Effect of the pseudorabies virus US3 protein on nuclear membrane localization of the UL34 protein and virus egress from the nucleus

The alphaherpesviruis UL34 protein is necessary for the primary envelopment of intranuclear capsids at the inner leaflet of the nuclear membrane. In h erpes simplex virus type 1, the UL34 protein is exclusively phosphorylated by the protein kinase encoded by the non-essential US3 gene. To investigate the effect of the pseudorabies virus (PrV) US3 product on the intracellula r localization of the UL34 protein and on virus morphogenesis, PrV US3 dele tion mutants were isolated and characterized. Immunofluorescence analyses d emonstrated that in the absence of the US3 protein, the localization of the UL34 polypeptide to the nuclear membrane was not as pronounced as that see n with US3, although immunoelectron microscopy indicated the presence of th e UL34 protein in both leaflets of the nuclear membrane. Ultrastructurally, an accumulation of enveloped virions in the perinuclear space in large inv aginations of the inner nuclear membrane was observed, which were shown by immunoelectron microscopy to contain the UL34 protein, but not glycoprotein s gB or gC. Thus, the US3 protein appears to be involved in the de-envelopm ent of perinuclear virions by fusion with the outer leaflet of the nuclear membrane. Surprisingly, no difference in the phosphorylation of the PrV UL3 4 protein was observed in the presence or absence of the US3 kinase. Theref ore, the observed effects of the PrV US3 protein on the intracellular local ization of the UL34 protein and on virus morphogenesis are probably not due to the phosphorylation of the UL34 protein by the US3 kinase.