Cytomegalovirus DNA replication and inversion

描述（申请人提供）：人巨细胞病毒（CMV）仍然是儿童先天性疾病的主要原因，也是免疫功能低下个体中的重要机会病原体，尽管使用抗病毒药物，仍会导致急性和慢性疾病后果。CMV在感染期间以影响对内在细胞防御途径的易感性的方式调节宿主细胞行为。程序性细胞死亡（PCD）、传感器触发的干扰素激活和信号级联都在感染期间的早期开始受到影响。重要的是，这些在与病毒成熟相一致的后期具有最大的影响。我们的工作表明，CMV编码的细胞死亡抑制因子和蛋白激酶在病毒复制过程的后期与细胞命运途径相互作用。假设细胞死亡抑制和磷酸化变化控制特定的病毒程序，称为cmvPCD，以终止复制周期。本修订项目将研究CMV编码的细胞死亡抑制因子和细胞周期失调对成熟的事件、过程和控制。提出的研究的第一个目标将主要集中在解剖一个内在的，压力相关的丝氨酸依赖性凋亡样途径，我们发现在CMV感染的细胞中是活跃的，评估发生在线粒体和线粒体下游的事件，以驱动细胞死亡。病毒功能促进细胞死亡，并且当细胞丝氨酸蛋白酶HtrA 2/Omi的水平增加时，这些功能在复制的晚期阶段是活跃的。我们将研究细胞周期和病毒蛋白激酶触发死亡的作用，使用感染的细胞为基础的测定，以寻求这些途径之间的联系。第二个目的是研究丝氨酸蛋白酶依赖性途径受β疱疹病毒保守的病毒性细胞凋亡抑制剂（vMIA）（人UL 37 x1基因的产物）控制的机制。我们将确定vMIA作用的关键病毒和细胞靶点，整合以前的证据，即这种病毒细胞死亡抑制因子需要与GADD 45家族蛋白相互作用才能发挥活性，并且抗凋亡家族蛋白Bcl-xL是相关的参与者。我们认为，这些病毒和细胞蛋白之间的相互作用通过感染细胞中的HtrA 2/Omi激活维持细胞活力，表明在发病机制中的作用。这一重要课题将作为第三个目标进行研究，通过研究感染小鼠CMV突变体的小鼠，这些突变体破坏功能同源基因（m38.5）或用人CMV基因取代小鼠CMV基因。通过这些努力，我们将获得一个完整的了解的过程，控制程序性细胞死亡的CMV复制细胞和一个完整的动物宿主。公共卫生相关性：人巨细胞病毒（CMV）仍然是儿童先天性疾病的主要原因，也是免疫功能低下个体的重要机会致病菌，尽管使用抗病毒药物，仍会导致急性和慢性疾病后果。病毒感染的细胞以一种被禁止的方式死亡，这种方式依赖于一种由线粒体驻留丝氨酸蛋白酶HtrA 2/Omi触发的新型宿主细胞途径，并由病毒UL 37 x1基因的产物控制，UL 37 x1基因是一种强大的细胞死亡抑制因子。这项调查将导致一个更完整的理解细胞和病毒功能的相互作用作为细胞命运的决定因素。

DESCRIPTION (provided by applicant): Human cytomegalovirus (CMV) remains a major cause of congenital disease in children as well as a significant opportunistic pathogen in immunocompromised individuals, causing acute and chronic disease consequences despite the use of antiviral drugs. CMV modulates host cell behavior during infection in ways that affects susceptibility to intrinsic cellular defense pathways. Programmed cell death (PCD), sensor-triggered interferon activation, and signaling cascades are all affected starting at early times during infection. Importantly, these have their greatest impact at late times that coincide with viral maturation. Our work has shown that CMV-encoded cell death suppressors and protein kinases interface with cellular fate pathways late in the course of viral replication. Cell death suppression and phosphorylation changes are hypothesized to control a specific viral program, termed cmvPCD to terminate the replication cycle. This revised project will investigate the events, process and control of maturation by CMV-encoded cell death suppressors and cell cycle dysregulation. The first aim of proposed investigations will focus primarily on the dissection of an intrinsic, stress-related serine-dependent apoptosis-like pathway we discovered to be active in CMV-infected cells, evaluating the events that occur at mitochondria and downstream of mitochondria to drive cell death. Viral functions promote cell death, and these are active during the late phase of replication when levels of the cellular serine protease HtrA2/Omi increase. We will investigate the role of cell cycle and viral protein kinase triggers of death using infected cell-based assays in order to seek a link between these pathways. The second aim will investigate the mechanism(s) through which this serine protease-dependent pathway is controlled by a betaherpesvirus-conserved viral mitochondrial-localized inhibitor of apoptosis (vMIA), the product of the human UL37x1 gene. We will identify critical viral and cellular targets of vMIA action, integrating previous evidence that this viral cell death suppressor requires an interaction with GADD45 family proteins for activity and that the anti-apoptotic family protein Bcl-xL is an associated player. We believe that the interplay between these viral and cellular proteins sustain cell viability through HtrA2/Omi activation in infected cells, suggesting a role in pathogenesis. This important topic will be studied as aim three, through studies in mice infected with murine CMV mutants that disrupt the functionally homologous gene (m38.5) or replace the murine CMV gene with the human CMV gene. Through these efforts we will gain a complete understanding of the processes that underlie control of programmed cell death in CMV replication in cells and in an intact animal host. PUBLIC HEALTH RELEVANCE: Human cytomegalovirus (CMV) remains a major cause of congenital disease in children as well as a significant opportunistic pathogen in immunocompromised individuals, causing acute and chronic disease consequences despite the use of antiviral drugs. Virus-infected cells die in a proscribed way that relies on a novel host cell pathway triggered by a mitochondrial resident serine protease HtrA2/Omi and is controlled by the product of the viral UL37x1 gene, a powerful suppressor of cell death. This investigation will lead to a more complete understanding of the interplay of cellular and viral functions as determinants of cell fate.