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

多功能蛋白酶在病毒复制和宿主免疫逃避中的结构和机制研究

• 批准号: RGPIN-2015-05310
• 负责人: Mark, Brian
• 金额: $ 3.28万
• 依托单位: University of Manitoba
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=613475
• 关键词: Structural; mechanistic; studies; multifunctional; proteases

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.

多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。多蛋白切割成功能单位对病毒至关重要，由病毒编码的蛋白酶执行，其中许多还以细胞蛋白为靶标，以扰乱宿主的抗病毒反应，并帮助病毒复制。我的NSERC研究项目探索了病毒蛋白酶在+RNA病毒的复制和宿主免疫逃避策略中多功能作用的分子基础。 我目前专注于Nidovirales目的+RNA病毒，其中包含对动物和人类重要的病毒，包括动脉病毒，如马动脉炎病毒(Eav)和猪繁殖与呼吸综合征病毒(PRRSv)，以及严重急性呼吸综合征(SARS)和新出现的中东呼吸综合征(MERS)冠状病毒。这些病毒表达两种多蛋白，其中包含许多非结构蛋白(NSP)，其中一部分包括活性蛋白酶域。PLP2和PLPro分别位于动脉病毒和冠状病毒的NSP2和NSP3中。值得注意的是，除了作为内肽酶在病毒多蛋白中裂解位点外，它们还作为异肽酶从细胞蛋白中移除翻译后修饰蛋白泛素(Ub)和ISG15，以抑制依赖Ub和ISG15的宿主抗病毒途径。相比之下，nsp1？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主要宿主蛋白PCBP2形成RRNA结合复合体来帮助病毒复制，以控制程序性核糖体移码事件，这些事件可能会在病毒基因组翻译过程中产生额外的病毒蛋白。 利用分子和结构生物学方法，我的目标是详细了解PLP2、PLPro和nsp1？在病毒生命周期中所扮演的不同角色。我将确定使PLP2和PLPro能够破坏依赖Ub和ISG15的抗病毒途径的结构特征，开发基于紧密结合的Ub突变体的PLP2和PLPro的抑制剂，以探索它们的底物结合特性和催化机制，并揭示nsp1？是如何与PCBP2形成RNA结合复合体来控制核糖体的移码。 这项研究将对病毒蛋白酶如何颠覆宿主的抗病毒反应产生重要的新见解，并指导病毒蛋白的非规范翻译，使其引起病毒学家、生物化学家和细胞生物学家的广泛兴趣。从事这项研究的受训者将从结构和分子生物学的高产出研究计划中受益。他们将获得所需的技能，成为学术界或工业中需要分子和结构生物学、生物化学和生物技术方面的实用知识的高素质人员。