Role of the Human Cytomegalovirus UL97 Proein Kinase in Viral Replication

描述(申请人提供)：这项申请旨在研究人类巨细胞病毒(HCMV)UL97蛋白激酶在病毒复制中的作用，明确的重点是核出口。UL97与层蛋白A/C的磷酸化有关，层蛋白A/C是一种细胞蛋白，被认为与疱疹病毒在核出口阶段的复制有关。UL97最初被认为是HCMV感染细胞中更昔洛韦激酶活性的来源，但LJL97似乎更有可能作为一种蛋白丝氨酸-苏氨酸激酶而不是核苷激酶发挥作用。Maribavir是UL97激酶活性的特异性抑制剂，在临床试验中显示出相当大的前景，证实了UL97作为治疗HCMV感染的抗病毒药物靶点的医学重要性。UL97缺失病毒(A97)的生长缺陷以前被认为是由于DNA复制和衣壳组装受损或核出口阶段的阻断所致。有趣的是，核膜的主要组成部分层蛋白A/C在巨细胞病毒感染过程中特异性地经历了UL97依赖的磷酸化模式的变化。由于A97病毒在核外流方面存在缺陷，而且不能诱导野生型HCMV感染细胞中常见的核膜典型变形，本项目将验证UL97协调层蛋白A/C聚合物的分解以促进核外流的假设。该项目将研究UL97是否能够在体外分解A型核层的聚合物，以及缺乏A型核层的细胞是否可以弥补A97病毒的生长缺陷。该项目还将测试UL97是否可以在感染HCMV期间直接导致核膜改变，或者是否也需要细胞激酶。最近发现UL97是在巨细胞病毒感染的细胞中负责视网膜母细胞瘤肿瘤抑制因子(RB)磷酸化的激酶。在正常细胞中，Rb的磷酸化可以引起几个宿主细胞基因的上调，这些基因可能有利于HCMV的复制，包括参与DNA复制和层蛋白A/C磷酸化的基因。因此，这一应用还将测试来自人乳头瘤病毒的Rb降解蛋白是否可以取代UL97来促进培养细胞中的HCMV复制。由于UL97与几种医学上重要的疱疹病毒之间保守的激酶有关，这一应用旨在增加我们对疱疹病毒生物学的基本了解。此外，由于人类巨细胞病毒是导致新生儿先天缺陷和美国人群免疫功能受损人群中危及生命的疾病的主要原因，这项研究也有助于更好地了解一种重要的治疗药物靶点。

DESCRIPTION (provided by applicant): This application aims to investigate the role of the UL97 protein kinase of human cytomegalovirus (HCMV) in viral replication, with an explicit focus on nuclear egress. UL97 has been implicated in the phosphorylation of lamin A/C, a cellular protein considered relevant to herpesvirus replication at the stage of nuclear egress. UL97 was originally identified as the source of ganciclovir kinase activity in HCMV-infected cells, but it appears that LJL97 is more likely to function as a protein serine-threonine kinase than as a nucleoside kinase. Maribavir, a specific inhibitor of UL97 kinase activity, has shown considerable promise in clinical trials, affirming the medical importance of UL97 as an antiviral drug target for treatment of HCMV infection. The growth defect of UL97 deletion virus (A97) has been previously attributed to impaired DNA replication and capsid assembly, or to a block at the stage of nuclear egress. Interestingly, lamin A/C, a major component of the nuclear lamina, specifically undergoes UL97-dependent changes in its phosphorylation pattern during HCMV infection. Since A97 virus has defects in nuclear egress, and moreover fails to induce characteristic deformations of the nuclear lamina commonly seen in cells infected with wild-type HCMV, this project will test the hypothesis that UL97 coordinates the disassembly of lamin A/C polymers to promote nuclear egress. The project will examine whether UL97 can disassemble polymers of A-type nuclear lamins in vitro, and whether cells lacking A-type lamins can complement the growth defect of A97 viruses. The project will also test if UL97 can directly cause nuclear lamina alterations during HCMV infection, or whether cellular kinases are also required. UL97 has recently been identified as the kinase responsible for phosphorylation of the retinoblastoma tumor suppressor (Rb) in HCMV-infected cells. In normal cells, Rb phosphorylation can cause upregulation of several host cell genes that may benefit HCMV replication, including genes involved in DNA replication and phosphorylation of lamin A/C. Therefore, this application will also test whether an Rb-degrading protein from human papillomavirus can substitute for UL97 to promote HCMV replication in cultured cells. As UL97 is related to kinases conserved among several medically important herpesviruses, this application aims to add to our basic understanding of herpesvirus biology. Furthermore, since HCMV is a leading cause of both congenital defects in newborn children, and of life-threatening disease in immunocompromised members of the U.S. population, this research also offers to contribute to a better understanding of an important therapeutic drug target.