. This hypothesis is based on our novel observations that (1) CoVs induce autophagy through a pathway independent of enzymes known to be required for traditional autophagy; and (2) CoV-encoded proteases target a number of host proteins involved in autophagy regulation for degradation. Despite these findings, the detailed mechanisms of how CoVs hijack the host autophagy pathway and the functional consequence of the cleavage of autophagic proteins remain unclear. To test this hypothesis, we will combine the use of cutting-edge proteomics and imaging approaches with conventional molecular and cellular technologies to study how CoVs manipulate the autophagy pathway and to identify the critical enzymes and target proteins within the autophagy pathway that regulates CoV infection. Our experience in investigating enteroviral subversion of the autophagy pathway places us in a unique position to understand the interplay between host autophagy and CoVs. This research, exploring a novel autophagy-based viral mechanism, will fill a key knowledge gap in our understanding of the mechanisms underlying effective CoV infection of host cells. Knowledge acquired from this research will also have broader implications for the study of host-pathogen interactions of other viruses in the same family. The proposed research program will also benefit Canada and the Canadian economy by training 3 PhD graduate and 5 undergraduate students in natural science for future careers in academic research and teaching institutions, medical laboratories, and biotechnology companies.