Molecular mechanisms of glycoprotein E in HSV cell-cell transmission

描述（由申请人提供）：人类疱疹病毒仍然是一个主要的公共卫生问题，并负责许多重要的人类疾病。这些病毒在体内传播的一个关键手段是细胞间传播，这是一个与病毒发病机制高度相关的过程。通过这种模式传播感染允许有效和快速的子代病毒粒子传播，并且还促进中和抗体的免疫逃避。在单纯疱疹病毒（HSV）的情况下，它对于在神经元细胞中成功建立潜伏感染也是至关重要的。本申请的长期目标是阐明HSV细胞间传播的分子机制，以便最终找到更好的策略和控制措施来预防感染的传播。本申请的重点是糖蛋白E（gE），这是一种膜蛋白，在所有α-疱疹病毒中具有同源物，长期以来一直被认为是细胞间传播所需的，尽管其机制仍然知之甚少。我们已经通过研究其蛋白质相互作用网络在了解这种分子方面取得了实质性进展。我们的研究已经揭示了三种被膜蛋白（UL 11，UL 16和UL 21）在gE的胞质尾部上的惊人的协调组装，这导致形成病毒诱导的细胞-细胞融合（合胞体形成）所需的功能复合物，这是一种特殊形式的细胞-细胞传播。为了进一步探究gE是如何工作的，我们设计了三个具体的目标。在第一个目标中，我们提出了一种无偏倚的遗传方法，其中我们将选择第二位点阻遏物，该阻遏物将合胞体表型恢复到由于缺乏gE尾或其结合配偶体而失去合胞体表型的突变体（例如，缺失突变体gE <$CT/gBsyn、<$UL11/gBsyn、<$UL16/gBsyn和<$UL21/gBsyn）。Syn表型具有选择性优势，因为合胞病毒可以通过引起细胞融合而更快地通过细胞培养物传递病毒基因组。因为UL 11、UL 16和UL 21是合胞体形成所必需的，所以这种方法将寻求“功能增益”。已经获得了gE <$CT/gBsyn的第二位点抑制子。第二个目的是通过一种新的体内试验来鉴定gE和其他病毒糖蛋白之间的相互作用。初步数据显示，与gD的相互作用，我们假设，直接与病毒融合机制的相互作用是必需的gE，以促进细胞间的传播和合胞体的形成。第三个目的是确定gE如何运输到细胞连接处的机制以及gE细胞表面表达与细胞间扩散的相关性。人们早就 已知gE在HSV感染后期重新分布到细胞连接处，并且这种运输特性对于gE促进病毒体分选到侧向细胞连接处至关重要，但其机制的细节尚不清楚。这三个目标将大大推进我们对gE在HSV细胞间传播中的分子机制的理解，这可能最终导致发现治疗HSV感染所需的抗病毒药物的新靶点。

DESCRIPTION (provided by applicant): Human herpesviruses remain a major public health concern and are responsible for many important human diseases. A critical means by which these viruses disseminate in vivo is cell-to-cell spread, a process that is highly relevant to vira pathogenesis. Spread of the infections by this mode allows efficient and rapid progeny virion dissemination, and also promotes immune evasion of neutralizing antibodies. In the case of herpes simplex virus (HSV), it is also critical for successful establishment of latent infections i neuronal cells. The long-term objective of this application is to elucidate the molecular mechanism of HSV cell-to-cell transmission so that better strategies and control measures can eventually be found to prevent the spread of infections. The focus of this application is glycoprotein E (gE), a membrane protein that has homologues among all alpha-herpesviruses and has long been known to be required for cell-to-cell spread, though the mechanism remains poorly understood. We have made substantial progress in understanding this molecule by investigating its protein-interaction network. Our studies have revealed a striking, coordinated assembly of three tegument proteins (UL11, UL16 and UL21) onto the cytoplasmic tail of gE, which leads to the formation of a functional complex required for virus-induced cell-cell fusion (syncytia formation), a special form of cell-to-cell spread. To pursue further clues of how gE works, we have designed three specific aims. In the first aim, we propose an unbiased genetic approach in which we will select for second-site repressors that restore the syncytia phenotype to mutants that have lost it because of the absence of the gE tail or its binding partners (e.g., deletion mutants gE¿CT/gBsyn, ¿UL11/gBsyn, ¿UL16/gBsyn, and ¿UL21/gBsyn). The Syn phenotype has a selective advantage because syncytial viruses can transmit viral genomes through the cell culture more quickly by causing cells to fuse. Because UL11, UL16 and UL21 are required for syncytia formation, this approach will be seeking a "gain of function". One second-site suppressor of gE¿CT/gBsyn has already been obtained. The second aim is to identify interactions between gE and other viral glycoproteins by means of a novel in vivo assay. Preliminary data have revealed an interaction with gD, and we hypothesize that direct interactions with the viral fusion machinery is required for gE to promote cell-to-cell spread and syncytia formation. The third aim is to determine the mechanism of how gE traffics to cell junctions and the relevance of gE cell surface expression for cell-to-cell spread. It has long been known that gE redistributes to cell junctions late in HSV infection, and this trafficking property s critical for gE to promote sorting of virions to lateral cell junctions, but details of the mechanim are not clear. These three aims will substantially advance our understanding of the molecular mechanisms of gE in HSV cell-to-cell transmission, which may eventually lead to discovery of novel targets for antiviral drugs that are needed for treatment of HSV infection.