U(L)31 and U(L)34 proteins of herpes simplex virus type 1 form a complex that accumulates at the nuclear rim and is required for envelopment of nucleocapsids

The herpes simplex virus type 1 (HSV-1) U(L)34 protein is likely a type II membrane protein that localizes within the nuclear membrane and is required for efficient envelopment of progeny virions at the nuclear envelope, whereas the U(L)31 gene product of HSV-1 is a nuclear matrix-associated phosphoprotein previously shown to interact with U(L)34 protein in HSV-1-infected cell lysates. For these studies, polyclonal antisera directed against purified fusion proteins containing U(L)31 protein fused to glutathione-S-transferase (U(L)31-GST) and U(L)34 protein fused to GST (U(L)34-GST) were demonstrated to specifically recognize the U(L)31 and U(L)34 proteins of approximately 34,000 and 30,000 Da, respectively. The U(L)31 and U(L)34 gene products colocalized in a smooth pattern throughout the nuclear rim of infected cells by 10 h postinfection. U(L)34 protein also accumulated in pleiomorphic cytoplasmic structures at early times and associated with an altered nuclear envelope late in infection. Localization of U(L)31 protein at the nuclear rim required the presence of U(L)34 protein, inasmuch as cells infected with a U(L)34 null mutant virus contained U(L)31 protein primarily in central intranuclear domains separate from the nuclear rim, and to a lesser extent in the cytoplasm. Conversely, localization of U(L)34 protein exclusively at the nuclear rim required the presence of the U(L)31 gene product, inasmuch as U(L)34 protein was detectable at the nuclear rim, in replication compartments, and in the cytoplasm of cells infected with a U(L)31 null virus. When transiently expressed in the absence of other viral factors, U(L)31 protein localized diffusely in the nucleoplasm, whereas U(L)34 protein localized primarily in the cytoplasm and at the nuclear rim. In contrast, coexpression of the U(L)31 and U(L)34 proteins was sufficient to target both proteins exclusively to the nuclear rim. The proteins were also shown to directly interact in vitro in the absence of other viral proteins. In cells infected with a virus lacking the U(S)3-encoded protein kinase, previously shown to phosphorylate the U(L)34 gene product, U(L)31 and U(L)34 proteins colocalized in small punctate areas that accumulated on the nuclear rim. Thus, U(S)3 kinase is required for even distribution of U(L)31 and U(L)34 proteins throughout the nuclear rim. Taken together with the similar phenotypes of the U(L)31 and U(L)34 deletion mutants, these data strongly suggest that the U(L)31 and U(L)34 proteins form a complex that accumulates at the nuclear membrane and plays an important role in nucleocapsid envelopment at the inner nuclear membrane.     

The U(L)15 gene of herpes simplex virus type 1 contains within its second exon a novel open reading frame that is translated in frame with the U(L)15 gene product.

The U(L)15 gene of herpes simplex virus type 1 is composed of two exons. A mutation previously shown to preclude viral DNA cleavage and packaging at the nonpermissive temperature was identified as a change from a highly conserved serine to proline at codon 653. Separate viral mutants that contained stop codons inserted into exon I of U(L)15 (designated S648) or an insertion of the Escherichia coli lacZ gene into a truncated U(L)15 exon II [designated HSV-1(delta U(L)15ExII)] were constructed. Recombinant viruses derived from S648 and HSV-1(delta U(L)15ExII) and containing restored U(L)15 genes were constructed and designated S648R and HSV-1(delta U(L)15ExIIR), respectively. Unlike HSV-1(delta U(L)15ExIIR) and S648R, the viruses containing mutant U(L)15 genes failed to cleave and package viral DNA when propagated on noncomplementing cells. As revealed by electron microscopy, large numbers of enveloped capsids lacking viral DNA accumulated within the cytoplasm of cells infected with either S648 or HSV-1(delta U(L)15ExII) but not in cells infected with HSV-1(delta U(L)15ExIIR) or S648R. Thus, one function of the U(L)15 gene is to effectively prevent immature particles lacking DNA from exiting the nucleus by envelopment at the inner lamella of the nuclear membrane. Cells infected with HSV-1(delta U(L)15ExII) did not express the 75,000- or 35,000-apparent-Mr proteins previously shown to be products of the U(L)15 open reading frame, whereas the 35,000-apparent-Mr protein was readily detectable in cells infected with S648. We conclude that at least the 75,000-Mr protein is required for viral DNA cleavage and packaging and hypothesize that the 35,000-Mr protein is derived from translation of a novel mRNA located partially or completely within the second exon of U(L)15.     

