Structural and mechanistic studies of multifunctional proteases in virus replication and host immune evasion

多聚蛋白的合成和翻译后切割是正链（+）RNA病毒常用的基因组表达策略。将多聚蛋白切割成功能单位对病毒是必不可少的，并由病毒编码的蛋白酶进行，其中许多蛋白酶也靶向细胞蛋白以破坏宿主的抗病毒反应并协助病毒复制。我的NSERC研究计划探索病毒蛋白酶在+RNA病毒的复制和宿主免疫逃避策略中的多功能作用的分子基础。我目前专注于+RNA病毒从订单Nidovirales，其中包含病毒的动物和人类的重要性，包括动脉炎病毒，如马动脉炎病毒（EAV）和猪生殖和呼吸综合征病毒（PRRSV），以及严重急性呼吸综合征（SARS）和新兴的中东呼吸综合征（MERS）冠状病毒。 这些病毒表达两种多聚蛋白，其含有许多非结构蛋白（nsps），其中一个子集包括活性蛋白酶结构域。PLP2和PLpro是分别位于动脉炎病毒和冠状病毒的nsp 2和nsp 3内的蛋白酶。值得注意的是，除了作为内肽酶切割病毒多聚蛋白中的位点外，它们还作为异肽酶从细胞蛋白中去除翻译后修饰蛋白泛素（Ub）和ISG15以抑制Ub和ISG15依赖性宿主抗病毒途径。相比之下，nsp1A是PRRSV的一种蛋白酶，也参与多蛋白切割，但其辅助功能是通过与宿主蛋白PCBP 2形成RNA结合复合物来控制程序化的核糖体移码事件，从而在病毒基因组翻译期间产生额外的病毒蛋白，从而协助病毒复制。使用分子和结构生物学方法，我的目标是提供一个详细的了解不同的作用，PLP2，PLpro和nsp1蛋白在病毒的生命周期中发挥作用。我将确定使PLP2和PLpro能够破坏Ub和ISG15依赖性抗病毒途径的结构特征，开发基于紧密结合Ub突变体的PLP2和PLpro抑制剂以探测其底物结合特性和催化机制，并揭示nsp1tR如何与PCBP2形成RNA结合复合物以控制核糖体移码。这项研究将产生重要的新见解，了解病毒蛋白酶如何破坏宿主的抗病毒反应，并指导病毒蛋白的非经典翻译，使其成为病毒学家，生物化学家和细胞生物学家的广泛兴趣。参与研究的受训人员将受益于结构和分子生物学的高生产力研究计划。他们将获得他们需要成为学术界或工业界的高素质人才的技能，其中需要分子和结构生物学，生物化学和生物技术的工作知识。

The synthesis and posttranslational cleavage of polyproteins is a common genome expression strategy used by positive-strand (+)RNA viruses. Cleavage of the polyproteins into functional units is essential to the virus and is carried out by virus-encoded proteases, many of which also target cellular proteins to disrupt host antiviral responses and to assist with virus replication. My NSERC research program explores the molecular basis underlying the multifunctional role of viral proteases in the replication and host immune evasion strategies of +RNA viruses.***I currently focus on +RNA viruses from the order Nidovirales, which contains viruses of animal and human importance, including arteriviruses, such as equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV), as well as the severe acute respiratory syndrome (SARS) and the emerging Middle-East respiratory syndrome (MERS) coronaviruses. These viruses express two polyproteins that contain a number of non-structural proteins (nsps), a subset of which include active protease domains. PLP2 and PLpro are proteases located within nsp2 and nsp3 of arteriviruses and coronaviruses, respectively. Remarkably, in addition to acting as endopeptidases that cleave sites in the viral polyproteins, they also act as isopeptidases to remove the posttranslational modifier proteins ubiquitin (Ub) and ISG15 from cellular proteins to suppress Ub- and ISG15-dependent host antiviral pathways. In contrast, nsp1ß is a protease of PRRSV that also participates in polyprotein cleavage, yet its ancillary function is to assist virus replication by forming an RNA binding complex with the host protein PCBP2 to control programmed ribosomal frameshifting events that yield additional viral proteins during virus genome translation.***Using molecular and structural biology methods, I aim to provide a detailed understanding of the diverse roles that PLP2, PLpro and nsp1ß play during the virus life cycle. I will identify the structural features that enable PLP2 and PLpro to disrupt Ub- and ISG15-dependent antiviral pathways, develop inhibitors of PLP2 and PLpro based on tight-binding Ub mutants to probe their substrate binding properties and catalytic mechanisms, and reveal how nsp1ß forms an RNA binding complex with PCBP2 to control ribosomal frameshifting.***The research will yield important new insights into how viral proteases subvert host antiviral responses and direct the non-canonical translation of viral proteins, making it of wide interest to virologists, biochemists and cell biologists. Trainees engaged in the research will benefit from a highly productive research program in structural and molecular biology. They will gain the skills they need to become highly qualified personnel in academia or industry where a working knowledge of molecular and structural biology, biochemistry and biotechnology is required.**