Visualization of herpes simplex virus type 1 virions using fluorescent colors

Our laboratory was one of the first to engineer a live fluorescent tag, enhanced green fluorescent protein (eGFP), that marked the capsid of herpes simplex virus type 1 (HSV-1) and subsequently maturing virus as the particle made its way to the cell surface. In the present study we sought to increase the repertoire of colors available as fusion to the small capsid protein, VP26, so that they can be used alone or in conjunction with other fluorescent tags (fused to other HSV proteins) to follow the virus as it enters and replicates within the cell. We have now generated viruses expressing VP26 fusions with Cerulean, Venus, mOrange, tdTomato, mCherry, and Dronpa3 fluorescent proteins. These fusions were made in a repaired UL35 gene (VP26) background. These fusions do not affect the replication properties of the virus expressing the fusion polypeptide and the fusion tag was stably associated with intranuclear capsids and mature virions. Of note we could not isolate viruses expressing fusions with fluorescent proteins that have a tendency to dimerize.