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。冠状病毒是正链RNA病毒，其引起一系列疾病，从相对轻微的呼吸道疾病（由于HCoV-NL 63、229 e或OC 43引起的哮吼和普通感冒样症状）到人类严重急性呼吸综合征（SARS）爆发。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。在此之前，我们确定了SARS-CoV的木瓜蛋白酶样蛋白酶（PLpro）作为病毒复制和致病的关键决定因素。在冠状病毒复制期间，输入基因组RNA被翻译以产生复制酶多蛋白，其必须被病毒木瓜蛋白酶样蛋白酶和3C样蛋白酶加工以产生复制复合物。我们发现，PLpro过程中的复制酶多聚蛋白在三个网站使用的共识识别位点LXGG，类似的共识序列识别的去泛素化酶（DUBs）。我们解决了PLpro的X射线晶体结构，并证明它确实是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，并且完全干扰素拮抗可能不需要催化活性。我们假设冠状病毒木瓜蛋白酶样蛋白酶结构域内的不同残基对于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。在这里，我们建议通过比较和对比SARS-CoV，HCoV-NL 63和鼠冠状病毒A59的木瓜蛋白酶样蛋白酶的活性来研究冠状病毒木瓜蛋白酶样蛋白酶的生物学。我们的具体目标是：2）确定冠状病毒木瓜蛋白酶样蛋白酶内对干扰素拮抗作用重要的残基，并鉴定与PLPs相互作用以阻断先天免疫的细胞蛋白; 3）确定木瓜蛋白酶样蛋白酶与其结合伴侣之间蛋白质-蛋白质相互作用所涉及的结构域、氨基酸和结合能;和4）确定PLpro或PLP 2结构域的突变是否改变泛素或ISG 15依赖性先天免疫应答的逃避。这项研究的结果将使我们能够确定介导木瓜蛋白酶样蛋白酶特异性的关键位点，并为先天免疫应答的发病机制和逃避提供新的见解。 公共卫生相关性：我们项目的目标是确定病毒的特定部分如何导致严重急性呼吸系统综合征（SARS）。我们建议使用生物化学和分子生物学的方法来研究SARS冠状病毒蛋白酶，并确定这种蛋白质的潜在“阿喀琉斯之踵”。我们的研究结果将使我们能够确定病毒蛋白酶中的位点，这些位点可能是抗病毒药物开发的靶点，也可能是改进疫苗开发的靶点。

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.