The UL31 and UL34 Gene Products of Herpes Simplex Virus 1 Are Required for Optimal Localization of Viral Glycoproteins D and M to the Inner Nuclear Membranes of Infected Cells

U_L31 and U_L34 of herpes simplex virus type 1 form a complex necessary for nucleocapsid budding at the inner nuclear membrane (INM). Previous examination by immunogold electron microscopy and electron tomography showed that pU_L31, pU_L34, and glycoproteins D and M are recruited to perinuclear virions and densely staining regions of the INM where nucleocapsids bud into the perinuclear space. We now show by quantitative immunogold electron microscopy coupled with analysis of variance that gD-specific immunoreactivity is significantly reduced at both the INM and outer nuclear membrane (ONM) of cells infected with a U_L34 null virus. While the amount of gM associated with the nuclear membrane (NM) was only slightly (P = 0.027) reduced in cells infected with the U_L34 null virus, enrichment of gM in the INM at the expense of that in the ONM was greatly dependent on U_L34 (P < 0.0001). pU_L34 also interacted directly or indirectly with immature forms of gD (species expected to reside in the endoplasmic reticulum or nuclear membrane) in lysates of infected cells and with the cytosolic tail of gD fused to glutathione S-transferase in rabbit reticulocyte lysates, suggesting a role for the pU_L34/gD interaction in recruiting gD to the NM. The effects of U_L34 on gD and gM localization were not a consequence of decreased total expression of gD and gM, as determined by flow cytometry. Separately, pU_L31 was dispensable for targeting gD and gM to the two leaflets of the NM but was required for (i) the proper INM-versus-ONM ratio of gD and gM in infected cells and (ii) the presence of electron-dense regions in the INM, representing nucleocapsid budding sites. We conclude that in addition to their roles in nucleocapsid envelopment and lamina alteration, U_L31 and U_L34 play separate but related roles in recruiting appropriate components to nucleocapsid budding sites at the INM.Herpesvirus virions comprise a nucleocapsid containing genomic viral DNA, a proteinaceous tegument layer surrounding the nucleocapsid, and a virion envelope surrounding the tegument. The envelope of extracellular herpes simplex virus (HSV) virions contains glycoproteins gB, gC, gD, gE, gI, gG, gH, gK, gL, and gM (23, 51).As viewed by electron microscopy, nascent virions form as the nucleocapsid buds through densely staining regions of the nuclear membrane (NM) (21, 41). Electron tomograms of HSV perinuclear virions compared to those of extracellular virions infer that the former contain glycoproteins of considerably less glycosylation and a relatively sparse tegument layer compared to their counterparts in mature extracellular virions (6). The lower levels of glycosylation in HSV perinuclear virions are consistent with the fact that the lumen of the perinuclear space is continuous with that of the endoplasmic reticulum. Thus, the polysaccharide moieties of virion glycoproteins become fully processed as virions access Golgi enzymes during their egress to the extracellular space. Although the full proteome of the nascent perinuclear virion is unknown, immunogold studies have shown that they contain at least pU_L31, pU_L34, pUS3, gB, gC, gD, gH, gM, and the VP16 and pU_L11 tegument proteins in addition to the proteins that comprise the viral capsid (4, 5, 15, 25, 37, 40, 47, 50, 55).The U_L31 and U_L34 gene products of HSV-1 (pU_L31 and pU_L34, respectively) form a complex that localizes at the inner and outer NMs (INM and ONM, respectively) of infected cells (40). Both proteins are essential for nucleocapsid envelopment at the INM and become incorporated into nascent virions when nucleocapsids bud through the INM into the perinuclear space (39, 40, 42). The proteins and their essential role in nucleocapsid envelopment are conserved in all herpesvirus subfamilies (14, 20, 32, 45). pU_L31 of HSV-1 is a mostly hydrophobic phosphoprotein that is held in close approximation to the nucleoplasmic face of the INM by interaction with pU_L34, an integral membrane protein of type II orientation (33, 40, 46, 56). The first 248 amino acids of pU_L34 are predicted to reside in the nucleoplasm or cytoplasm, depending on whether the protein localizes in the INM or ONM, respectively. This is followed by an approximately 22-amino acid transmembrane domain with up to 5 amino acids residing in the perinuclear space or lumen of the endoplasmic reticulum.In the most prominent model of herpesvirion egress, the envelope of the perinuclear virion fuses with the ONM, releasing the deenveloped nucleocapsid into the cytoplasm, where it subsequently buds into cytoplasmic membranous organelles such as the Golgi or trans-Golgi network (34, 49). This model is supported by the observation that pU_L31 and pU_L34 are located in the perinuclear virion but not extracellular virions (18, 40). Thus, these proteins are lost from the virion upon fusion of the virion envelope with the ONM. Also supporting this egress model is the observation that deletion of both gB and gH causes virions to accumulate aberrantly in the perinuclear space (15). The involvement of gH and gB is potentially satisfying because these proteins comprise essential components of the machinery that mediates fusion of the virion envelope with the plasma or endosomal membranes during the initiation of infection (9, 12, 16, 44, 52). Moreover, expression of a combination of gB, gD, gH, and gL is sufficient to mediate fusion of cell membranes, whereas coexpression with gM or gK inhibits this fusion (3, 8, 11). Although the mechanism of fusion is unclear, gD is known to bind viral receptors on cell surfaces, and the structure of gB indicates features reminiscent of other viral fusion proteins (24, 35, 48). gD has been shown to interact with gB and gH at least transiently, suggesting that these interactions may be important for the fusion reaction (1, 2). Thus, fusion between the nascent and mature virion envelopes with target membranes may share mechanistic similarities.On the other hand, it is likely that the two fusion events are mechanistically distinct because (i) single deletion of either gH or gB precludes viral entry and cell/cell fusion but does not cause nascent virions to accumulate in the perinuclear space (9, 16, 31, 43) and (ii) the activity of a viral kinase encoded by U_S3 is dispensable for entry but believed to promote fusion of the perinuclear virion and ONM (28, 40). Moreover, the lack of glycoproteins from the pseudorabies virus perinuclear virion suggests that fusion is mediated by an entirely different mechanism in this system (26).The current study focuses on how glycoproteins are incorporated into the nascent virion. We show that optimal recruitment of gD to both leaflets of the NM and gM to the INM requires pU_L34 and pU_L31. We also show that immature gD interacts with pU_L34, suggesting a mechanism by which pU_L34 might recruit gD to the NM.