Herpes simplex virus type 1 infection induces activation and recruitment of protein kinase C to the nuclear membrane and increased phosphorylation of lamin B

We report that herpes simplex virus type 1 (HSV-1) infection leads to the recruitment of protein kinase C (PKC) to the nuclear rim. In HEp-2 cells, PKC recruitment to the nuclear rim was initiated between 8 h and 12 h postinfection. PKCdelta, a proapoptotic kinase, was completely recruited to the nuclear rim upon infection with HSV-1. PKCalpha was less dramatically relocalized mostly at the nuclear rim upon infection, although some PKCalpha remained in the cytoplasm. PKCzeta-specific immunofluorescence was not significantly relocated to the nuclear rim. The UL34 and UL31 proteins, as well as their association, were each required for PKC recruitment to the nuclear rim. The HSV-1 US3 protein product, a kinase which regulates the phosphorylation state and localization of UL34, was not required for PKC recruitment to the nuclear rim; however, it was required for proper localization along the nuclear rim, as PKC appeared unevenly distributed along the nuclear rim of cells infected with US3 null and kinase-dead mutants. HSV-1 infection induced the phosphorylation of both lamin B and PKC. Elevated lamin B phosphorylation in HSV-1-infected cells was partially reduced by inhibitors of PKC. The data suggest a model in which kinases that normally disassemble the nuclear lamina during apoptosis are recruited to the nuclear membrane through functions requiring UL31 and UL34. We hypothesize that the recruitment of PKC functions to phosphorylate lamin B to help modify the nuclear lamina and promote budding of nucleocapsids at the inner nuclear membrane.     

Conformational changes in the nuclear lamina induced by herpes simplex virus type 1 require genes U(L)31 and U(L)34

The herpes simplex virus type 1 (HSV-1) U(L)31 and U(L)34 proteins are dependent on each other for proper targeting to the nuclear membrane and are required for efficient envelopment of nucleocapsids at the inner nuclear membrane. In this work, we show that whereas the solubility of lamins A and C (lamin A/C) was not markedly increased, HSV induced conformational changes in the nuclear lamina of infected cells, as viewed after staining with three different lamin A/C-specific antibodies. In one case, reactivity with a monoclonal antibody that recognizes an epitope in the lamin tail domain was greatly reduced in HSV-infected cells. This apparent HSV-induced epitope masking required both U(L)31 and U(L)34, but these proteins were not sufficient to mask the epitope in uninfected cells, indicating that other HSV proteins are also required. In the second case, staining with a rabbit polyclonal antibody that primarily recognizes epitopes in the lamin A/C rod domain revealed that U(L)34 is required for HSV-induced decreased availability of epitopes for reaction with the antibody, whereas U(L)31 protein was dispensable for this effect. Still another polyclonal antibody indicated virtually no difference in lamin A/C staining in infected versus uninfected cells, indicating that the HSV-induced changes are more conformational than the result of lamin depletion at the nuclear rim. Further evidence supporting an interaction between the nuclear lamina and the U(L)31/U(L)34 protein complex includes the observations that (i) overexpression of the U(L)31 protein in uninfected cells was sufficient to relocalize lamin A/C from the nuclear rim into nucleoplasmic aggregates, (ii) overexpression of U(L)34 was sufficient to relocalize some lamin A/C into the cytoplasm, and (iii) both U(L)31 and U(L)34 could directly bind lamin A/C in vitro. These studies suggest that the U(L)31 and U(L)34 proteins modify the conformation of the nuclear lamina in infected cells, possibly by direct interaction with lamin A/C, and that other proteins are also likely involved. Given that the nuclear lamina potentially excludes nucleocapsids from envelopment sites at the inner nuclear membrane, the lamina alteration may reflect a role of the U(L)31/U(L)34 protein complex in perturbing the lamina to promote nucleocapsid egress from the nucleus. Alternatively, the data are compatible with a role of the lamina in targeting the U(L)31/U(L)34 protein complex to the nuclear membrane.     

