SUPPRESSION OF APOPTOSIS BY HUMAN CYTOMEGALOVIRUS INFECTION

描述(申请人提供)：人巨细胞病毒(HCMV)是一种重要的人类病原体。人巨细胞病毒的原发感染和重新激活会导致新生儿和免疫功能低下的人患上严重疾病。在此之前，我们已经克隆了HCMV AD169株和TOLEDO株作为感染性细菌人工染色体(BAC)，开发了基于BAC的高效的HCMV遗传系统，并利用系统的突变方案构建了一套完整的HCMV AD169突变体。对巨细胞病毒突变体库的初步鉴定已经产生了大量信息，使我们能够开始描述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在病毒感染过程中抑制细胞程序性死亡的机制。细胞凋亡是应对病毒感染、消除病毒复制和传播的关键宿主防御措施之一；由于其在宿主中的复制、持续和传播，人类巨细胞病毒已经进化出了拮抗细胞凋亡的策略。一些HCMV蛋白(即IE1、IE2、pUL36和pUL37x1)的过表达已被证明具有抑制细胞凋亡的作用。我们利用AD169突变体文库直接鉴定了HCMV蛋白pUL38是一种新的细胞死亡抑制因子，是有效感染人成纤维细胞病毒所必需的。我们推测，pUL38在防止细胞死亡和促进病毒在不同细胞类型中的复制方面发挥重要作用，有助于HCMV的致病。在特定的目标1中，我们将确定pUL38及其小鼠巨细胞病毒同系物(即pM38)在不同细胞培养模型中在病毒感染中的生物学作用。在特定的目标2中，我们将使用突变方法来确定pUL38在病毒感染中阻止细胞死亡的能力的序列基础。在特定的目标3中，我们将研究pUL38干扰细胞信号通路以阻断细胞凋亡的机制。这些研究将有助于更好地理解CMV感染过程中复杂的细胞凋亡模式，并为利用小鼠MCMV感染模型研究pUL38在体内CMV致病中的作用奠定基础。最终，这些病毒编码的凋亡抑制因子有可能成为开发新的治疗干预措施以控制这种全球重要病原体的候选靶点。

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.