Comparative analysis of functional domains and motifs within glycoprotein gH of Epstein-Barr Virus and Pseudorabies Virus, and their functional role in the herpesviral fusion process

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Herpesviruses are amongst the most complex viruses and herpes virions consist of more than 40 proteins. Three subfamilies, alpha-, beta-, and gamma-Herpesvirinae, have been classified within the Herpesviridae based on biological properties and sequence comparison. Infection of host cells starts after attachment either by direct fusion of the viral envelope with the plasma membrane or, after endo-cytic uptake, with the endosomal membrane. While membrane fusion in many viruses is accomplished by one or two viral proteins, herpesvirus entry requires the concerted action of four different proteins. The first step in penetration is binding of subfamily-specific viral proteins to host cell receptors. Stable binding triggers fusion which relies on a conserved core fusion machinery consisting of glycoproteins (g)B and gH/gL. Mutants lacking either of these proteins are unable to enter cells. The crystal structure of gB shows signatures of class III fusion proteins, however, gB is not sufficient to mediate efficient fusion but requires the heterodimeric gH/gL complex. The role of gH and gL in the fusion process is still enigmatic. The recently solved crystal structures for gH/gL of Herpes Simplex Virus-2 and Epstein-Barr virus (EBV), as well as of a core domain of Pseudorabies virus (PrV) gH revealed surprising similarity with four clearly separable gH domains. This proposal aims at the comparison of functional domains and motifs within gH of PrV and EBV, and their role in the fusion process. To this end, the different gH domains will be swapped between gH of PrV, a member of the y-herpesviruses and of EBV, which belongs to the y-Herpesvirinae, and identified motifs as well as key amino acids will be mutated. The chimeras and mutated gH molecules will be tested in fusion assays, for interaction with gL and gB, and for complementation of corresponding deletion mutants. These results will help to understand the fusion process and could lead to new antiviral strategies.