Pseudorabies virus envelope glycoproteins gp50 and gII are essential for virus penetration, but only gII is involved in membrane fusion.

To investigate the function of the envelope glycoproteins gp50 and gII of pseudorabies virus in the entry of the virus into cells, we used linker insertion mutagenesis to construct mutant viruses that are unable to express these proteins. In contrast to gD mutants of herpes simplex virus, gp50 mutants, isolated from complementing cells, were able to form plaques on noncomplementing cells. However, progeny virus released from these cells was noninfectious, although the virus was able to adsorb to cells. Thus, the virus requires gp50 to penetrate cells but does not require it in order to spread by cell fusion. This finding indicates that fusion of the virus envelope with the cell membrane is not identical to fusion of the cell membranes of infected and uninfected cells. In contrast to the gp50 mutants, the gII mutant was unable to produce plaques on noncomplementing cells. Examination by electron microscopy of cells infected by the gII mutant revealed that enveloped virus particles accumulated between the inner and outer nuclear membranes. Few noninfectious virus particles were released from the cell, and infected cells did not fuse with uninfected cells. These observations indicate that gII is involved in several membrane fusion events, such as (i) fusion of the viral envelope with the cell membrane during penetration, (ii) fusion of enveloped virus particles with the outer nuclear membrane during the release of nucleocapsids into the cytoplasm, and (iii) fusion of the cell membranes of infected and uninfected cells.     

Effects of lamin A/C, lamin B1, and viral US3 kinase activity on viral infectivity, virion egress, and the targeting of herpes simplex virus U(L)34-encoded protein to the inner nuclear membrane

Previous results indicated that the U(L)34 protein (pU(L)34) of herpes simplex virus 1 (HSV-1) is targeted to the nuclear membrane and is essential for nuclear egress of nucleocapsids. The normal localization of pU(L)34 and virions requires the U(S)3-encoded kinase that phosphorylates U(L)34 and lamin A/C. Moreover, pU(L)34 was shown to interact with lamin A in vitro. In the present study, glutathione S-transferase/pU(L)34 was shown to specifically pull down lamin A and lamin B1 from cellular lysates. To determine the role of these interactions on viral infectivity and pU(L)34 targeting to the inner nuclear membrane (INM), the localization of pU(L)34 was determined in LmnA(-/-) and LmnB1(-/-) mouse embryonic fibroblasts (MEFs) by indirect immunofluorescence and immunogold electron microscopy in the presence or absence of U(S)3 kinase activity. While pU(L)34 INM targeting was not affected by the absence of lamin B1 in MEFs infected with wild-type HSV as viewed by indirect immunofluorescence, it localized in densely staining scalloped-shaped distortions of the nuclear membrane in lamin B1 knockout cells infected with a U(S)3 kinase-dead virus. Lamin B1 knockout cells were relatively less permissive for viral replication than wild-type MEFs, with viral titers decreased at least 10-fold. The absence of lamin A (i) caused clustering of pU(L)34 in the nuclear rim of cells infected with wild-type virus, (ii) produced extensions of the INM bearing pU(L)34 protein in cells infected with a U(S)3 kinase-dead mutant, (iii) precluded accumulation of virions in the perinuclear space of cells infected with this mutant, and (iv) partially restored replication of this virus. The latter observation suggests that lamin A normally impedes viral infectivity and that U(S)3 kinase activity partially alleviates this impediment. On the other hand, lamin B1 is necessary for optimal viral replication, probably through its well-documented effects on many cellular pathways. Finally, neither lamin A nor B1 was absolutely required for targeting pU(L)34 to the INM, suggesting that this targeting is mediated by redundant functions or can be mediated by other proteins.     

