SUPPRESSION OF APOPTOSIS BY HUMAN CYTOMEGALOVIRUS INFECTION

描述（由申请人提供）：人类巨细胞病毒（HCMV）是一种重要的人类病原体。HCMV的原发感染和再激活在新生儿和免疫功能低下的个体中引起严重疾病。在此之前，我们已经克隆了HCMV菌株AD 169和Toledo作为感染性细菌人工染色体（BAC），建立了一个高效的基于BAC的HCMV遗传系统，并利用系统诱变方案构建了HCMV AD 169突变体的综合收集。HCMV突变文库的初步表征已经产生了大量信息，使我们能够开始描绘许多以前未表征的病毒基因的功能。本研究项目的长期目标是了解HCMV在病毒感染中抑制程序性细胞死亡（即细胞凋亡）的机制。细胞凋亡是宿主应对病毒感染的关键防御手段之一，可消除病毒的复制和传播；HCMV已经进化出对抗细胞凋亡的策略，从而在宿主体内进行复制、持续和传播。几种HCMV蛋白（即IE1， IE2， pUL36和pUL37x1）的过表达已被证明可以抑制细胞凋亡。我们利用AD169突变文库直接鉴定出HCMV蛋白pUL38是一种新的细胞死亡抑制因子，它是人类成纤维细胞有效感染病毒所必需的。我们假设pUL38在预防细胞死亡和促进病毒复制中发挥重要作用，有助于HCMV发病的各种细胞类型。在特异性目标1中，我们将定义pUL38及其小鼠巨细胞病毒同源物（即pM38）在各种细胞培养模型中病毒感染中的生物学作用。在Specific Aim 2中，我们将使用突变方法定义pUL38在病毒感染中阻断细胞死亡能力的序列基础。在Specific Aim 3中，我们将研究pUL38通过干扰细胞信号通路来阻止细胞凋亡的机制。这些研究将有助于更好地理解巨细胞病毒感染过程中的复杂细胞凋亡模式，并为使用MCMV感染小鼠模型作为研究pUL38在巨细胞病毒体内发病机制的有价值的替代方法奠定基础。最终，这些病毒编码的细胞凋亡抑制因子有潜力作为开发新的治疗干预措施的候选靶点来控制这种全球重要的病原体。

DESCRIPTION (provided by applicant): Human-cytomegalovirus (HCMV) is a significant human pathogen. Primary infection and reactivation of HCMV causes severe diseases in neonates and immuno-compromised individuals. Previously, we have cloned HCMV strains AD 169 and Toledo as infectious bacterial artificial chromosomes (BAC), developed an efficient BAC-based HCMV genetic system and constructed a comprehensive collection of HCMV AD 169 mutants using a systematic mutagenesis scheme. Initial characterization of the HCMV mutant library has generated a body of information that allows us to start to delineate functions of many previously uncharacterized viral genes. The long term goal of this research project is to understand the mechanisms by which HCMV inhibits programmed cell death, i.e. apoptosis, in virus infection. Apoptosis is one of the critical host defenses in response to virus infection to eliminate virus replication and spread; HCMV has evolved strategies to antagonize apoptosis for its replication, persistence and dissemination in the host. Over-expressions of several HCMV proteins (i.e. IE1, IE2, pUL36 and pUL37x1) have been shown to inhibit apoptosis. We have taken advantage of the AD169 mutant library to directly identify the HCMV protein pUL38 as a novel cell death suppressor that is required for efficient virus infection in human fibroblasts. We hypothesize that pUL38 plays an important role in preventing cell death and facilitating virus replication in various cell types instrumental to HCMV pathogenesis. In Specific Aim 1, we will define the biological role of pUL38 and its murine cytomegalovirus homolog (i.e., pM38) in virus infection in various cell culture models. In Specific Aim 2, we will use the mutational approach to define the sequence basis for the ability of pUL38 to block cell death in virus infection. In Specific Aim 3, we will investigate the mechanisms by which pUL38 interferes with cellular signaling pathways to block apoptosis. The proposed studies will lead to a better understanding of the complex apoptosis paradigm during CMV infection and set the stage for using the mouse model of MCMV infection as a valuable alternative to study pUL38 in CMV pathogenesis in vivo. Ultimately, these virus-encoded apoptosis suppressors have the potential to serve as candidate targets for developing novel therapeutic interventions to control this globally important pathogen.