SUPPRESSION OF APOPTOSIS BY HUMAN CYTOMEGALOVIRUS INFECTION

描述（由申请方提供）：人巨细胞病毒（HCMV）是一种重要的人类病原体。HCMV的原发感染和再活化导致新生儿和免疫功能低下个体的严重疾病。在此之前，我们已经克隆了HCMV株AD 169和Toledo作为感染性细菌人工染色体（BAC），开发了一个有效的基于BAC的HCMV遗传系统，并使用系统诱变方案构建了一个全面的收集HCMV AD 169突变体。HCMV突变体库的初步鉴定已经产生了大量的信息，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（即凋亡）的机制。细胞凋亡是宿主抵抗病毒感染以消除病毒复制和传播的关键防御机制之一; HCMV已经进化出对抗细胞凋亡的策略以用于其在宿主中的复制、持续和传播。几种HCMV蛋白（即IE 1、IE 2、pUL 36和pUL 37 x1）的过表达已显示抑制细胞凋亡。我们利用AD 169突变体文库直接鉴定HCMV蛋白pUL 38作为一种新的细胞死亡抑制因子，该抑制因子是病毒有效感染人成纤维细胞所必需的。我们推测pUL 38在防止细胞死亡和促进病毒在各种细胞类型中复制中起重要作用，这些细胞类型有助于HCMV发病。在具体目标1中，我们将定义pUL 38及其鼠巨细胞病毒同系物的生物学作用（即，pM 38）在各种细胞培养模型中的病毒感染。在具体目标2中，我们将使用突变方法来定义pUL 38在病毒感染中阻断细胞死亡的能力的序列基础。在具体目标3中，我们将研究pUL 38干扰细胞信号通路以阻断细胞凋亡的机制。所提出的研究将导致更好地理解CMV感染过程中的复杂的细胞凋亡范例，并为使用MCMV感染的小鼠模型作为研究pUL 38在体内CMV发病机制中的有价值的替代方案奠定基础。最终，这些病毒编码的凋亡抑制因子有可能成为开发新型治疗干预措施以控制这种全球重要病原体的候选靶标。

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.