Cells infected with wild-type herpes simplex virus type 1 (HSV-1) show disruption of the organization of the nuclear lamina that underlies the nuclear envelope. This disruption is reflected in changes in the localization and phosphorylation of lamin proteins. Here, we show that HSV-1 infection causes relocalization of the LEM domain protein emerin. In cells infected with wild-type virus, emerin becomes more mobile in the nuclear membrane, and in cells infected with viruses that fail to express UL34 protein (pUL34) and US3 protein (pUS3), emerin no longer colocalizes with lamins, suggesting that infection causes a loss of connection between emerin and the lamina. Infection causes hyperphosphorylation of emerin in a manner dependent upon both pUL34 and pUS3. Some emerin hyperphosphorylation can be inhibited by the protein kinase C␦ (PKC␦) inhibitor rottlerin. Emerin and pUL34 interact physically, as shown by pull-down and coimmunoprecipitation assays. Emerin expression is not, however, necessary for infection, since virus growth is not impaired in cells derived from emerin-null transgenic mice. The results suggest a model in which pUS3 and PKC␦ that has been recruited by pUL34 hyperphosphorylate emerin, leading to disruption of its connections with lamin proteins and contributing to the disruption of the nuclear lamina. Changes in emerin localization, nuclear shape, and lamin organization characteristic of cells infected with wild-type HSV-1 also occur in cells infected with recombinant virus that does not make viral capsids, suggesting that these changes occur independently of capsid envelopment.During primary envelopment, herpes simplex virus type 1 (HSV-1) nucleocapsids translocate from the nucleus to the cytoplasm by budding into the inner nuclear membrane and then fusing with the outer nuclear membrane. The capsid does not, however, have unimpeded access to the inner nuclear membrane. Lining the inside of the inner nuclear membrane is the nuclear lamina, which is composed of a meshwork of proteins with spaces too small for the capsid to move through without some disruption (2,19,65). The lamina meshwork is made up of intermediate filament family proteins called lamins that are linked to the inner nuclear membrane and to intranuclear proteins by association with lamin-associated proteins (LAPs) (reviewed in reference 65). Connection of the network of lamin proteins to the inner nuclear membrane is mediated by integral membrane LAPs, including emerin, lamin B receptor, LAP2-␤, and MAN-1 (26).Emerin is a member of a family of nuclear envelope proteins that share a common sequence called the LEM domain that mediates association with BAF (barrier to autointegration factor) and is important for the assembly of LEM domain proteins into the re-forming nuclear envelope following mitosis (21,37,39). Emerin also contains a lamin-binding domain that helps retain it in the interphase nuclear envelope (6,14,25,37). Emerin is ubiquitously expressed but is not essential for the viability of cells in culture (36). Failure to ...

Cells infected with wild type HSV-1 showed significant lamin A/C and lamin B rearrangement, while UL34-null virus-infected cells exhibited few changes in lamin localization, indicating that UL34 is necessary for lamin disruption. During HSV infection, US3 limited the development of disruptions in the lamina, since cells infected with a US3-null virus developed large perforations in the lamin layer. US3 regulation of lamin disruption does not correlate with the induction of apoptosis. Expression of either UL34 or US3 proteins alone disrupted lamin A/C and lamin B localization. Expression of UL34 and US3 together had little effect on lamin A/C localization, suggesting a regulatory interaction between the two proteins. The data presented in this paper argue for crucial roles for both UL34 and US3 in regulating the state of the nuclear lamina during viral infection.

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...

Herpes simplex virus type 1 (HSV-1) is a DNA virus that acquires an envelope by budding into the inner nuclear membrane of an infected cell. Recombinant HSV-1 lacking the U L 34 gene cannot undergo this event. U L 34 and U L 31, another viral protein, colocalize in an infected cell and are necessary and sufficient to target both proteins to the inner nuclear envelope. In order to define and characterize sequences of U L 34 that are necessary for primary envelopment to occur, a library of 19 U L 34 charged cluster mutants and a truncation mutant lacking the putative transmembrane domain (⌬TM) were generated. Mutants in this library were analyzed in a complementation assay for their ability to function in the production of infectious virus. Herpes simplex virus type 1 (HSV-1) undergoes primary envelopment, budding from the interior of the nucleus into the perinuclear space, as one of the first steps in egress from the cell (10,29,34). Herpesvirus egress is a complex process involving many virus-encoded proteins (2-4, 12, 13, 18, 21, 26), but only the U L 34 protein and its homologs have been shown to be required for primary envelopment in HSV-1 (35), pseudorabies virus (22), herpes simplex virus type 2 (HSV-2) (39), and most recently in equine herpesvirus type 1 (28). The HSV-1 U L 34 gene product is a 30-kDa membrane-associated phosphoprotein that is a substrate for the virus-encoded protein kinase U S 3 (31, 32). The U L 34 sequence contains a putative transmembrane domain of 14 to 17 amino acids near the C terminus of the protein. While localization and membrane association of the U L 34 protein suggest that U L 34 contains a transmembrane anchor, biochemical tests have not been done to verify the existence of such a domain. In addition, the interaction of U L 34 with another HSV-1 protein, the U L 31 gene product, is necessary and sufficient to target both proteins to the nuclear membrane, where they are thought to form a complex (33). Colocalization of U L 34 and U L 31 at the nuclear membrane is also seen in HSV-2 and pseudorabies virus (16,43). Since the U L 34 protein coding sequence is well conserved in alphaherpesviruses for which sequences are available (11,15,40), it seems likely that U L 34 protein function is also widely conserved among herpesviruses.Efficient primary envelopment in alphaherpesviruses is linked to complete packaging of DNA into the capsid. Many viral mutants that synthesize capsids but fail to correctly package their DNA are unable to envelop the empty capsids (1,6,7,19,25,30,36). Once genome packaging is complete, the nucleocapsid must gain access to the inner nuclear membrane, and this, in turn, likely requires a mechanism to break down the nuclear lamina. It is currently unknown how HSV-1 gains access to the nuclear membrane, but several lines of evidence suggest that herpesvirus infection leads to alterations of nuclear envelope structure. Infection with HSV-1 has been reported to lead to an increase in soluble lamin A/C, suggesting at least limited disassembly of the lamina (38)...