Mechanisms of viral proteases in coronavirus replication and pathogenesis

描述（由申请人提供）：我们研究的长期目标是确定病毒蛋白酶和干扰素拮抗活性在冠状病毒复制和发病机制中的作用。冠状病毒是阳性链RNA病毒，可引起一系列疾病，从相对轻微的呼吸道疾病（由HCoV- NL63、229e或OC43引起的群感冒和普通感冒样症状）到人类严重急性呼吸道综合征（SARS）暴发。迄今为止，还没有有效的疫苗或抗病毒药物来限制任何冠状病毒感染的发病机制。显然，我们需要识别和了解有助于有效病毒复制和发病机制的病毒和宿主因素，以帮助开发新的治疗方法。之前，我们发现sars冠状病毒的木瓜蛋白酶（PLpro）是病毒复制和发病的关键决定因素。在冠状病毒复制过程中，输入的基因组RNA被翻译成一种复制酶多蛋白，这种多蛋白必须经过病毒木瓜蛋白酶和3c样蛋白酶的加工才能产生复制复合体。我们发现PLpro使用LXGG的共识识别位点在三个位点处理复制酶多蛋白，类似于去泛素化酶（DUBs）识别的共识序列。我们解决了PLpro的x射线晶体结构，并证明它确实是一个DUB。然而，其病毒DUB活性在发病机制中的作用仍有待确定。此外，我们发现PLpro具有干扰素拮抗活性，而完全拮抗干扰素可能不需要催化活性。我们假设冠状病毒的木瓜蛋白酶结构域内的不同残基对介导底物特异性和干扰素拮抗作用至关重要，并且这些位点的鉴定将为抗病毒干预提供新的靶点。本研究拟通过比较SARS-CoV、HCoV-NL63和小鼠冠状病毒A59的木瓜蛋白酶活性，探讨冠状病毒木瓜蛋白酶的生物学特性。我们的具体目标是：1)确定木瓜蛋白酶结构域内的位点，这些位点对多蛋白加工、去泛素化和去isgylation活性至关重要；2)确定冠状病毒木瓜蛋白酶中对干扰素拮抗重要的残基，确定与PLPs相互作用阻断先天免疫的细胞蛋白；3)确定木瓜蛋白酶及其结合伙伴之间蛋白-蛋白相互作用的结构域、氨基酸和结合能；4)确定PLpro或PLP2结构域的突变是否会改变泛素或isg15依赖性先天免疫反应的逃避。这项研究的结果将使我们能够确定介导木瓜蛋白酶特异性的关键位点，并为病毒的发病机制和逃避先天免疫反应提供新的见解。

DESCRIPTION (provided by applicant): The long term goal of our research is to determine the role of viral protease and interferon antagonism activity in coronavirus replication and pathogenesis. Coronaviruses are positive strand RNA viruses which cause a range of illness, from relatively mild respiratory disease (croup and common cold like symptoms due to HCoV- NL63, 229e or OC43) to severe acute respiratory syndrome (SARS) outbreaks in humans. To date, there are no effective vaccines or antiviral drugs to limit the pathogenesis of any coronavirus infection. Clearly, we need to identify and understand the viral and host factors that contribute to efficient viral replication and pathogenesis to aid in the development of new therapeutics. Previously, we identified the papain-like protease (PLpro) of SARS-CoV as a critical determinant of viral replication and pathogenesis. During coronavirus replication, the input genomic RNA is translated to produce a replicase polyprotein which must be processed by viral papain-like and 3C-like proteases to generate the replication complex. We showed that PLpro processes the replicase polyprotein at three sites using a consensus recognition site of LXGG, similar to the consensus sequence recognized by de-ubiquitinating enzymes (DUBs). We solved the X-ray crystal structure of PLpro and demonstrated that it is indeed a DUB. However, the role of its viral DUB activity in pathogenesis remains to be determined. In addition, we found that PLpro exhibits interferon antagonism activity and that catalytic activity may not be required for full interferon antagonism. We hypothesize that distinct residues within the papain-like protease domain of coronaviruses are critical for mediating substrate specificity and interferon antagonism and that identification of these sites will provide novel targets for antiviral intervention. Here, we propose to investigate the biology of coronavirus papain-like proteases by comparing and contrasting the activity of papain-like proteases of SARS-CoV, HCoV-NL63, and murine coronavirus A59. Our specific aims are to: 1) Identify sites within the papain-like protease domains that are critical for polyprotein processing, deubiquitinating and deISGylating activity; 2) Determine residues within coronavirus papain-like proteases that are important for interferon antagonism and identify cellular proteins that interact with PLPs to block innate immunity; 3) Determine the domains, amino acids, and binding energies involved in protein-protein interactions between papain-like proteases and their binding partners; and 4) Determine if mutation of the PLpro or PLP2 domain alters evasion of ubiquitin- or ISG15-dependent innate immune responses. The results from this research will allow us to identify critical sites that mediate specificity of papain-like proteases and provide new insight into viral mechanisms for pathogenesis and evasion of the innate immune response. PUBLIC HEALTH RELEVANCE: The goal of our project is to determine how specific parts of a virus contribute to causing severe acute respiratory syndrome (SARS). We propose to use biochemical and molecular methods to study the SARS coronavirus protease and identify potential "Achilles' heels" of this protein. The results from our research will allow us to identify sites in the viral protease that may be targeted for antiviral drug development or modified to improve vaccine development.