Determining mechanisms of innate immune modulation by ADP-ribosylation

项目总结 ADP-核糖化是一种重要的翻译后修饰，直接影响几种 包括癌症、过敏和传染病在内的生物过程。ADP-核糖可以添加到蛋白质中，作为 ADP-核糖基转移酶的一个或多个连续单位，也称为PAPS，导致单-或多- ADP-核糖化(MAR或PAR)。大多数核糖化蛋白被干扰素(IFN)上调 据预测，感染和几种病毒具有抗病毒功能。此外，几个病毒家族，包括 冠状病毒科和冠状病毒科，编码具有单-ADP-核糖水解酶的大结构域蛋白 (ARH)活动。这种活动使这些病毒能够特别地中和MAR的影响，进一步 这表明MAR参与了哺乳动物的抗病毒反应。尽管有这些发现，但已知的只有几个 已知MAR可抑制病毒复制的例子。这在很大程度上是由于缺乏细胞培养模型 可以特别控制细胞的MAR状态的病毒感染，例如使用突变的模型 具有显著表型的病毒或PARP基因敲除细胞。重要的是，私家侦探建立了一种病毒 使用模型冠状病毒小鼠肝炎病毒(MHV)的感染系统，其中缺乏ARH活性的病毒 I)病毒复制显著受损，ii)独立地诱导强烈的干扰素反应。这些 表型被PARP抑制剂逆转，使MAR成为驱动这种抗病毒反应的关键因素。 研究人员的长期目标是从机制上确定MAR如何抑制病毒复制和 增强病毒感染后的先天免疫反应。这一知识差距将通过以下方式解决 回答以下问题：1)MAR如何抑制MHV感染？它能抑制进入吗，核糖核酸 MHV的复制、蛋白质翻译、组装或释放？2)干扰素诱导途径的哪一步(S) 被MAR增强，MAR是否也影响蝙蝠的干扰素反应，已知蝙蝠体内有许多 高致病性病毒？3)病毒感染后，PARPS会修饰哪些蛋白质？ 底物与特定的表型有关吗？这项研究的基本原理是它将增强我们的 了解MAR，包括其调节蛋白质功能的能力，并将发现新的细胞蛋白质 或调节病毒复制的过程。这项工作具有创新性，因为：i)它将架起一座意义重大的 ADP-核糖生物学、先天免疫反应和病毒复制之间的知识差距；ii)它利用 利用突变病毒和PARP基因敲除细胞的独特感染模型；以及iii)将是第一个 阐述MAR在蝙蝠中的作用。最后，这些项目对NIGMS的任务意义重大且相关 因为它们将提供对MAR如何影响可能产生的抗病毒反应的透彻理解 为在治疗受MAR影响的病毒感染或其他人类疾病方面取得进展奠定基础。

PROJECT SUMMARY ADP-ribosylation is an important post-translational modification that directly influences several biological processes including cancer, allergy, and infectious disease. ADP-ribose can be added to proteins as one or more consecutive units by ADP-ribosyltransferases, also termed PARPs, resulting in mono- or poly- ADP-ribosylation (mAR or pAR). Most mARylating PARPs are upregulated by interferon (IFN) upon virus infection and several are predicted to have antiviral functions. In addition, several viral families, including the Coronaviridae and Togaviridae, encode for macrodomain proteins that have mono-ADP-ribosyl hydrolase (ARH) activity. This activity allows these viruses the ability to specifically counteract the effects of mAR, further implicating mAR in the mammalian antiviral response. Despite these findings, there are only a few known examples where mAR is known to inhibit virus replication. This is largely due to the lack of cell culture models of virus infections where the mAR status of a cell can be specifically controlled, such as models using mutant viruses or PARP knockout cells that have significant phenotypes. Importantly, the PI has established a virus infection system using a model coronavirus, Murine Hepatitis Virus (MHV), where virus lacking ARH activity is i) significantly impaired in virus replication and ii) independently induces a robust IFN response. These phenotypes are reversed by PARP inhibitors, establishing mAR as a key factor driving this anti-viral response. The investigator’s long-term goal is to determine mechanistically how mAR inhibits virus replication and enhances the innate immune response following virus infection. This gap in knowledge will be resolved by answering the following questions: 1) How does mAR inhibit MHV infection? Does it inhibit the entry, RNA replication, protein translation, assembly, or release of MHV? 2) What step(s) of the IFN induction pathway is enhanced by mAR, and does mAR also affect the IFN response in bats, which are known to harbor many highly pathogenic viruses? 3) What proteins are modified by PARPs following virus infection and which substrates are relevant for specific phenotypes? The rationale for this research is that it will enhance our understanding of mAR, including its ability to modulate protein function and will uncover novel cellular proteins or processes that mediate virus replication. The work is innovative because: i) it will bridge a significant knowledge gap between ADP-ribose biology, the innate immune response, and virus replication; ii) it utilizes unique models of infection utilizing both mutant viruses and PARP knockout cells; and iii) will be the first to address the role of mAR in bats. Finally, these projects are significant and relevant to the NIGMS mission because they will provide a thorough understanding of how mAR impacts the anti-viral response that could lay the foundation for advances in the treatment of virus infections or other human diseases impacted by mAR.