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Engagement of Cellular Mitotic and Antiviral Signaling by Poxviral Kinases

痘病毒激酶参与细胞有丝分裂和抗病毒信号传导

基本信息

批准号：
10458234
负责人：
MATTHEW S WIEBE
金额：
$ 42.58万
依托单位：
UNIVERSITY OF NEBRASKA LINCOLN
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-12 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10458234
关键词：
Address Affinity Antiviral Agents Antiviral Response Binding Biology Cell Nucleus Cell division Cells Communicable Diseases Comparative Study Cultured Cells Cytoplasm DNA DNA biosynthesis Data Development Diagnostic Elements Enzymes Eukaryota Exhibits Family Future Genes Genetic Epistasis Genetic Transcription Genome Goals Histone H2A Histones Host Defense Immunity In Vitro Infection Investigation Knowledge Laboratory Study Masks Mediating Mitosis Mitotic Modeling Modification Molecular Mutation Analysis Names Nuclear Outcome Pathway interactions Phosphoproteins Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Physical condensation Play Post-Translational Protein Processing Poxviridae Poxviridae Infections Process Property Protein Family Protein Kinase Protein Phosphatase 2A Regulatory Subunit PR53 Protein phosphatase Proteins Regulation Repression Research Signal Transduction Structure Testing Vaccinia Viral Viral Genome Virus Work base cellular targeting ds-DNA extrachromosomal DNA fascinate innovation insight loss of function member novel targeted treatment tool transcription factor viral DNA virus host interaction

项目摘要

Viral manipulation of mitotic and antiviral signal transduction determines the outcome of infection but remains poorly understood. To address this knowledge gap, our laboratory studies a family of protein kinases comprised of homologs widely expressed in poxviruses and in all multicellular eukaryotes. The long term goal of our research is to determine how poxviruses usurp and redirect signaling cascades governing mitotic and host defense effectors responsive to foreign DNA. Mammalian poxviruses express two proteins, B1 and B12, which are homologous to each other and to three eukaryotic protein kinases named vaccinia related kinases (VRKs). Comparative studies of B1 and VRK1 revealed that both enzymes directly modify the cellular protein BAF. Importantly, BAF acts as both a mitotic regulator and antiviral effector by binding and compacting dsDNA, a property that is inactivated via phosphorylation by B1 or VRK1. Our new data argue that B1 and cellular VRKs co-regulate other pathways as well, including an antiviral pathway activated by the B12 protein. Our data indicate that B12 directs strong repression of vaccinia DNA replication via an unknown mechanism governed by B1. Intriguingly, B12 is a nuclear poxviral protein and a non-catalytic kinase or ‘pseudokinase’, which are of key innovative importance for this proposal. Pseudokinases are members of the pseudoenzyme family, about which little is known in viruses. It is our central hypothesis that vaccinia B1 and B12 form a novel signaling axis that supplants and redirects cellular VRK pathways regulating BAF and other VRK substrates such as histones. To test our hypothesis, we propose three aims. AIM 1) Determine how B1 and B12 remodel VRK1- responsive BAF and histone H2A signaling during poxvirus infection. This Aim tests the hypothesis that B12 interacts with VRKs in the nucleus, thereby altering H2A and BAF phosphorylation, and augmenting BAF:DNA interaction throughout the cell. Characterization of B12 interaction with BAF, B1, and cellular VRKs in vitro and in cultured cells will be achieved. AIM 2) Determine the molecular mechanisms governing B12 repression of poxvirus DNA replication. This Aim tests the hypothesis that B12 possesses ATP binding and other functional elements of active kinases that play key roles in how B12 dysregulates signaling. Structure/function analysis of B12 through targeted mutational analysis, novel loss of function screens, and investigation of B12 phosphoregulation are outlined in this Aim. AIM 3) Determine how viral/cellular pseudokinases mediate repression of the poxvirus lifecycle and converge with protein phosphatase signaling. This Aim will test the hypotheses that B1 and VRK2 kinases regulate B12 via direct phosphorylation while VRK3 and the phosphatase PP2A control dynamic regulation of B1/VRK1 substrates, leading to manipulation of downstream antiviral responses. The completion of this work will: fill gaps in our understanding of poxvirus manipulation of nuclear processes, yield broadly relevant insights to the field of kinase-pseudokinase biology, and provide needed information of how mitotic and antiviral signaling interweave.

病毒操纵有丝分裂和抗病毒信号转导决定了感染的结果，但仍然知之甚少。为了解决这一知识差距，我们的实验室研究了一个由同源物组成的蛋白激酶家族，这些同源物在痘病毒和所有多细胞真核生物中广泛表达。我们研究的长期目标是确定痘病毒如何篡夺和重定向控制有丝分裂和宿主防御效应物对外来DNA反应的信号级联。哺乳动物痘病毒表达两种蛋白B1和B12，它们彼此同源，并与三种真核蛋白激酶同源，称为牛痘相关激酶（VRKs）。对B1和VRK1的比较研究表明，这两种酶都能直接修饰细胞蛋白BAF。重要的是，BAF通过结合和压实dsDNA作为有丝分裂调节剂和抗病毒效应剂，dsDNA通过B1或VRK1的磷酸化而失活。我们的新数据表明B1和细胞vrk也共同调节其他途径，包括由B12蛋白激活的抗病毒途径。我们的数据表明，B12通过一种未知的由B1控制的机制强烈抑制牛痘DNA复制。有趣的是，B12是一种核痘病毒蛋白和一种非催化激酶或“假激酶”，这对该提案具有关键的创新意义。假激酶是假酶家族的成员，在病毒中所知甚少。我们的中心假设是，牛痘B1和B12形成了一个新的信号轴，取代和重定向调节BAF和其他VRK底物（如组蛋白）的细胞VRK通路。为了验证我们的假设，我们提出了三个目标。目的1)确定在痘病毒感染期间B1和B12如何重塑VRK1响应的BAF和组蛋白H2A信号。本实验验证了B12与细胞核内的vrk相互作用，从而改变H2A和BAF的磷酸化，并在整个细胞内增强BAF:DNA相互作用的假设。将在体外和培养细胞中表征B12与BAF、B1和细胞vrk的相互作用。2)确定B12抑制痘病毒DNA复制的分子机制。这一目的验证了B12具有ATP结合和其他活性激酶的功能元件的假设，这些活性激酶在B12如何失调信号传导中起关键作用。本文概述了通过靶向突变分析、新的功能丧失筛选和B12磷酸化调控研究来分析B12的结构/功能。目的3)确定病毒/细胞假激酶如何介导痘病毒生命周期的抑制并与蛋白磷酸酶信号汇合。本研究将验证B1和VRK2激酶通过直接磷酸化调节B12，而VRK3和磷酸酶PP2A控制B1/VRK1底物的动态调节，从而操纵下游抗病毒反应的假设。这项工作的完成将填补我们对痘病毒操纵核过程的理解空白，为激酶-假激酶生物学领域提供广泛相关的见解，并提供有丝分裂和抗病毒信号如何交织的所需信息。