Human Cytomegalovirus Entry into Cells Mediated by Pentamer and Trimer Complexes

人巨细胞病毒(HCMV)是一种普遍存在的、通常是良性的病毒。然而，HCMV经常导致移植患者的器官排斥反应，并导致全身疾病和新生儿中枢神经系统的发育缺陷。人巨细胞病毒感染多种不同的细胞类型，包括上皮细胞和内皮细胞、胶质细胞、成纤维细胞和单核巨噬细胞。这种广泛的趋向性是由于能够通过涉及不同病毒糖蛋白的不同进入途径进入不同类型的细胞，包括：Gh/gl/UL128-131，表示五聚体，Gh/gl/Go，三聚体。我们关于巨细胞病毒如何进入上皮细胞和内皮细胞的模型表明，巨细胞病毒三聚体与细胞表面受体结合，例如，PDGFRα病毒内化，五聚体作用于内体，促进GB介导的在成纤维细胞中的融合，然后病毒粒子与细胞膜的融合。还有第三种形式的Gh/gl，Gh/gl与Gb，Gb-Gh/gl的复合体，我们不知道Gb-Gh/gl是否促进病毒进入。鉴于它们在病毒侵入中的重要性，三聚体和五聚体也是抗体的重要靶点，被认为是设计巨细胞病毒疫苗的关键角色。目的1.研究人巨细胞病毒进入成纤维细胞、上皮细胞和内皮细胞的途径，并确定三聚体和五聚体的功能。这一目标将检验这一假说，即三聚体与细胞受体结合导致细胞表面交通，随后病毒颗粒内化到细胞内，下游五聚体介导的效应促进病毒从内体进入细胞质。目的2.确定三聚体和三聚体的结构：PDGFRα，确定三聚体结构/功能关系，并鉴定其他三聚体受体。我们已经有了一个带有其受体PDGFR的三聚体的初步结构，我们将扩大这些结构研究，并使用定点突变来测试功能。其他研究将确定对进入上皮细胞和内皮细胞至关重要的三聚体受体。目的3.研究人巨细胞病毒gB-Gh/gl复合体的功能。我们将通过使用Gb和Gh/gl的突变形式来阻止Gb-Gh/gl的组装来检验Gb-Gh/gl对HCMV进入的重要性的假设。目的4.鉴定人血清中三聚体特异性抗体，并与五聚体抗体进行比较。我们发现，人血清中的三聚体和五聚体特异性抗体可以协同作用中和人巨细胞病毒。我们将扩展这些研究，表征三聚体特异性抗体的流行率和效力，并确定这些抗体识别的三聚体表位。

Human cytomegalovirus (HCMV) is a ubiquitous, usually benign virus. Nevertheless, HCMV frequently contributes to rejection of organs in transplant patients and causes systemic disease and defects in the development of the CNS in neonates. HCMV infects many different cell types including epithelial and endothelial, glial cells, fibroblasts and monocyte-macrophages. This broad tropism is facilitated by a capacity to enter different cell types via distinct entry pathways involving different viral glycoproteins including: gH/gL/UL128-131, denoted the pentamer, gH/gL/gO, the trimer. Our model for how HCMV enters epithelial and endothelial cells suggests that HCMV trimers bind to cell surface receptors, e.g. PDGFRα viruses are internalized and pentamer acts in endosomes to promote gB-mediated fusion in fibroblasts, then of the virion envelope with cellular membranes. There is a third form of gH/gL, a complex of gH/gL with gB, gB-gH/gL and we do not know whether gB-gH/gL promotes in virus entry. Given their importance in virus entry, trimer and pentamer are also important targets of antibodies (Abs) and are considered key players in the design of HCMV vaccines. Four aims are proposed: Aim 1. To characterize pathways of HCMV entry into fibroblasts and epithelial and endothelial cells and determine where trimer and pentamer function. This aim will test the hypothesis that trimer binding to cellular receptors leads to cell surface traffic followed by internalization of virus particles into cells and downstream pentamer-mediated effects promoting virus exit from endosomes into the cytoplasm. Aim 2. To determine the structures of trimer and trimer:PDGFRα, define trimer structure/function relationships and identify other trimer receptors. We have a preliminary structure of trimer with its receptor PDGFR We will extend these structural studies and use site directed mutants to test function. Other studies will identify trimer receptors important for entry into epithelial and endothelial cells. Aim 3. To investigate how HCMV gB-gH/gL complexes function. We will test the hypothesis that gB- gH/gL is important for HCMV entry by using mutant forms of gB and gH/gL to block assembly of gB-gH/gL. Aim 4. To characterize trimer- specific Abs in human sera and compare to pentamer Abs. We made striking observations that trimer- and pentamer-specific Abs in human sera synergize to neutralize HCMV. We will extend these studies characterize the prevalence and potency of trimer-specific Abs and identify the epitopes in trimer recognized by these Ab.