Interaction between pUL34 and pUL31 is essential for targeting both proteins to the inner nuclear membrane (INM). Sequences mediating the targeting interaction have been mapped by others with both proteins. We have previously reported identification of charge cluster mutants of herpes simplex virus type 1 UL34 that localize properly to the inner nuclear membrane, indicating interaction with UL31, but fail to complement a UL34 deletion. We have characterized one mutation (CL04) that alters a charge cluster near the N terminus of pUL34 and observed the following. Egress of herpesvirus capsids from the nucleus occurs by envelopment of capsids at the inner nuclear membrane (INM) and is followed by de-envelopment at the outer nuclear membrane (ONM). This process can be broken down into a pathway of discrete steps that begin with recruitment of the viral envelopment apparatus to the INM. Herpes simplex virus type 1 (HSV-1) UL34 and UL31 and their homologs in other herpesviruses are required for efficient envelopment at the INM (7,13,22,23,29). HSV-1 pUL31 and pUL34 are targeted specifically to the INM by a mechanism that requires their interaction with each other (27,28), and this mutual dependence is a conserved feature of herpesvirus envelopment (9,14,27,28,32,33,39). Localization of these two proteins at the INM results in the recruitment of other proteins, including protein kinase C delta and pUS3, to the nuclear membrane (22,24,30). The sequences in HSV-1 pUL34 that mediate interaction with UL31 and that lead to nuclear envelope targeting were mapped to amino acids (aa) 137 to 181 (16). The sequences in the murine cytomegalovirus (MCMV) homolog of UL31, M53, that mediate the nuclear envelope targeting interaction with the UL34 homolog, M50, were mapped to the N-terminal third of the protein in the first of four conserved regions (17), and Schnee et al. subsequently showed that this same region of pUL31 homologs from other families of herpesviruses mediates interaction with the corresponding pUL34 homologs (33).After the targeting of the pUL34/pUL31 complex to the INM, subsequent steps in nuclear egress include, it is thought, (i) local disruption of the nuclear lamina to allow capsid access to the INM, (ii) recognition and docking of capsids by the envelopment apparatus at the INM, (iii) curvature of the inner and outer nuclear membranes around the capsid, (iv) scission of the INM to create an enveloped virion in the space between the INM and ONM, (v) fusion of the virion envelope with the outer nuclear membrane, and (vi) capsid release into the cytoplasm.At least some of the viral and cellular factors critical for nuclear lamina disruption and for de-envelopment fusion have been identified. pUL34, pUL31, and pUS3 of HSV-1 have all been implicated in changes in localization, interaction, and phosphorylation of nuclear lamina components, including lamins A/C and B and the lamina-associated protein, emerin (3,15,19,20,24,26,34,35). pUS3, pUL31, and glycoproteins B and H have been implicated in de-envelopment of pri...

The herpes simplex virus 1 (HSV-1) UL51 gene encodes a 244-amino-acid (aa) palmitoylated protein that is conserved in all herpesviruses. The alphaherpesvirus UL51 (pUL51) protein has been reported to function in nuclear egress and cytoplasmic envelopment. No complete deletion has been generated because of the overlap of the UL51 coding sequence 5= end with the UL52 promoter sequences, but partial deletions generated in HSV and pseudorabies virus (PrV) suggest an additional function in epithelial cell-to-cell spread. Here we show partial uncoupling of the replication, release, and cell-to-cell spread functions of HSV-1 pUL51 in two ways. Viruses in which aa 73 to 244 were deleted from pUL51 or in which a conserved YXX⌽ motif near the N terminus was altered showed cell-specific defects in spread that cannot be accounted for by defects in replication and virus release. Also, a cell line that expresses C-terminally enhanced green fluorescent protein (EGFP)-tagged pUL51 supported normal virus replication and release into the medium but the formation of only small plaques. This cell line also failed to support normal localization of gE to cell junctions. gE and pUL51 partially colocalized in infected cells, and these two proteins could be coimmunoprecipitated from infected cells, suggesting that they can form a complex during infection. The cell-to-cell spread defect associated with the pUL51 mutation was more severe than that associated with gE-null virus, suggesting that pUL51 has gE-independent functions in epithelial cell spread. IMPORTANCEHerpesviruses establish and reactivate from lifelong latency in their hosts. When they reactivate, they are able to spread within their hosts despite the presence of a potent immune response that includes neutralizing antibody. This ability is derived in part from a specialized mechanism for virus spread between cells. Cell-to-cell spread is a conserved property of herpesviruses that likely relies on conserved viral genes. An understanding of their function may aid in the design of vaccines and therapeutics. Here we show that one of the conserved viral genes, UL51, has an important role in cell-to-cell spread in addition to its previously demonstrated role in virus assembly. We find that its function depends on the type of cell that is infected, and we show that it interacts with and modulates the function of another viral spread factor, gE.

