Viral and cellular mechanisms of HSV fusion and entry

疱疹病毒是一种普遍存在的病原体，在世界范围内会导致严重的发病率和死亡率。单纯疱疹病毒(HSV)会导致唇疱疹和性传播感染。HSV感染的严重后果包括新生儿疾病、失明和免疫低下者的播散性感染。我们的长期目标是全面了解这一重要病原体的进入机制。虽然已经取得了很大的进展，但疱疹病毒融合和进入的复杂机制仍然是个谜。这对开发临床上有效的进入抑制剂构成了障碍。单纯疱疹病毒进入大多数培养的哺乳动物细胞，并占据多种进入途径。疱疹病毒学中的一个新概念是，宿主细胞的内体pH是病毒进入所需的，通常是以细胞类型特有的方式。然而，低pH值在疱疹病毒侵入中所起的作用机制尚不清楚。HSV囊膜蛋白Gb、Gd和Gh/Gl异源二聚体是必需的，但可能不足以在HSV感染的情况下进行膜融合和进入。囊膜蛋白GC是病毒进入细胞表面时附着于细胞表面硫酸乙酰肝素蛋白多糖的主要介体。然而，这种相互作用对于病毒进入细胞培养是必不可少的，也不能解释GC缺失病毒的感染缺陷。我们最近在HSV Entry中发现了一个新的GC连接后功能。GC影响融合蛋白GB，并促进HSV在进入内体时有效地穿透。GC提高了融合蛋白GB中融合相关构象变化的pH阈值。我们制定了互补但独立的目标来描述HSV-1 gC以前未被识别的功能。在特定的目标1中，我们将通过在所需的HSV包膜蛋白和细胞受体的背景下采用一系列分析来阐明GC在融合和进入中的作用。具体目标2包括与上皮细胞进入和感染相关的GC的结构和功能研究，上皮细胞是HSV初次和复发感染的主要靶点。我们的实验设计采用了细胞生物学、生物化学和分子病毒学技术。这些目标的完成将揭示对HSV-1 GC多功能本质的详细洞察，并将填补有关疱疹病毒的复杂融合机制如何在病理生理相关的细胞类型中被触发的关键知识空白。这些研究将有助于开发新的HSV预防和治疗方法。

Herpesviruses are ubiquitous pathogens that cause significant morbidity and mortality worldwide. Herpes simplex virus (HSV) causes cold sores and sexually transmitted infections. Grave outcomes of HSV infection include neonatal disease, blindness, and disseminated infections of the immunocompromised. Our long-term goal is a comprehensive understanding of the entry mechanisms of this important group of pathogens. Although much progress has been made, the complex mechanisms of herpesvirus fusion and entry remain enigmatic. This has posed a roadblock to developing clinically effective entry inhibitors. HSV enters most cultured mammalian cells and commandeers diverse entry pathways. An emerging concept in herpesvirology is that endosomal pH of the host cell is required for viral entry, often in a cell type specific manner. However, the mechanistic role that low pH plays in herpesviral entry is not clear. The HSV envelope proteins gB, gD, and the gH/gL heterodimer are required but are likely not sufficient for membrane fusion and entry in the context of HSV infection. Envelope protein gC is the principal mediator of viral attachment to cell surface heparan sulfate proteoglycans during entry. However, this interaction is dispensable for viral entry in cell culture and does not explain the infection defect of gC-deleted viruses. We recently revealed a new, post-attachment function for gC in HSV entry. gC influences the fusion protein gB and promotes efficient penetration of HSV from endosomes during entry. gC elevates the pH-threshold of fusion- associated conformational changes in the fusion protein gB. We formulated complementary yet independent aims to delineate previously unrecognized functions of HSV-1 gC. In Specific Aim 1, we will elucidate gC's role in fusion and entry by employing a battery of assays in the context of the required HSV envelope proteins and cellular receptors. Specific Aim 2 encompasses structure-function studies of gC pertinent to entry and infection of epithelial cells, the main target of primary and recurrent HSV infection. Our experimental design employs techniques of cell biology, biochemistry, and molecular virology. Completion of the aims will reveal detailed insight into the multifunctional nature of HSV-1 gC and will fill critical knowledge gaps about how the complex fusion mechanism of herpesviruses is triggered in pathophysiologically relevant cell types. These studies will aid in the development of new preventions and therapeutics for HSV.