The herpes simplex virus 1 U(L)34 protein interacts with a cytoplasmic dynein intermediate chain and targets nuclear membrane

To express the function encoded in its genome, the herpes simplex virus 1 capsid-tegument structure released by deenvelopment during entry into cells must be transported retrograde to the nuclear pore where viral DNA is released into the nucleus. This path is essential in the case of virus entering axons of dorsal root ganglia. The objective of the study was to identify the viral proteins that may be involved in the transport. We report the following findings. (i) The neuronal isoform of the intermediate chain (IC-1a) of the dynein complex pulled down, from lysates of [(35)S]methionine-labeled infected cells, two viral proteins identified as the products of U(L)34 and U(L)31 open reading frames, respectively. U(L)34 protein is a virion protein associated with cellular membranes and phosphorylated by the viral kinase U(S)3. U(L)31 protein is a largely insoluble, evenly dispersed nuclear phosphoprotein required for optimal processing and packaging of viral DNA into preformed capsids. Reciprocal pulldown experiments verified the interaction of IC-1a and U(L)34 protein. In similar experiments, U(L)34 protein was found to interact with U(L)31 protein and the major capsid protein ICP5. (ii) To determine whether U(L)34 protein is transported to the nuclear membrane, a requirement if it is involved in transport, the U(L)34 protein was inserted into a baculovirus vector under the cytomegalovirus major early promoter. Cells infected with the recombinant baculovirus expressed U(L)34 protein in a dose-dependent manner, and the U(L)34 protein localized primarily in the nuclear membrane. An unexpected finding was that U(L)34-expressing cells showed a dissociation of the inner and outer nuclear membranes reminiscent of the morphologic changes seen in cells productively infected with herpes simplex virus 1. U(L)34, like many other viral proteins, may have multiple functions expressed both early and late in infection.     

UL31 of herpes simplex virus 1 is necessary for optimal NF-kappaB activation and expression of viral gene products

Previous results suggested that the U(L)31 gene of herpes simplex virus 1 (HSV-1) is required for envelopment of nucleocapsids at the inner nuclear membrane and optimal viral DNA synthesis and DNA packaging. In the current study, viral gene expression and NF-κB and c-Jun N-terminal kinase (JNK) activation of a herpes simplex virus mutant lacking the U(L)31 gene, designated ΔU(L)31, and its genetic repair construct, designated ΔU(L)31-R, were studied in various cell lines. In Hep2 and Vero cells infected with ΔU(L)31, expression of the immediate-early protein ICP4, early protein ICP8, and late protein glycoprotein C (gC) were delayed significantly. In Hep2 cells, expression of these proteins failed to reach levels seen in cells infected with ΔU(L)31-R or wild-type HSV-1(F) even after 18 h. The defect in protein accumulation correlated with poor or no activation of NF-κB and JNK upon infection with ΔU(L)31 compared to wild-type virus infection. The protein expression defects of the U(L)31 deletion mutant were not explainable by a failure to enter nonpermissive cells and were not complemented in an ICP27-expressing cell line. These data suggest that pU(L)31 facilitates initiation of infection and/or accelerates the onset of viral gene expression in a manner that correlates with NF-κB activation and is independent of the transactivator ICP27. The effects on very early events in expression are surprising in light of the fact that U(L)31 is designated a late gene and pU(L)31 is not a virion component. We show herein that while most pUL31 is expressed late in infection, low levels of pU(L)31 are detectable as early as 2 h postinfection, consistent with an early role in HSV-1 infection.     

Emerin is hyperphosphorylated and redistributed in herpes simplex virus type 1-infected cells in a manner dependent on both UL34 and US3

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 Cdelta (PKCdelta) 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 PKCdelta 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.     

Herpes simplex virus type 1 primary envelopment: UL34 protein modification and the US3-UL34 catalytic relationship

The herpes simplex virus type 1 (HSV-1) US3 kinase is likely important for primary envelopment of progeny nucleocapsids since it localizes to the nuclear envelope of infected cells and largely determines the phosphorylation state and localization of the necessary primary envelopment factor, the UL34 protein. In HEp-2 cells, the production of infectious US3 null progeny is delayed and decreased relative to that of the parental strain, HSV-1(F). Furthermore, the US3 kinase affects the morphology of primary envelopment such that in its absence, UL34 protein-containing enveloped virions accumulate within membrane-bound vesicles. These vesicles are most often found along the interior periphery of the nucleus and may be derived from the inner nuclear membrane. Since the US3 and UL34 proteins comprise a kinase-substrate pair, a reasonable hypothesis is that the US3 kinase influences these replication parameters by direct phosphorylation of the UL34 protein. For this report, recombinant viruses were constructed to determine the significance of UL34 protein phosphorylation and US3 catalytic activity on UL34 protein localization, single-step growth, and envelopment morphology in both HEp-2 and Vero cells. The data presented suggest that the significance of UL34 phosphorylation is cell type dependent and that efficient viral morphogenesis requires US3-mediated phosphorylation of an infected cell protein other than UL34.     

