Effects of human cytomegalovirus on monocyte survival and differentiation

描述(申请人提供)：人巨细胞病毒(HCMV)感染在免疫能力正常的个体中通常是无症状的，尽管在包括动脉粥样硬化和炎症性肠病在内的几种慢性炎症性疾病的病因学中，HCMV是主要的候选病毒。在免疫功能低下的个体中，如新生儿、艾滋病患者和移植受者，HCMV感染可导致急性多器官炎症，导致显著的发病率和死亡率。与巨细胞病毒感染相关的炎症性器官疾病是在无症状或有症状的感染期间发生的系统性病毒传播到多个器官部位并对其进行感染的直接后果。单核细胞负责将病毒运送到组织中，并在炎症状态中发挥核心作用 受感染的器官。然而，由于抗凋亡病毒蛋白在感染的早期阶段不表达，目前尚不清楚HCMV如何同时促进这些短暂细胞的存活和促炎分化。我们假设，HCMV刺激了一种独特的细胞抗凋亡重编程，专为满足分化感染单核细胞的生存需要而设计，这将在三个不同的目标中进行测试。目标 1将描述在病毒感染期间发生的病毒糖蛋白和细胞受体的相互作用 进入是导致单核细胞凋亡快速阻断的原因。中和抗体和药物抑制剂将单独或以不同的组合使用，以剖析每个病毒配体和细胞受体相互作用在促进HCMV感染的单核细胞存活中的作用。目的2将确定HCMV如何同时驱动感染单核细胞的长期存活和促炎分化。突变病毒和抗miRs[绑定和抑制microRNAs(MiRNAs)]将被用来中和单核细胞感染过程中病毒和细胞miRNAs的活性。单核细胞的存活和分化将分别通过Annexin V染色和Affymetrix微阵列基因分析进行检测。目的3明确靶向抗细胞凋亡的Bcl2家族蛋白在单核细胞感染巨细胞病毒过程中的作用机制和治疗潜力。为了确定病毒诱导的细胞抗凋亡蛋白的作用模式，将进行免疫共沉淀实验和质谱仪，以全局识别新的和独特的针对HCMV感染的单核细胞的蛋白质-蛋白质相互作用。BH3图谱，一种用于预测细胞对小分子抑制剂对抗凋亡的Bcl-2家族成员的敏感性的新技术，将 被用来确定靶向细胞中的Bcl-2家族生存蛋白的抗病毒潜力，以引导受感染的单核细胞走向细胞死亡。这些研究将增加我们对HCMV发病机制和传播的了解，评估针对感染细胞而不是直接针对病毒的新的抗病毒策略，并为使用BH3图谱来预测bc l-2家族小分子拮抗剂的疗效提供原理证据。 消除特定的病毒和细胞感染组合。

DESCRIPTION (provided by applicant): Human cytomegalovirus (HCMV) infection is generally asymptomatic in immunocompetent individuals, although HCMV is a primary viral candidate in the etiology of several chronic inflammatory diseases including atherosclerosis and inflammatory bowel disease. In immunocompromised individuals, such as neonates, AIDS patients, and transplant recipients, HCMV infection can lead to acute multi-organ inflammation resulting in significant morbidity and mortality. Inflammatory organ diseases associated with a HCMV infection is a direct consequence of the systemic viral spread to and infection of multiple organ sites that occur during either asymptomatic or symptomatic infections. Monocytes are responsible for delivering the virus into tissues and play a central role in the inflammatory state of infected organs. However, because anti-apoptotic viral proteins are not expressed during the early stages of infection, it remains unclear how HCMV simultaneously promotes the survival and pro-inflammatory differentiation of these short-lived cells. We hypothesize that HCMV stimulates a unique cellular anti-apoptotic reprogramming specifically designed to meet the viability needs of differentiating infected monocytes, which will be tested in 3 separate aims. Aim 1 will delineate which viral glycoprotein and cellular receptor interactions occurring during viral entry are responsible for the rapid block of monocyte apoptosis. Neutralizing antibodies and pharmaceutical inhibitors will be used individually or in different combinations to dissect the rol of each viral ligand and cellular receptor interaction in promoting the survival of HCMV-infected monocytes. Aim 2 will determine how HCMV concurrently drives the long-term survival and the pro-inflammatory differentiation of infected monocytes. Mutant viruses and anti-miRs [bind and inhibit microRNAs (miRNAs)] will be used to neutralize the activity of viral and cellular miRNAs during infection of monocytes. Monocyte survival and differentiation will then be examined by Annexin V staining and Affymetrix microarray gene analysis, respectively. Aim 3 will identify the molecular mechanism of action and therapeutic potential of targeting cellular anti-apoptotic Bcl-2 family proteins induced during HCMV infection of monocytes. To define the mode of action of virally induced cellular anti-apoptotic proteins, co-immunoprecipitation experiments will be done along with mass spectrometry to globally identify new and unique protein-protein interactions specific to HCMV-infected monocytes. BH3 profiling, a new technique used for predicting the sensitivity of cells to small-molecule inhibitors against anti-apoptotic Bcl-2 family members, will be used to determine the antiviral potential of targeting cellular Bcl-2 family survival proteins i order to direct infected monocytes towards cell death. These studies will increase our understanding on the pathogenesis and dissemination of HCMV, evaluate new antiviral strategies that target infected cells rather than the virus directly, and provide a proof-of-princile for the use of BH3 profiling to predict the efficacy of Bcl- 2 family small-molecule antagonists at eliminating specific virus and cell infection combinations.