Structure and function of HCMV gHgL complexes

人巨细胞病毒（HCMV）是疱疹病毒家族的一员，广泛分布于人群中，并造成重大健康问题，特别是在免疫功能低下的个体和在妊娠或分娩期间暴露于该病毒的新生儿中。了解对HCMV感染的免疫反应，特别是中和抗体反应的主要靶点，是非常重要的，因为开发有效疫苗的努力是高度优先的。最近的研究表明，中和抗体反应的最有效靶点之一是5种HCMV糖蛋白gH、gL、UL128、UL130和UL131的组装，形成“五聚体”复合物，负责扩大病毒对内皮细胞、上皮细胞和髓细胞的感染性。gH和gL蛋白是核心疱疹病毒膜融合和进入机制的一部分，而UL蛋白代表调节细胞趋向性的病毒特异性接头。已经报道了HCMV gHgL、五聚体和抗体复合物的低分辨率电镜研究，提供了对五聚体整体形状和中和抗体表位位置的深入了解。然而，由于没有确定高分辨率的结构，限制了我们对gHgL五聚体的整体折叠和结构以及中和抗体表位与中和效力之间关系的了解。在这里，我们将确定HCMV五聚体与代表性中和抗体复合物的结构。此外，我们将利用细胞表面文库展示方法，绘制参与五聚体组装、抗体结合和gHgL功能的关键gHgL和UL蛋白残基。本研究结果将加深我们对HCMV进入机制的理解，促进HCMV进入受体的鉴定，并为开发疫苗抗原和小分子抑制剂来预防HCMV感染提供关键的基础知识。

Human cytomegalovirus (HCMV) is member of the herpesvirus family that is widely distributed in the human population and responsible for significant health problems, particularly in immune-compromised individuals and in newborns exposed to the virus during pregnancy or birth. Understanding the immune response to HCMV infection, and in particular the major targets of the neutralizing antibody response, is of great interest as efforts to develop an effective vaccine are of high priority. Recent studies have shown that one of the most potent targets of the neutralizing antibody response is an assembly of 5 HCMV glycoproteins, gH, gL, UL128, UL130 and UL131, into a `pentameric' complex, which is responsible for broadening the infectivity of the virus to endothelial, epithelial and myeloid cells. The gH and gL proteins are part of the core herpesvirus membrane fusion and entry machinery, while the UL proteins represent virus-specific adaptors that modulate cell tropism. Low resolution electron microscopy studies of HCMV gHgL, pentamer and antibody complexes have been reported, providing insight into the overall shape of the pentamer and the locations of neutralizing antibody epitopes. However, no high resolution structures have been determined, limiting our insights into the overall folding and architecture of the gHgL pentamer as well as the relationship between neutralizing antibody epitopes and neutralization potency. Here we will determine the structures of HCMV pentamer in complex with representative neutralizing antibodies. In addition, we will map key gHgL and UL protein residues involved in pentamer assembly, antibody binding and gHgL functions, using cell surface library display approaches. Results generated from this proposal will deepen our understanding of the HCMV entry machinery, facilitate the identification of HCMV entry receptors, and provide key fundamental knowledge for the development of vaccine antigens and small molecule inhibitors to prevent HCMV infection.