Enveloped viruses enter cells by fusing their envelope with the host cell membrane. Herpes simplex virus 1 (HSV-1) entry requires the coordinated interaction of several viral glycoproteins, including gH/gL and gB. gH/gL and gB are essential for virus replication and both proteins are targets of neutralizing antibodies. gB fuses the membranes after being activated by gH/gL, but the details of how gH/gL activates gB are not known.
Enveloped virus entry requires fusion of the viral envelope with a host cell membrane. Herpes simplex virus type 1 (HSV-1) entry is mediated by a set of glycoproteins that interact to trigger the viral fusion protein glycoprotein B (gB). In the current model, receptor-binding by gD signals a gH/gL heterodimer to trigger a refolding event in gB that fuses the membranes. To explore functional interactions between gB and gH/gL, we used a bacterial artificial chromosome (BAC) to generate two HSV-1 mutants that show a small plaque phenotype due to changes in gB. We passaged the viruses to select for restoration of plaque size and analyzed second-site mutations that arose in gH. HSV-1 gB was replaced either by gB from saimiriine herpesvirus type 1 (SaHV-1) or by a mutant form of HSV-1 gB with three alanine substitutions in domain V (gB3A). To shift the selective pressure away from gB, the gB3A virus was passaged in cells expressing gB3A. Sequencing of passaged viruses identified two interesting mutations in gH, including gH-H789 in domain IV and gH-S830N in the cytoplasmic tail (CT). Characterization of these gH mutations indicated they are responsible for the enhanced plaque size. Rather than being globally hyperfusogenic, both gH mutations partially rescued function of the specific gB version present during their selection. These sites may represent functional interaction sites on gH/gL for gB. gH-H789 may alter the positioning of a membrane-proximal flap in the gH ectodomain, whereas gH-S830 may contribute to an interaction between the gB and gH CTs.
During virus entry into cells, herpesviruses rely on the coordinated action of multiple viral glycoproteins. For herpes simplex virus type 1 (HSV1), four glycoproteins (gD, gH, gL, and gB) are required to fuse the viral and host membranes. gB functions as a fusion protein and is conserved in the herpesvirus family. It mediates viral entry by undergoing a conformational change that results in fusion viral and host membrane. This conformational change is triggered by both gD, the receptor‐binding protein, and gHgL, a heterodimer that modulates fusion. Capturing a physical interaction among these glycoproteins has been challenging, most likely because the interactions are low affinity and/or transient. The goal of this study is to characterize the interactions between gB and other herpesvirus proteins using natural selection. We previously generated a fusion‐deficient gB mutant. By serially passaging HSV1 carrying this gB mutant, we are selecting for mutations that restore fusion function. To shift the selective pressure away from second‐site mutations within gB, we generated a virus that is deleted for the gB gene but carries the mutant gB protein in its membrane. After six rounds of passage, the virus showed enhanced replication and the viral genome was submitted for next‐generation sequencing. The sequence results will be analyzed to determine the locations of novel mutations. The impact of these mutations on virus entry will be examined by mapping them to existing structures and analyzing mutant function in cell‐cell fusion assays. Support or Funding Information NIH 1R01AI148478‐01
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.