Structure-Function Analysis of Herpesvirus Glycoprotein H

疱疹病毒利用受体结合组件和融合诱导复合体的机制感染细胞。虽然受体结合主要涉及亚家族特异性蛋白，如α疱疹病毒糖蛋白gD，但参与病毒粒子被膜和细胞膜融合的蛋白质在整个疱疹病毒科中是保守的。其中包括结构类似于水泡性口炎病毒融合基因G蛋白的gB，以及Gh和gl的异源二聚体复合体。根据最近获得的三种疱疹病毒Gh蛋白的晶体结构，在第一阶段，我们进行了定点突变，分析了Gh的不同结构域和结构特征对融合的贡献，包括一个可以遮盖或暴露疏水表面的可移动的活瓣和一个类合成素的束。在第二种方法中，我们使用gl缺失的伪狂犬病病毒的逆转分析来选择补偿突变，这产生了关于融合过程调节的信息。该项目第二阶段的目标是继续这些研究，并具体分析参与膜融合的甲型疱疹病毒糖蛋白之间的相互作用，即Gd、Gh和Gb。我们使用特征良好的甲型疱疹病毒PRV作为模型，因为它指定了几个有吸引力的表型。(I)在瞬时融合试验中，PRV能够在没有受体结合成分Gd的情况下介导膜融合；(Ii)在gB和/或gD和Gh存在补偿突变的情况下，PRV能够在没有gl的情况下介导有效的膜融合；(Iii)关于Gh和gB的结构信息是可用的。

Herpesviruses infect cells using a machinery of receptor-binding components and the fusion-inducing complex. Whereas receptor-binding primarily involves subfamily-specific proteins, such as alphaherpesvirus glycoprotein gD, proteins involved in fusion between virion envelope and cellular plasma membrane are conserved throughout the Herpesviridae. These include gB whose structure is similar to that of the fusogenic G protein of vesicular stomatitis virus, and a heterodimeric complex of gH and gL. Based on crystal structures of gH proteins of three herpesviruses which became available recently, in the first period we performed site-directed mutagenesis to analyze the contributions of different domains and structural features of gH to fusion including a mobile flap which may shield or expose a hydrophobic surface and a syntaxin-like bundle. In a second approach we used reversion analysis of gL-deleted pseudorabies virus to select for compensatory mutations which yielded information on the regulation of the fusion process. The goal in this second period of the project is to continue these studies and to specifically analyze the interaction between the alphaherpesvirus glycoproteins involved in membrane fusion, i.e. gD, gH and gB. We use the well-characterized alphaherpesvirus PrV as a model since it specifies several attractive phenotypes. (i) PrV is able to mediate membrane fusion without the receptor binding component gD in transient fusion assays, in gD-independent infectivity, and in direct cell-to-cell spread; (ii) PrV can mediate efficient membrane fusion without gL in the presence of compensatory mutations in gB and/or gD and gH; (iii) structural information on gH as well as gB is available.