Subversion of Cellular Mitotic and Antiviral Signaling by Poxviral Kinases

病毒操纵有丝分裂和抗病毒信号转导决定感染的结果，但仍然知之甚少。为了解决这一知识缺口，我们的实验室研究了一个蛋白激酶家族，该家族由痘病毒和所有多细胞真核生物中广泛表达的同系物组成。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源DNA反应的信号级联。哺乳动物痘病毒表达两种蛋白质，B1和B12，它们彼此同源，并且与三种称为牛痘相关激酶（Vaccinia related kinases，VRKs）的真核蛋白激酶同源。B1和VRK 1的比较研究表明，这两种酶直接修饰细胞蛋白BAF。重要的是，BAF通过结合和压缩dsDNA（通过B1或VRK 1磷酸化失活的特性）作为有丝分裂调节剂和抗病毒效应物。我们的新数据表明，B1和细胞VRKs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通过部分未知的机制，也由B1的牛痘病毒DNA复制的强烈抑制。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”，这对该提议具有关键的创新重要性。假激酶是假酶家族的成员，对病毒中的假酶家族知之甚少。我们的中心假设是，牛痘B1和B12形成一种新的信号传导轴，取代和重定向细胞VRK途径，调节BAF和其他VRK底物，如组蛋白和HUSH（人类沉默枢纽）复合物。为了验证我们的假设，我们提出了三个目标。目的1）确定B1和B12如何在痘病毒感染期间重塑VRK 1应答信号。该目的测试了B12与细胞核中的VRKs相互作用，从而改变H2 A，BAF和HUSH调节的假设。将在体外和培养细胞中实现B12与BAF、B1和细胞VRKs相互作用的表征。目的2）探讨B12抑制痘病毒DNA复制的分子机制。该目的检验了B12/VRK 1复合物在B12信号失调机制中起关键作用的假设。B12的结构/功能分析，通过有针对性的突变分析，新的功能丧失的屏幕，和调查B12磷酸化调节概述了这个目的。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转导会聚。该目的将测试B1和VRK 2激酶通过直接磷酸化调节B12，而VRK 3和磷酸酶PP 2A控制B1/VRK 1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：填补我们理解痘病毒操纵核过程的空白，产生广泛相关的激酶-假激酶生物学领域的见解，并提供有丝分裂和抗病毒信号交织所需的信息。

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 partly unknown mechanisms also 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 and the HUSH (Human Silencing Hub) complex. To test our hypothesis, we propose three aims. AIM 1) Determine how B1 and B12 remodel VRK1-responsive signaling during poxvirus infection. This Aim tests the hypothesis that B12 interacts with VRKs in the nucleus, thereby altering H2A, BAF, and HUSH regulation. 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 the B12/VRK1 complex plays key roles in the mechanism of B12 signaling dysregulation. 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.