Engagement of Cellular Mitotic and Antiviral Signaling by Poxviral Kinases

病毒对有丝分裂和抗病毒信号转导的操纵决定了感染的结局，但仍知之甚少。为了解决这一知识鸿沟，我们的实验室研究了一系列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器对外来DNA的反应的信号级联。哺乳动物痘病毒表达两种蛋白B1和B12，这两种蛋白彼此同源，并与三种被称为牛痘相关蛋白的真核蛋白激酶同源。对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通过结合和压缩dsDNA同时作为有丝分裂调节器和抗病毒效应器发挥作用，这一特性通过B1或VRK1的磷酸化而失活。我们的新数据认为，B1和细胞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通过一种由B1控制的未知机制来强烈抑制牛痘DNA复制。耐人寻味的是，B12是一种核痘病毒蛋白和一种非催化激酶或“假激酶”，这对这一提议具有关键的创新意义。假酶是伪酶家族的成员，在病毒中对其知之甚少。我们的中心假设是，痘苗病毒B1和B12形成了一个新的信号轴，取代并重定向调节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。目的1)确定在痘病毒感染过程中，B1和B12如何重塑VRK1应答的BAF和组蛋白H_2A信号。这一目的验证了B12与细胞核中的VRKs相互作用，从而改变H2A和BAF磷酸化，并增强整个细胞中BAF：DNA相互作用的假设。在体外和培养细胞中，B12与BAF、B1和细胞VRKs的相互作用的特征将被实现。目的2)探讨B12抑制痘病毒DNA复制的分子机制。这一目标验证了B12具有ATP结合和活性激酶的其他功能元件的假说，这些元件在B12如何失调信号调节中发挥关键作用。本文综述了通过靶向突变分析来分析B12的结构/功能、新的功能缺失筛选以及B12的磷酸化调节研究。目的3)确定病毒/细胞假蛋白激酶如何介导痘病毒生命周期的抑制以及与蛋白磷酸酶信号通路的融合。这一目标将检验以下假设：B1和VRK2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，从而操纵下游的抗病毒反应。这项工作的完成将：填补我们对痘病毒操纵核过程的理解的空白，产生与激酶-假性激酶生物学领域广泛相关的见解，并提供有丝分裂和抗病毒信号如何交织的必要信息。

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.