Human cytomegalovirus entry into epithelial and endothelial cells

描述（由申请人提供）：人类巨细胞病毒（HCMV）是一种普遍存在的良性病毒，具有终身持久性和/或潜伏期。然而，免疫缺陷或免疫抑制患者的感染往往导致严重的疾病，包括肺炎、胃肠道综合征、肝炎、视网膜炎和脑炎。重要的是，HCMV经常导致移植器官的排斥反应。在免疫系统未成熟的儿童中，HCMV可引起中枢神经系统发育缺陷，特别是耳聋。此外，临床确认的幼儿HCMV病例可能只是“冰山一角”，许多其他在子宫内HCMV不太明显的婴儿可能会出现更微妙的发育缺陷。HCMV引起多种疾病，部分原因与感染多种细胞类型的能力有关，包括上皮细胞、内皮细胞和胶质细胞、成纤维细胞、单核巨噬细胞（M/M）和神经元。为了感染所有这些不同类型的细胞，HCMV必须结合到细胞表面并进入细胞。这是HCMV复制中至关重要的第一步，在病毒的趋向性和发病机制中起着重要作用，但人们对其知之甚少。在某种程度上，理解HCMV进入的差距与广泛研究的HCMV实验室菌株不能进入M/M细胞、上皮细胞和内皮细胞有关。HCMV临床毒株进入所有这些不同的细胞类型，显然是因为这些病毒表达两种进入糖蛋白：进入上皮细胞和内皮细胞所需的gH/gL/UL128-131和进入成纤维细胞所需的gH/gL/gO。HCMV实验室株（在成纤维细胞上长期增殖）仅表达gH/gL/gO。当我们诱导上皮细胞表达gH/gL/UL128-131时，HCMV进入细胞被阻断。这种对病毒进入的“干扰”为我们的假设提供了主要支持，即gH/gL/UL128-131具有结合上皮细胞受体的功能。我们正在测试gH/gL/gO结合成纤维细胞受体的相关假设。我们还发现gH/gL（不含UL128-131）和第二种HCMV糖蛋白gB的表达引起上皮细胞融合，令人惊讶的是，即使以反式表达，即gB在一组细胞中表达，gH/gL在另一组细胞中表达。这些观察结果对病毒蛋白如何融合膜的模型具有重要的概念意义。我们正在进行的研究重点是了解两种HCMV糖蛋白gH/gL/UL128-131和gH/gL/gO如何组装并结合到病毒颗粒中，以及这些蛋白如何在病毒进入中发挥作用。此外，我们将研究gB和gH/ gl介导的膜融合的分子机制。其他的努力将集中在鉴定新的细胞受体，这将解释临床HCMV进入上皮细胞和内皮细胞。关于病毒和细胞进入介质的信息是更好地了解HCMV发病机制以及设计疫苗和抗病毒药物的关键。公共卫生相关性：人类巨细胞病毒（HCMV）在几个脆弱人群中引起实质性疾病：发育中的儿童、移植患者和艾滋病患者。HCMV可以感染非常广泛的人类细胞类型，包括视网膜和肠道上皮细胞、内皮细胞、肝细胞、单核巨噬细胞和神经胶质细胞。我们的研究将为HCMV如何进入生物学相关的上皮细胞和内皮细胞提供更好的图像，以促进设计更好的抗HCMV药物和疫苗。

DESCRIPTION (provided by applicant): Human cytomegalovirus (HCMV) is a ubiquitous, normally benign virus that establishes lifelong persistence and/or latency. However, infections in immunodeficient or immunosuppressed patients often cause severe disease including pneumonia, gastrointestinal syndromes, hepatitis, retinitis, and encephalitis. Importantly, HCMV frequently contributes to the rejection of transplanted organs. In immunologically naive children, HCMV can cause developmental defects of the CNS, especially deafness. Moreover, clinically recognized cases of HCMV in young children may represent only the "tip of the iceberg", many other infants with less well pronounced HCMV in utero may suffer more subtle developmental defects. HCMV causes a diverse spectrum of diseases, in part, related to a capacity to infect a broad array of cell types including epithelial, endothelial and glial cells, fibroblasts, monocyte-macrophages (M/M) and neurons. In order to infect all these different cell types, HCMV must bind onto cell surfaces and enter the cells. This fundamentally important first step in HCMV replication play an important role in virus tropism and pathogenesis, yet is poorly understood. In part, gaps in understanding HCMV entry, relate to the fact that extensively studied laboratory strains of HCMV fail to enter M/M, epithelial and endothelial cells. Clinical strains of HCMV enter all these diverse cell types, apparently because these viruses express two entry glycoproteins: gH/gL/UL128-131, required for entry into epithelial and endothelial cells and gH/gL/gO, required for entry into fibroblasts. Lab strains of HCMV (propagated long term on fibroblasts) express only gH/gL/gO. When we transduced epithelial cells to cause expression of gH/gL/UL128-131, HCMV entry into the cells was blocked. This "interference" with virus entry provides principal support for our hypothesis that gH/gL/UL128-131 functions to bind epithelial cell receptors. We are testing the related hypothesis that gH/gL/gO acts to bind fibroblast receptors. We also found that expression of gH/gL (without UL128-131) and a second HCMV glycoprotein, gB, caused fusion of epithelial cells, surprisingly, even when expressed in trans, i.e. gB in one set of cells and gH/gL in other cells. These observations have important conceptual implications for models of how viral proteins fuse membranes. Our ongoing research is focused on understanding how the two HCMV glycoproteins gH/gL/UL128-131 and gH/gL/gO are assembled and incorporated into virus particles, and how these proteins function in virus entry. Moreover, we will study the molecular mechanisms of gB and gH/gL-mediated membrane fusion. Other efforts will focus on the identification of novel cellular receptors that will explain clinical HCMV entry into epithelial and endothelial cells. Information about viral and cellular entry mediators is key to a better understanding of HCMV pathogenesis and for the design of vaccines and anti-virals. PUBLIC HEALTH RELEVANCE: Human cytomegalovirus (HCMV) causes substantial disease in several vulnerable populations: developing children, transplant patients, and AIDS patients. HCMV can infect a very broad spectrum of human cell types including retinal and gut epithelial cells, endothelial cells, hepatocytes, monocyte-macrophages and glial cells. Our studies will provide a better picture of how HCMV gains entry into biologically relevant epithelial and endothelial cells to facilitate the design of better anti-HCMV drugs and vaccines.