Mechanisms of viral proteases in coronavirus replication and pathogenesis

描述(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。冠状病毒是一种正链RNA病毒，可导致一系列疾病，从相对轻微的呼吸道疾病(由HCoV-NL63、229E或OC43引起的喉咙和普通感冒症状)到人类爆发的严重急性呼吸综合征(SARS)。到目前为止，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病毒有效复制和致病的病毒和宿主因素，以帮助开发新的治疗方法。在此之前，我们发现SARS冠状病毒的木瓜蛋白酶(PLPro)是病毒复制和致病的关键决定因素。在冠状病毒复制过程中，输入的基因组RNA被翻译成复制酶多蛋白，该复制酶多蛋白必须被病毒类木瓜蛋白酶和类3C蛋白酶处理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，证明了它确实是一种配音。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。我们假设冠状病毒类木瓜蛋白酶结构域中的不同残基对于介导底物特异性和干扰素拮抗至关重要，这些位点的识别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-NL63和小鼠冠状病毒A59的木瓜蛋白样酶的活性来研究冠状病毒木瓜蛋白样蛋白酶的生物学特性。我们的具体目标是：1)确定木瓜蛋白样蛋白酶域中对多蛋白加工、去泛素化和去ISGyl化活性至关重要的位置；2)确定冠状病毒木瓜蛋白样蛋白酶中对干扰素拮抗重要的残基，并确定与PLP相互作用以阻断先天免疫的细胞蛋白；3)确定木瓜蛋白样蛋白酶及其结合伙伴之间的蛋白-蛋白质相互作用所涉及的结构域、氨基酸和结合能；以及4)确定PLPro或PLP2结构域的突变是否改变依赖泛素或ISG15的先天免疫反应。这项研究的结果将使我们能够确定关键的位置，中介木瓜蛋白酶样酶的特异性，并为病毒致病机制和逃避天然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.