Mutations in Pseudorabies Virus Glycoproteins gB, gD, and gH Functionally Compensate for the Absence of gL

Entry of herpesviruses depends on the combined action of viral glycoproteins (g)B and the heterodimeric gH/gL complex which are activated by binding of the virion to specific cellular receptors. Although gB carries signatures of a bona fide fusion protein, efficient membrane fusion requires gH/gL. However, although gB and gH/gL are essential for entry, the alphaherpesvirus Pseudorabies Virus (PrV) is capable of limited cell-to-cell spread in the absence of gL. To understand gH/gL function in more detail, the limited spread of PrV-ΔgL was used for reversion analyses by serial cell culture passages. In a first experiment an infectious gL-negative mutant was isolated in which gL function was substituted by generation of a gD-gH hybrid protein (Klupp and Mettenleiter, 1999). In a second, independent experiment PrV-ΔgLPassB4.1 was isolated, which also replicated productively without gL. Sequence analysis revealed mutations in gH, but also in gB and gD. In a transfection based fusion assay two amino acid substitutions in the N-terminal part of gHB4.1 (L70P; W103R) were found to be sufficient to compensate for lack of gL, while mutations present in gBB4.1 enhanced fusogenicity. Co-expression of gBB4.1 with the homologous gHB4.1 resulted in strongly enhanced syncytium formation, which was further augmented by truncation of the gBB4.1 C-terminal 29 amino acids. Nevertheless, gH was still required for membrane fusion. Surprisingly, co-expression of gDB4.1 blocked syncytium formation in the fusion assays, which could be attributed to a V106A substitution within the ectodomain of gDB4.1.