Analysis of HCMV Infection of Monocytes and Macrophages

 描述(申请人提供)：人类巨细胞病毒(HCMV)是一种特定物种的疱疹病毒，在免疫功能正常的宿主中会导致发病率和死亡率，尤其是在免疫功能低下的宿主中。巨细胞病毒病可累及多个器官系统，因此，感染的一个关键特征是病毒的血源性传播。我们已经证明，单核/巨噬细胞可能是导致HCMV在初次感染后传播到周围器官的主要细胞类型，也是病毒持续存在的关键储存库，这表明这些细胞是初次感染后最初传播到器官组织和在宿主中观察到的长期病毒持续的来源。利用单核细胞和巨噬细胞进行病毒传播和持续的病毒策略将HCMV感染与病毒介导的致病机制联系起来。我们的工作和其他人的工作为病毒传播提供了以下模式。单核细胞在血液中被感染，尽管在最初感染时不会产生病毒，并通过病毒与同源受体的结合诱导单核细胞渗入各种组织。在那里，它们分化成长寿的巨噬细胞，支持原始输入病毒的复制，允许病毒在目标器官中持续存在。驱动单核细胞外渗和单核细胞向巨噬细胞分化的能力似乎是该病毒的一项基本功能。我们最近的研究首次证明，表皮生长因子受体(EGFR)和β1和β3整合素是单核细胞上真正的病毒受体，它们在病毒结合过程中的结合(分别通过糖蛋白B(GB)和Gh/G1/UL128-131复合体)是直接将单核细胞的感染和进入与病毒持续相关的不同功能变化联系起来的关键触发因素。这些识别的病毒受体/配体相互作用促进单核细胞发生独特的生化和分子变化，集中在改变/增强的运动性、生存和分化上，这是其他病毒/病原体或已知的单核细胞激活剂所不能模仿的。这些分子研究与我们新的基于生物信息学的荟萃分析相一致，表明与用各种细胞因子/趋化因子和病原体处理的单核细胞相比，感染HCMV的单核细胞表现出不同的基因表达模式，并且这种独特的巨噬细胞图谱在感染过程中(从几周到几个月)保持不变。对于病毒来说，这种由巨细胞病毒引导的单核细胞生物重编程的高潮是生产性感染。我们的新数据表明，病毒通过一种类似内吞作用的改良过程进入体内， 在感染的单核/巨噬细胞中，病毒的转运(成熟的颗粒在最终的核转位前保留在细胞质的各种囊泡中几天)、脱壳和基因表达谱与感染其他类型细胞时观察到的不同。此外，我们自最初提交以来的新数据现在有力地表明，延迟的运输、脱涂层和核转位是由病毒结合过程中EGFR和整合素的特定参与所指导的。根据我们的数据，病毒受体/配体的相互作用和随后的信号转导成为病毒的需要(即进入、核转位)满足特定功能的分子聚合点。 将单核细胞用作传播和持久的血管所需的单核细胞的变化(即运动、存活和分化)。基于这些新的发现，我们假设HCMV介导的受体/配体相互作用的独特组合触发了感染单核细胞的明显变化，从而促进了病毒的持续。为了验证我们的假设，我们提出了以下具体目标：1)研究受体/配体介导的信号如何触发与HCMV感染相关的早期生物学变化；2)研究HCMV诱导的信号促进病毒在感染的单核/巨噬细胞中转运的调节；以及3)确定受体/配体介导的信号如何诱导感染单核细胞分化的机制。这项拟议研究的结果将深入了解HCMV感染如何启动受感染的单核细胞和巨噬细胞的分子变化，这是病毒传播和持续所需的。由于我们还提供了对可能控制髓系细胞生物学的生化和分子过程的新理解，我们的研究有可能对理解髓系细胞具有广泛的免疫学意义。此外，破译单核细胞改变和感染所需的关键调控通路将为深入了解HCMV致病的潜在原因提供分子基础，并为治疗干预寻找新的潜在靶向途径。

 DESCRIPTION (provided by applicant): Human cytomegalovirus (HCMV) is a species-specific herpesvirus that causes morbidity and mortality in immunocompetent and, especially, in immunocompromised hosts. HCMV disease can involve multiple organ systems and a critical feature of infections is, therefore, the hematogenous dissemination of the virus. We have documented that monocytes/macrophages are likely the principal cell types responsible for HCMV spread to peripheral organs following primary infection and are key reservoirs for viral persistence, suggesting these cells are the source of the initial spread to organ tissue following primary infection and for the long-term viral persistence observed in the host. The viral strategy of utilizing monocytes and macrophages for viral dissemination and persistence links HCMV infection to viral-mediated pathogenesis. Our work and the work of others suggest the following model for viral spread. Monocytes are infected in the blood, although not productively at the time of initial infection, and are induced by viral binding to cognate receptors to extravasate int various tissues. There they differentiate into long-lived macrophages, which support replication of the original input virus, allowing for viral persistence in target organs. The ability to drive monocyte extravasation and monocyte-to-macrophage differentiation appears to be an essential function of the virus. Our recent studies documented for the first time that the epidermal growth factor receptor (EGFR) and β1 and β3 integrins are bona fide viral receptors on monocytes and that their engagement during viral binding (via glycoprotein B (gB) and the gH/gL/UL128-131 complex, respectively) is the key trigger