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

• 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
• 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

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.

有丝分裂和抗病毒信号转导的病毒操作决定了感染的结果，但仍知之甚少。为了解决这一知识空白，我们的实验室研究了由在痘病毒和所有多细胞真核生物中广泛表达的同源物组成的蛋白激酶家族。我们的研究是确定痘病毒如何篡夺和重定向控制有丝分裂和对外来 DNA 做出反应的宿主防御效应器的信号级联，它们表达两种蛋白质：B1 和 B1。 B12 彼此同源，并且与称为牛痘相关激酶 (VRK) 的三种真核蛋白激酶同源。B1 和 VRK1 的比较研究表明，这两种酶都直接修饰细胞蛋白 BAF。重要的是，BAF 既充当有丝分裂调节剂又具有抗病毒作用。我们的新数据表明，B1 和细胞通过结合和压缩 dsDNA 来结合和压缩效应子，这种特性可通过 B1 或 VRK1 磷酸化而失活。 VRK 还共同调节其他途径，包括由 B12 蛋白激活的抗病毒途径。我们的数据表明，B12 通过 B1 控制的未知机制直接强烈抑制痘苗 DNA 复制。有趣的是，B12 是一种核痘病毒蛋白，并且是一种非痘病毒蛋白。 -催化激酶或“假激酶”，对于该提议具有关键的创新重要性。假激酶是假酶家族的成员，对此知之甚少。我们的中心假设是痘苗 B1 和 B12 形成一种新的信号轴，可取代和重定向调节 BAF 和其他 VRK 底物（如组蛋白）的细胞 VRK 通路。为了检验我们的假设，我们提出了三个目标 1) 确定。 B1 和 B12 如何在痘病毒感染过程中重塑 VRK1 响应性 BAF 和组蛋白 H2A 信号传导。该目的测试了 B12 与细胞核中的 VRK 相互作用的假设。改变 H2A 和 BAF 磷酸化，并增强整个细胞中的 BAF：DNA 相互作用。 将实现体外和培养细胞中 B12 与 BAF、B1 和细胞 VRK 相互作用的表征 2) 确定控制痘病毒的 B12 抑制的分子机制。 DNA 复制。该目标测试了以下假设：B12 具有 ATP 结合和活性激酶的其他功能元件，这些元件在 B12 信号传导失调中发挥着关键作用。本目标 3) 概述了通过靶向突变分析、新型功能丧失筛选和 B12 磷酸调节研究对 B12 进行结构/功能分析，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传导相融合。该目标将检验以下假设：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。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-伪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。

项目成果

Vaccinia Virus Arrests and Shifts the Cell Cycle.

痘苗病毒阻止并改变细胞周期。

• 发表时间: 2022-02-19
• 作者: Martin, Caroline K;Samolej, Jerzy;Olson, Annabel T;Bertoli, Cosetta;Wiebe, Matthew S;de Bruin, Robertus A M;Mercer, Jason
• 通讯作者: Mercer, Jason

Dysregulation of Cellular VRK1, BAF, and Innate Immune Signaling by the Vaccinia Virus B12 Pseudokinase.

痘苗病毒 B12 假激酶导致细胞 VRK1、BAF 和先天免疫信号传导失调。

• 发表时间: 2022-06-08
• 作者: Linville, Alexandria C;Rico, Amber B;Teague, Helena;Binsted, Lucy E;Smith, Geoffrey L;Albarnaz, Jonas D;Wiebe, Matthew S
• 通讯作者: Wiebe, Matthew S