Mechanisms of viral proteases in coronavirus replication and pathogenesis

 描述(申请人提供)：我们研究的目标是确定病毒蛋白水解酶如何在冠状病毒(CoV)的复制和发病机制中发挥作用。冠状病毒是一类正链RNA病毒，包括严重急性呼吸综合征(SARS)冠状病毒和中东呼吸综合征(MERS)冠状病毒，它们是人类具有大流行潜力的重要病原体。先前，我们剖析了冠状病毒类木瓜蛋白酶(PLP)的多功能性质，发现PLP通过去除与蛋白质上赖氨酸残基结合的泛素(Ub)或ISG15来切割病毒复制酶多蛋白，作为脱泛素酶(DUBS)和脱糖酶(DeISGs)，并且PLP可能通过去泛素化信号分子来拮抗先天免疫反应。利用详细的生化和PLP-Ub共晶结构分析，我们在体外和基于细胞的检测中确定了冠状病毒PLP中对酶活性有不同影响的残基。我们最初的研究是用SARS-CoV PLPro进行的。在这里，我们提供了初步的体外和结构数据，证明这些结果可以扩展到BSL-2模型小鼠肝炎病毒冠状病毒(MHV-A59)木瓜蛋白样蛋白酶。我们假设多功能PLP/DUB活性在病毒致病中起作用，选择性地干扰DUB活性将激活先天免疫，降低病毒致病能力。为了验证这一假设，我们将确定修饰的PLP/DUB酶活性是否改变了病毒复制、先天免疫反应或致病机制。我们将使用反向遗传学来产生编码PLP的小鼠CoV，这些PLP具有不同的酶谱，例如DUB缺陷、脱糖基化缺陷或过度活性的蛋白酶。这些新型病毒将在细胞培养和小鼠体内进行评估，以研究病毒RNA合成的动力学、感染性病毒的产生以及天然免疫反应的激活动力学。为了将这些研究扩展到其他冠状病毒，我们将测定包括BAT冠状病毒PLP在内的α和β冠状病毒木瓜蛋白酶样酶的酶谱(EP)和酶指纹(EF)。我们将表达来自10个不同冠状病毒物种的PLP结构域，并确定每个酶的多肽切割活性、去泛素活性、去ISGyl化活性和赖氨酸连接偏好。有了这个概况，我们将使用现有的和新的X射线结构相结合，以指导突变实验，以差异地破坏DUB活性，并识别与DUB活性降低相关的指纹。我们还将确定差异活动在调节蝙蝠细胞的先天免疫反应中的作用。此外，我们还确定了一个 这些研究将揭示冠状病毒ADP-核糖-1“-磷酸酶(ADRP)结构域与PLP的相互作用，以及这种相互作用对酶活性、病毒复制和致病机制的影响。这些研究将揭示有关病毒蛋白酶/DUB活性的新信息，这些信息将有助于设计针对冠状病毒和其他蛋白酶/DUB编码病毒的抗病毒疗法和疫苗。

 DESCRIPTION (provided by applicant): The goal of our research is to determine how viral proteases function in the replication and pathogenesis of coronaviruses (CoVs). CoVs are a family of positive strand RNA viruses and include Severe Acute Respiratory Syndrome (SARS) CoV and Middle East Respiratory Syndrome (MERS) CoV which are significant human pathogens with pandemic potential. Previously, we dissected the multifunctional nature of CoV papain-like proteases (PLPs) and found that CoV PLPs cleave the viral replicase polyprotein, act as deubiquitinases (DUBs) and deISGylases (deISGs) by removing ubiquitin (Ub) or ISG15 conjugated to lysine residues on proteins, and that PLPs can antagonize the innate immune response, likely by deubiquitylating signaling molecules. Using detailed biochemical and PLP-Ub co-crystal structural analysis, we identified residues within CoV PLPs that differentially affec enzymatic activity in vitro and in cell-based assays. Our initial studies were performed using SARS-CoV PLpro. Here we provide preliminary in vitro and structural data demonstrating that these results can be extended to the BSL-2 model coronavirus mouse hepatitis virus (MHV-A59) papain-like protease. We hypothesize that multifunctional PLP/DUB activity contributes to viral pathogenesis and that selectively disrupting DUB activity will allow activation of innate immunity and reduced viral pathogenesis. To test this hypothesis, we will determine if a modified PLP/DUB enzymatic activity alters viral replication, innate immune response or pathogenesis. We will use reverse genetics to generate murine CoVs encoding PLPs with distinct enzymatic profiles such as DUB deficient, deISGylation deficient, or hyperactive protease. These novel viruses will be evaluated in cell culture and in mice for kinetics of viral RNA synthesis, production of infectious virus, and kinetics of activation of innate immune responses. To extend these studies to other CoVs, we will determine the enzymatic profile (EP) and enzymatic fingerprint (EF) of alpha- and beta-CoV papain-like proteases including bat CoV PLPs. We will express the PLP domain from 10 different CoV species and determine the peptide cleavage activity, deubiquitinating activity, deISGylating activity, and lysine-linkage preferences for each enzyme. With this profile in hand, we will use existing and new X-ray structures combined to guide mutagenesis experiments to differentially disrupt DUB activity and identify the fingerprint associated with reduced DUB activity. We will also determine the role of differential activity in regulating the innate immune response in bat cells. Also, we identified an interaction of the CoV ADP-ribose-1"-phosphatase (ADRP) domain with PLP and we will determine the effect of modifying this interaction on enzymatic activity, viral replication and pathogenesis. These studies will reveal new information on viral protease/DUB activity that will be useful for designing antiviral therapies and vaccines for coronaviruses and other protease/DUB-encoding viruses.