that directly links infection and entry of monocytes to the distinct functional changes associated with viral persistence. These identified viral receptor/ligand interactions promote unique biochemical and molecular changes in monocytes focused on altered/enhanced motility, survival and differentiation that are not mimicked by other viruses/pathogens or known monocyte-activating agents. These molecular studies are consistent with our new bioinformatics-based meta-analyses showing that HCMV-infected monocytes exhibit a distinct gene expression pattern when compared to that observed with monocytes treated with a variety of cytokines/chemokines and pathogens and that this unique macrophage profile remains for the course of infection (from weeks to months). For the virus, the culmination of this HCMV-directed biological reprogramming of monocytes is productive infection. Our new data identifies that viral entry occurs via a modified endocytosis-like process, and that viral trafficking (the mature particle is retained for days in various vesicles in the cytoplasm prior to eventual nuclear translocation ~4 days post infection), uncoating and gene expression profiles in infected monocytes/macrophages is distinct from that observed during infection of other cell types. Furthermore, our new data since the original submission now shows strongly that the delayed trafficking, uncoating and nuclear translocation is directed by the specific engagement of EGFR and integrins during viral binding. From our data, viral receptor/ligand interactions and the ensuing signaling emerges as the molecular convergence point where the needs of the virus (i.e. entry, nuclear translocation) meet the specific functional changes in monocytes (i.e. motility, survival, and differentiation) required for the use of these cells as vessels for spread and persistence. Based on these new findings, we hypothesize that a unique combination of HCMV-mediated receptor/ligand interactions triggers the distinct changes in infected monocytes that promote viral persistence. To test our hypothesis, we propose the following specific aims - 1) to examine how receptor/ligand-mediated signaling triggers the early biological changes associated with HCMV infection; 2) to investigate the regulation by which HCMV-induced signaling promotes viral trafficking in infected monocytes/macrophages; and, 3) to determine the mechanisms for how receptor/ligand-mediated signaling induces differentiation of infected monocytes. The results from this proposed study will provide insight into how HCMV infection initiates the molecular changes in infected monocytes and macrophages required for viral spread and persistence. Because we are also providing a new understanding about the biochemical and molecular processes that may control myeloid cell biology, our studies have the potential to have broad immunological implications for the understanding of myeloid cells. Furthermore, the decoding of the key regulatory pathways required for changes and infection of monocytes will provide molecular insight into the underlying causes of HCMV pathogenesis, as well as identify new potential target pathways for therapeutic intervention.