Cytomegalovirus DNA replication and inversion

描述(申请人提供)：人类巨细胞病毒(CMV)仍然是儿童先天性疾病的主要原因，也是免疫受损个人的重要机会性病原体，尽管使用了抗病毒药物，但仍会导致急性和慢性疾病后果。CMV在感染过程中调节宿主细胞的行为，从而影响对固有细胞防御途径的敏感性。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从感染的早期开始受到影响。重要的是，这些病毒在病毒成熟的后期影响最大。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通路相互作用。细胞死亡抑制和磷酸化变化被认为是为了控制名为cmvPCD的特定病毒程序来终止复制周期。这个修订的项目将研究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。拟议研究的第一个目标将主要集中在解剖我们发现在CMV感染的细胞中活跃的内在的、与应激相关的丝氨酸依赖的类凋亡途径，评估发生在线粒体和线粒体下游的驱动细胞死亡的事件。病毒功能促进细胞死亡，在复制的后期，当细胞丝氨酸蛋白酶HtrA2/Omi水平增加时，这些功能是活跃的。我们将使用基于感染细胞的分析方法来研究细胞周期和病毒蛋白激酶死亡触发因素的作用，以寻找这些途径之间的联系。第二个目标将研究这种丝氨酸蛋白酶依赖的途径受人类UL37x1基因产物--β疱疹病毒保守的病毒线粒体定位的凋亡抑制物(VMIA)控制的机制(S)。我们将确定VMIA作用的关键病毒和细胞靶点，整合先前的证据，即这种病毒细胞死亡抑制因子需要与GADD45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是一个相关的参与者。我们认为，这些病毒和细胞蛋白之间的相互作用通过激活感染细胞中的HtrA2/Omi来维持细胞的存活，这表明在发病机制中发挥了作用。这一重要的课题将作为目标三进行研究，通过对感染小鼠CMV突变体的研究，这些突变体破坏功能上的同源基因(m38.5)或用人CMV基因替换小鼠CMV基因。通过这些努力，我们将对CMV在细胞内和在完整的动物宿主中复制时控制细胞程序性死亡的过程有一个完整的了解。公共卫生相关性：人类巨细胞病毒(CMV)仍然是儿童先天性疾病的主要原因，也是免疫受损个体的重要机会性病原体，尽管使用了抗病毒药物，但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，这种方式依赖于线粒体驻留丝氨酸蛋白酶HtrA2/Omi触发的一种新的宿主细胞途径，并受病毒UL37x1基因的产物控制，UL37x1基因是一种强大的细胞死亡抑制因子。这项研究将导致对细胞和病毒功能作为细胞命运决定因素的相互作用的更全面的理解。

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.