Extragenic suppression of an HSV-1 UL34 nuclear egress mutant reveals role for pUS9 as an inhibitor of epithelial cell-to-cell spread

The herpes simplex virus pUL34 component of the nuclear egress complex functions in both nuclear egress and cell-to-cell spread. A R158A/R161A double substitution in pUL34 has been shown to strongly inhibit both nuclear egress and spread. Selection for extragenic suppressors of UL34_R158A/R161A on the background of a virus that expresses monomeric red fluorescent protein-tagged pUL35 yielded two viruses, SUP7 and SUP9, that both replicated and spread much better than the parental virus. The two viruses carried both unique and common genetic changes. The most striking unique genetic change was a large rearrangement of the US region of SUP7 resulting in duplication of ICP22 and deletion of part of US3 and all of US9-12. The two common genetic changes were a frame shift that eliminated expression of pUL35, and mutations that either eliminated or diminished expression of pUS9. The mutations in pUL35 significantly improved replication but had only a small effect on spread. We observed that mutations that diminish or eliminate detectable pUS9 expression significantly improved spread on Vero cells of viruses that were impaired in the spread function of either pUL34 or pUL51, but did not enhance spread of an otherwise wild-type virus. A similar enhancement was observed on U2OS, but not HaCaT cells, suggesting that the spread-inhibitory function of pUS9 differs between cell lines. IMPORTANCE Herpesviruses are able to disseminate in infected hosts despite development of a strong immune response. Their ability to do this relies on a specialized process called cell-to-cell spread in which newly assembled virus particles are trafficked to plasma membrane surfaces that abut adjacent uninfected cells. The mechanism of cell-to-cell spread is obscure, and little is known about whether or how it is regulated in different cells. We show here that a viral protein with a well-characterized role in promoting spread from neurons has an opposite, inhibitory role in other cells.