pUL21 regulation of pUs3 kinase activity influences the nature of nuclear envelope deformation by the HSV-2 nuclear egress complex

It is well established that the herpesvirus nuclear egress complex (NEC) has an intrinsic ability to deform membranes. During viral infection, the membrane-deformation activity of the NEC must be precisely regulated to ensure efficient nuclear egress of capsids. One viral protein known to regulate herpes simplex virus type 2 (HSV-2) NEC activity is the tegument protein pUL21. Cells infected with an HSV-2 mutant lacking pUL21 (ΔUL21) produced a slower migrating species of the viral serine/threonine kinase pUs3 that was shown to be a hyperphosphorylated form of the enzyme. Investigation of the pUs3 substrate profile in ΔUL21-infected cells revealed a prominent band with a molecular weight consistent with that of the NEC components pUL31 and pUL34. Phosphatase sensitivity and retarded mobility in phos-tag SDS-PAGE confirmed that both pUL31 and pUL34 were hyperphosphorylated by pUs3 in the absence of pUL21. To gain insight into the consequences of increased phosphorylation of NEC components, the architecture of the nuclear envelope in cells producing the HSV-2 NEC in the presence or absence of pUs3 was examined. In cells with robust NEC production, invaginations of the inner nuclear membrane were observed that contained budded vesicles of uniform size. By contrast, nuclear envelope deformations protruding outwards from the nucleus, were observed when pUs3 was included in transfections with the HSV-2 NEC. Finally, when pUL21 was included in transfections with the HSV-2 NEC and pUs3, decreased phosphorylation of NEC components was observed in comparison to transfections lacking pUL21. These results demonstrate that pUL21 influences the phosphorylation status of pUs3 and the HSV-2 NEC and that this has consequences for the architecture of the nuclear envelope.     

Identification of an essential domain in the herpes simplex virus 1 UL34 protein that is necessary and sufficient to interact with UL31 protein

Previous results have indicated that the herpes simplex virus 1 UL31 and UL34 proteins interact and form a complex at the inner nuclear membranes of infected cells, where both play important roles in the envelopment of nucleocapsids at the inner nuclear membrane. In the work described here, mapping studies using glutathione S-transferase pull-down assays indicated that amino acids 137 to 181 of the UL34 protein are sufficient to mediate an interaction with the UL31 protein. A recombinant virus (v3480) lacking UL34 codons 138 to 181 was constructed. Similar to a UL34 null virus, v3480 failed to replicate on Vero cells and grew to a limited extent on rabbit skin cells. A UL34-expressing cell line restored v3480 growth and plaque formation. Similar to the localization of UL31 protein in cells infected with a UL34 null virus, the UL31 protein was present in the nuclei of Hep2 cells infected with v3480. Hep2 cells infected with v3480 contained the UL34 protein in the cytoplasm, the nucleus, and the nuclear membrane, and this was noted to be similar to the appearance of cells infected with a UL31 null virus. In transient expression assays, the interaction between UL34 amino acids 137 to 181 and the UL31 protein was sufficiently robust to target green fluorescent protein and emerin to intranuclear sites that contained the UL31 protein. These data indicate that amino acids 137 to 181 of the UL34 protein are (i) sufficient to mediate interactions with the UL31 protein in vitro and in vivo, (ii) necessary for the colocalization of UL31 and UL34 in infected cells, and (iii) essential for normal viral replication.     

