Determining mechanisms of innate immune modulation by ADP-ribosylation

项目摘要 ADP-核糖基化是一种重要的翻译后修饰，其直接影响多个细胞的功能。 生物过程，包括癌症、过敏和传染病。ADP-核糖可以添加到蛋白质中， 一个或多个连续的单位由ADP-核糖基转移酶，也称为PARP，导致单-或多- ADP-核糖基化（mAR或pAR）。病毒感染后，干扰素（IFN）可上调大多数马来酰化PARP。 感染和几个被预测具有抗病毒功能。此外，一些病毒家族，包括 冠状病毒科和披膜病毒科编码具有单ADP核糖基水解酶的大结构域蛋白 (ARH)活动这种活性使这些病毒能够特异性地抵消mAR的作用，进一步 表明mAR参与了哺乳动物的抗病毒反应。尽管有这些发现， 已知mAR抑制病毒复制的实例。这在很大程度上是由于缺乏细胞培养模型 在病毒感染中，细胞的mAR状态可以被特异性控制，例如使用突变体 病毒或具有显著表型的PARP敲除细胞。重要的是PI已经建立了一种病毒 使用模型冠状病毒，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，和ii）独立诱导强IFN应答。这些 表型被PARP抑制剂逆转，确立mAR为驱动这种抗病毒应答的关键因素。 研究者的长期目标是确定mAR如何在机制上抑制病毒复制， 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得到解决： 回答以下问题：1）mAR如何抑制MHV感染？它能抑制RNA进入 MHV的复制、蛋白质翻译、组装或释放？2)IFN诱导途径的步骤是什么 增强mAR，mAR是否也影响蝙蝠的IFN反应，这是众所周知的港口许多 高致病性病毒3)病毒感染后PARP修饰了哪些蛋白质， 底物与特定表型相关？这项研究的基本原理是，它将提高我们的 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 或介导病毒复制的过程。这项工作是创新的，因为：i）它将弥合一个重大的 ADP-核糖生物学、先天免疫应答和病毒复制之间的知识差距; ii）它利用 利用突变病毒和PARP敲除细胞的独特感染模型; iii）将是第一个 解决mAR在蝙蝠中的作用。最后，这些项目对国家地理测量系统的使命具有重要意义和相关性 因为他们将提供一个彻底的了解mAR如何影响抗病毒反应， 为治疗病毒感染或其他受mAR影响的人类疾病的进展奠定了基础。

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.