| Summary: | Glycoprotein B (gB) is the conserved herpesvirus fusion protein, and it is required for the entry of herpesviruses. The structure of the postfusion conformation of gB has been solved for several herpesviruses; however, the gB prefusion crystal structure and the details of how the protein refolds from a prefusion to a postfusion form to mediate fusion have not been determined. Using structure-based mutagenesis, we previously reported that three mutations (I671A, H681A, and F683A) in the C-terminal arm of the gB ectodomain greatly reduced cell-cell fusion. This fusion deficit could be rescued by the addition of a hyperfusogenic mutation, suggesting that the gB triple mutant was not misfolded. Using a bacterial artificial chromosome (BAC), we constructed two independent herpes simplex virus 1 mutant strains (gB 3A) carrying the three arm mutations. The gB 3A viruses have 200-fold smaller plaques than the wild-type virus and demonstrate remarkably delayed entry into cells. Single-step and multistep growth curves show that gB 3A viruses have delayed replication kinetics. Interestingly, incubation at 40°C promoted the entry of the gB 3A viruses. We propose that the gB 3A viruses’ entry deficit is due to a loss of interactions between residues in the gB C-terminal arm and the coiled-coil core of gB. The results suggest that the triple alanine mutation may destabilize the postfusion gB conformation and/or stabilize the prefusion gB conformation and that exposure to elevated temperatures can overcome the defect in gB 3A viruses.
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