Herpes simplex virus (HSV) pUL34 plays a critical role in virus replication by mediating egress of nucleocapsids from the infected cell nucleus. We have identified a mutation in pUL34 (Y68A) that produces a major defect in virus replication and impaired nuclear egress but also profoundly inhibits cell-to-cell spread and trafficking of gE. Virion release to the extracellular medium is not affected by the Y68A mutation, indicating that the mutation specifically inhibits cell-to-cell spread. We isolated extragenic suppressors of the Y68A plaque formation defect and mapped them by a combination of high-throughput Illumina sequencing and PCR-based screening. We found that suppression is highly correlated with a nonsense mutation in the US9 gene, which plays a critical role in cell-to-cell spread of HSV-1 in neurons. The US9 mutation alone is not sufficient to suppress the Y68A spread phenotype, indicating a likely role for multiple viral factors.Dissemination of herpes simplex virus (HSV) during recurrent disease in the host is dependent upon efficient viral replication and on the ability of the virus to spread from cell to cell in the face of the host innate and adaptive immune defenses. Cytoplasmic envelopment of HSV-1 virions is followed by vesicular transport of virions to the cell surface and secretion by fusion of the vesicle membrane with the plasma membrane (8,23,44). Transport of virions to cell membranes in contact with the extracellular medium results in release of free virions. Transport to surfaces apposed to other cells results in cell-tocell spread of virus infection. The mechanism by which virions are sorted to junctional or basolateral surfaces in epithelial and fibroblast cells is poorly characterized. About half of the virusencoded proteins play critical roles in virus replication, but relatively few have been found to have specific functions in cell-to-cell spread of virus. The essential components of the virion entry apparatus, gB, gD, and gH/gL, are required for cell-to-cell spread (7,17,30,52). It is likely that this is because cell-to-cell spread requires interaction of the virus entry proteins with cellular receptors and subsequent fusion of the virion envelope with the plasma membrane of the naïve host cell. A few additional viral proteins have been shown to be required for efficient cell-to-cell spread at least in some cell types. HSV-1 gE and gI form a heterodimeric complex that is required for efficient cell-to-cell spread in the nervous system in vivo (10,11,21,22,36). The gE/gI complex is also required for spread in cultured neuronal cells and in epithelial and fibroblast cells that form well-defined cell junctions (1, 10, 12, 36). The gE spread phenotype in epithelial cells requires sequences in the cytoplasmic tail of gE and also requires sorting of gE to basolateral cell surfaces and adherens junctions, where it colocalizes with ␤-catenin (12, 16, 37, 64). Deletion of US9 in pseudorabies virus (PRV) is associated with failure of viral spread in neuronal cultures and in vivo (5, 33). ...

Late in infection herpesviruses move DNA-filled capsids from the nucleus to the cytoplasm by enveloping DNA-containing capsids at the inner nuclear membrane (INM) and deenveloping them at the outer nuclear membrane. This process requires two conserved herpesvirus proteins, pUL31 and pUL34. Interaction between pUL34 and pUL31 is essential for targeting both proteins to the nuclear envelope (NE), and sequences that mediate the targeting interaction have been mapped in both proteins. Here, we show that a mutation in the INM-targeting domain of pUL34 fails to support production of infectious virus or plaque formation. The mutation results in multiple defects, including impaired interaction between pUL34 and pUL31, poor NE targeting of pUL34, and misregulated, capsid-independent budding of the NE. The mutant defects in virus production, plaque formation, and pUL31 interaction can be suppressed by other mutations in the INMtargeting domain of pUL31 and by additional mutations in the pUL34 coding sequence.Efficient nuclear egress of herpesviruses requires formation of a nuclear envelopment complex (NEC) consisting of viral and cellular proteins (24,26,34). The NEC contains homologs of the herpes simplex virus (HSV) UL31 and UL34 proteins (called pUL31 and pUL34), and these are critical for nuclear egress in all herpesviruses tested (9,15,24,25,33). pUL31 and pUL34 homologs interact with each other, and formation of a pUL31/pUL34 complex is required for proper targeting of the complex to the nuclear envelope (NE) (10,16,30,31,36,37,41). The interactions that underlie complex formation are therefore critical for assembly and egress of all herpesviruses.Despite the conservation of a pUL31-pUL34 interaction, it is not clear that the structural basis for that interaction is completely conserved. The sequence of pUL31 and its homologs can be divided into four conserved regions (CRs) (Fig. 1B) (37). The most N-terminal of these regions (CR1) has been shown to mediate interaction with pUL34 homologs in examples from all herpesvirus subfamilies (19, 37). The situation with pUL34 homologs is less clear. For HSV-1 pUL34, the sequence that interacts with pUL31 and that is required for nuclear envelope targeting was mapped by deletion and domain swapping to amino acids (aa) 137 to 181 (18). This corresponds to the third of three CRs in the pUL34 sequence (Fig. 1A). Consistent with this, a construct containing CR1, CR2, and most of CR3 (aa 1 to 161) of pseudorabies virus (PRV) pUL34 was sufficient to interact with pUL31 in a yeast two-hybrid assay (10). In mouse cytomegalovirus (MCMV), on the other hand, use of small insertions and point mutations implicated a different region of the UL34 homolog, M50, in binding to the UL31 homolog, M53 (4,19,28). The interaction region is located in a highly conserved stretch of residues at the N terminus of M50 CR2. Whether the differences between MCMV M50 and HSV pUL34 reflect a very different structural basis for interaction is not yet clear.At the NE, pUL34 and pUL31 mediate subsequent steps ...

Herpesvirus infection reorganizes components of the nuclear lamina usually without loss of integrity of the nuclear membranes. We report that wild-type HSV infection can cause dissolution of the nuclear envelope in transformed mouse embryonic fibroblasts that do not express torsinA. Nuclear envelope breakdown is accompanied by an eight-fold inhibition of virus replication. Breakdown of the membrane is much more limited during infection with viruses that lack the gB and gH genes, suggesting that breakdown involves factors that promote fusion at the nuclear membrane. Nuclear envelope breakdown is also inhibited during infection with virus that does not express UL34, but is enhanced when the US3 gene is deleted, suggesting that envelope breakdown may be enhanced by nuclear lamina disruption. Nuclear envelope breakdown cannot compensate for deletion of the UL34 gene suggesting that mixing of nuclear and cytoplasmic contents is insufficient to bypass loss of the normal nuclear egress pathway.