Selection of Mutations that Affect Herpesvirus Entry into Cells

Abstract

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 InformationNIH 1R01AI148478‐01