Primary envelopment of pseudorabies virus at the nuclear membrane requiresthe UL34 gene product

Primary envelopment of several herpesviruses has been shown to occur by bud ding of intranuclear capsids through the inner nuclear membrane. By subsequ ent fusion of the primary envelope with the outer nuclear membrane, capsids are released into the cytoplasm and gain their final envelope by budding i nto vesicles in the trans-Golgi area. We show here that the product of the UL34 gene of pseudorabies virus, an alphaherpesvirus of swine, is localized in transfected and infected cells in the nuclear membrane. It is also dete cted in the envelope of virions in the perinuclear space but is undetectabl e in intracytoplasmic and extracellular enveloped virus particles. Converse ly, the tegument protein UL49 is present in mature virus particles and abse nt from perinuclear virions. In the absence of the UL34 protein, acquisitio n of the primary envelope is blocked and neither virus particles in the per inuclear space nor intracytoplasmic capsids or virions are observed. Howeve r, light particles which label with the anti-UL49 serum are formed in the c ytoplasm. We conclude that the UL34 protein is required for primary envelop ment, that the primary envelope is biochemically different from the final e nvelope in that it contains the UL34 protein, and that perinuclear virions lack the tegument protein UL49, which is present in mature virions. Thus, w e provide additional evidence for a two-step envelopment process in herpesv iruses.