Structure-Function Analysis of Herpesvirus Glycoprotein H

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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.