Structure-Based Mutations in the Herpes Simplex Virus 1 Glycoprotein B Ectodomain Arm Impart a Slow-Entry Phenotype

Area of Science:

• Virology
• Structural Biology
• Molecular Biology

Background:

• Herpesvirus entry relies on the conserved fusion protein, glycoprotein B (gB).
• The mechanism of gB-mediated membrane fusion, particularly the prefusion to postfusion conformational change, remains incompletely understood.
• Previous studies identified key mutations in the gB C-terminal arm affecting cell-cell fusion.

Purpose of the Study:

• To investigate the role of the gB C-terminal arm in herpesvirus entry and fusion.
• To elucidate the functional consequences of specific gB mutations on viral replication and entry kinetics.
• To understand the structural basis of gB refolding and its impact on herpesvirus-mediated membrane fusion.

Main Methods:

• Structure-based mutagenesis was employed to create three mutations (I671A, H681A, F683A) in the gB ectodomain's C-terminal arm.
• Herpes simplex virus 1 (HSV-1) mutant strains (gB 3A) were constructed using a bacterial artificial chromosome (BAC) system.
• Viral entry, plaque formation, replication kinetics, and rescue by temperature were assessed for gB 3A viruses.

Main Results:

• The gB 3A viruses exhibited significantly smaller plaques (200-fold) and markedly delayed entry into cells compared to wild-type HSV-1.
• Replication kinetics were delayed in gB 3A viruses, as shown by single-step and multistep growth curves.
• Incubation at 40°C rescued the entry defect of gB 3A viruses, suggesting altered energetics of gB refolding.

Conclusions:

• The C-terminal arm of gB is critical for efficient herpesvirus entry, likely through interactions with the coiled-coil core.
• The triple alanine mutations destabilize the postfusion conformation and/or stabilize the prefusion conformation of gB.
• Elevated temperatures can overcome the fusion defect, providing insights into the conformational dynamics of gB during viral entry.