THE UL45 GENE-PRODUCT IS REQUIRED FOR HERPES-SIMPLEX VIRUS TYPE-1 GLYCOPROTEIN B-INDUCED FUSION

Herpes simplex virus type 1 (HSV-1) syncytial (syn) mutants cause form ation of giant polykaryocytes and have been utilized to identify genes promoting or suppressing cell fusion. We previously described an HSV- 1 recombinant, F1 (J. L. Goodman, M. L. Cook, F. Sederati, K. Izumi, a nd J. G. Stevens, J. Virol. 63:1153-1161, 1989), which has unique viru lence properties and a syn mutation in the carboxy terminus of glycopr otein B (gB). We attempted to replace this single-base-pair syn mutati on through cotransfection with a 379-bp PCR-generated fragment of wild -type gB. The nonsyncytial viruses isolated were shown by DNA sequenci ng not to have acquired the expected wild-type gB sequence. Instead, t hey had lost their fell-cell fusion properties because of alterations mapping to the UL45 gene. The mutant UL45 gene in one nonsyncytial der ivative of F1, A4B, was found to have a deletion of a C at UL45 nucleo tide 230, resulting in a predicted frame shift and termination at 92 r ather than 172 amino acids. Northern (RNA) analysis showed that the mu tant UL45 gene was normally transcribed. However, Western immunoblotti ng showed no detectable UL45 gene product from A4B or from another sim ilarly isolated nonsyncytial F1 derivative, A61B, while another such v irus, 1ACSS, expressed reduced amounts of UL45. When A4B was cotransfe cted with the wild-type UL45 gene, restoration of UL45 expression corr elated with restoration of syncytium formation. Conversely, cloned DNA fragments containing the mutant A4B UL45 gene transferred the loss of cell-cell fusion to other gB syn mutants, rendering them UL45 negativ e and nonsyncytial. We conclude that normal UL45 expression is require d to allow cell fusion induced by gB syn mutants and that the nonessen tial UL45 protein may play an important role as a mediator of fusion e vents during HSV-1 infection.