Effects of human cytomegalovirus on monocyte survival and differentiation

描述（由申请方提供）：人巨细胞病毒（HCMV）感染在免疫功能正常的个体中通常无症状，尽管HCMV是几种慢性炎症性疾病（包括动脉粥样硬化和炎症性肠病）病因学中的主要病毒候选者。在免疫功能低下的个体中，如新生儿、AIDS患者和移植受者，HCMV感染可导致急性多器官炎症，从而导致显著的发病率和死亡率。与HCMV感染相关的炎性器官疾病是在无症状或有症状感染期间发生的全身性病毒扩散至多个器官部位并感染的直接后果。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 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，因此仍然不清楚HCMV如何同时促进这些短寿命细胞的存活和促炎分化。我们假设HCMV刺激一种独特的细胞抗凋亡重编程，专门设计用于满足分化感染单核细胞的生存力需求，这将在3个单独的目标中进行测试。目的 1将描述在病毒感染期间发生的病毒糖蛋白和细胞受体相互作用。 进入负责单核细胞凋亡的快速阻断。中和抗体和药物抑制剂将单独使用或以不同组合使用，以剖析每种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如何同时驱动受感染单核细胞的长期存活和促炎分化。突变病毒和抗miR [结合并抑制微小RNA（miRNA）]将用于中和单核细胞感染期间病毒和细胞miRNA的活性。然后将分别通过膜联蛋白V染色和Affyssin微阵列基因分析来检查单核细胞存活和分化。目的3将确定在HCMV感染单核细胞期间诱导的靶向细胞抗凋亡Bcl-2家族蛋白的分子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，将沿着质谱法进行免疫共沉淀实验，以全面鉴定HCMV感染的单核细胞特异性的新的和独特的蛋白质-蛋白质相互作用。BH 3谱是一种用于预测细胞对抗凋亡Bcl-2家族成员的小分子抑制剂敏感性的新技术， 用于确定靶向细胞Bcl-2家族存活蛋白的抗病毒潜力，以将感染的单核细胞导向细胞死亡。这些研究将增加我们对HCMV发病机制和传播的理解，评估新的靶向感染细胞而不是病毒的抗病毒策略，并为使用BH 3谱预测Bcl- 2家族小分子拮抗剂在HCMV感染中的疗效提供原理证明。 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.