Human Cytomegalovirus Entry into Cells Mediated by Pentamer and Trimer Complexes

五聚体和三聚体复合物介导的人巨细胞病毒进入细胞

• 批准号: 10120270
• 负责人: Theodore S Jardetzky
• 金额: $ 76.77万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-17 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10120270
• 关键词: Address; Affect; Antibodies; Antibody Response; Benign; Binding; Biochemical; Blood Vessels; Capsid; Cell Surface Receptors; Cell surface; Cells; Cellular Membrane; Complex; Cytomegalovirus; Cytomegalovirus Vaccines; Cytoplasm; Data; Defect; Development; Disease; Endosomes; Endothelial Cells; Endothelium; Epithelial; Epithelial Cells; Epithelium; Epitopes; Fetal Development; Fibroblasts; Follow-Up Studies; Foundations; Glycoproteins; Grant; Hepatitis; Hepatocyte; Herpesviridae; Human; Image; Immune Targeting; Immune system; Immunity; Immunize; Infection; Maps; Mediating; Membrane Fusion; Modeling; Molecular Conformation; Mutagenesis; Nervous system structure; Neuroglia; Newborn Infant; Organ; Oryctolagus cuniculus; PDGFRB gene; Pathology; Pathway interactions; Peptides; Platelet-Derived Growth Factor alpha Receptor; Pneumonia; Prevalence; Production; Property; Proteins; Receptor Cell; Role; Site; Structure; Structure-Activity Relationship; Systemic disease; Testing; Transplant Recipients; Tropism; Vaccines; Viral; Viral Fusion Proteins; Virion; Virus; Virus Diseases; base; cell type; design; experimental study; graft failure; human imaging; immunosuppressed; in vivo; interest; macrophage; monocyte; mutant; neonate; particle; receptor; trafficking

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.

人类巨细胞病毒 (HCMV) 是一种普遍存在的、通常是良性的病毒。然而，HCMV 经常导致移植患者器官排斥，并导致新生儿全身性疾病和中枢神经系统发育缺陷。 HCMV 感染许多不同的细胞类型，包括上皮细胞和内皮细胞、神经胶质细胞、成纤维细胞和单核巨噬细胞。通过涉及不同病毒糖蛋白的不同进入途径进入不同细胞类型的能力促进了这种广泛的向性，包括：gH/gL/UL128-131，表示五聚体，gH/gL/gO，表示三聚体。我们的 HCMV 如何进入上皮细胞和内皮细胞的模型表明 HCMV 三聚体与细胞表面受体结合，例如PDGFRα病毒被内化，五聚体在内体中发挥作用，促进成纤维细胞中gB介导的融合，然后促进病毒体包膜与细胞膜的融合。 gH/gL 还有第三种形式，即 gH/gL 与 gB 的复合物，gB-gH/gL，我们不知道 gB-gH/gL 是否促进病毒进入。鉴于三聚体和五聚体在病毒进入中的重要性，三聚体和五聚体也是抗体 (Ab) 的重要靶标，并被认为是 HCMV 疫苗设计中的关键角色。提出了四个目标： 目标 1. 表征 HCMV 进入成纤维细胞以及上皮细胞和内皮细胞的途径，并确定三聚体和五聚体在何处发挥作用。该目标将检验以下假设：三聚体与细胞受体的结合导致细胞表面运输，随后病毒颗粒内化到细胞中，以及下游五聚体介导的效应促进病毒从内体进入细胞质。目标 2. 确定三聚体和三聚体：PDGFRα 的结构，定义三聚体结构/功能关系并鉴定其他三聚体受体。我们已经有了三聚体及其受体 PDGFR 的初步结构。我们将扩展这些结构研究并使用定点突变体来测试功能。其他研究将鉴定对于进入上皮细胞和内皮细胞很重要的三聚体受体。目标 3. 研究 HCMV gB-gH/gL 复合物如何发挥作用。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 对于 HCMV 进入很重要的假设。目标 4. 表征人血清中三聚体特异性抗体并与五聚体抗体进行比较。我们惊人地观察到，人血清中的三聚体和五聚体特异性抗体可协同中和 HCMV。我们将扩展这些研究，表征三聚体特异性抗体的普遍性和效力，并鉴定这些抗体识别的三聚体中的表位。