The Interacting UL31 and UL34 Gene Products of Pseudorabies Virus Are Involved in Egress from the Host-Cell Nucleus and Represent Components of Primary Enveloped but Not Mature Virions

A 2.6-kbp fragment of the pseudorabies virus (PrV) genome was sequenced and shown to contain the homologues of the highly conserved herpesvirus genes UL31 and UL32. By use of a monospecific antiserum, the UL31 gene product was identified as a nuclear protein with an apparent molecular mass of 29 kDa. For functional analysis, UL31 was deleted by mutagenesis in Escherichia coli of an infectious full-length clone of the PrV genome. The resulting virus mutants were deficient in plaque formation, and titers were reduced more than 100-fold from those of wild-type PrV. Ultrastructural analyses demonstrated that capsid maturation and DNA packaging were not affected. However, neither budding at the inner nuclear membrane nor cytoplasmic or extracellular virus particles were observed. These replication defects were similar to those of a UL34 deletion mutant (B. G. Klupp, H. Granzow, and T. C. Mettenleiter, J. Virol. 74:10063-10073, 2000) and could be completely repaired in a cell line which constitutively expresses the UL31 protein. Yeast two-hybrid studies revealed that a UL31 fusion protein specifically interacts with plasmids of a PrV genome library expressing the N-terminal part of UL34. Vice versa, UL34 selected UL31-encoding plasmids from the library. Immunofluorescence studies and immune electron microscopy demonstrated that in cells infected with wild-type PrV, both proteins accumulate at the nuclear membrane, whereas in the absence of UL34 the UL31 protein is dispersed throughout the nucleus. Like the UL34 protein, the UL31 gene product is a component of enveloped virus particles within the perinuclear space and absent from mature virions. Our findings suggest that physical interaction between these two virus proteins might be a prerequisite for primary envelopment of PrV at the inner nuclear membrane and that this envelope is removed by fusion with the outer nuclear membrane.     

The null mutant of the U(L)31 gene of herpes simplex virus 1: construction and phenotype in infected cells.

Earlier studies have shown that the U(L)31 protein is homogeneously distributed throughout the nucleus and cofractionates with nuclear matrix. We report the construction from an appropriate cosmid library a deletion mutant which replicates in rabbit skin cells carrying the U(L)31 gene under a late (gamma1) viral promoter. The mutant virus exhibits cytopathic effects and yields 0.01 to 0.1% of the yield of wild-type parent virus in noncomplementing cells but amounts of virus 10- to 1,000-fold higher than those recovered from the same cells 3 h after infection. Electron microscopic studies indicate the presence of small numbers of full capsids but a lack of enveloped virions. Viral DNA extracted from the cytoplasm of infected cells exhibits free termini indicating cleavage/packaging of viral DNA from concatemers for packaging into virions, but analyses of viral DNAs by pulsed-field electrophoresis indicate that at 16 h after infection, both the yields of viral DNA and cleavage of viral DNA for packaging are decreased. The repaired virus cannot be differentiated from the wild-type parent. These results suggest the possibility that U(L)31 protein forms a network to enable the anchorage of viral products for the synthesis and/or packaging of viral DNA into virions.     

Herpes simplex virus nucleocapsids mature to progeny virions by an envelopment --> deenvelopment --> reenvelopment pathway

Herpes simplex virus (HSV) nucleocapsids acquire an envelope by budding through the inner nuclear membrane, but it is uncertain whether this envelope is retained during virus maturation and egress or whether mature progeny virions are derived by deenvelopment at the outer nuclear membrane followed by reenvelopment in a cytoplasmic compartment. To resolve this issue, we used immunogold electron microscopy to examine the distribution of glycoprotein D (gD) in cells infected with HSV-1 encoding a wild-type gD or a gD which is retrieved to the endoplasmic reticulum (ER). In cells infected with wild-type HSV-1, extracellular virions and virions in the perinuclear space bound approximately equal amounts of gD antibody. In cells infected with HSV-1 encoding an ER-retrieved gD, the inner and outer nuclear membranes were heavily gold labeled, as were perinuclear enveloped virions. Extracellular virions exhibited very little gold decoration (10- to 30-fold less than perinuclear virions). We conclude that the envelope of perinuclear virions must be lost during maturation and egress and that mature progeny virions must acquire an envelope from a post-ER cytoplasmic compartment. We noted also that gD appears to be excluded from the plasma membrane in cells infected with wild-type virus.     

Fate of the inner nuclear membrane protein lamin B receptor and nuclear lamins in herpes simplex virus type 1 infection

During herpesvirus egress, capsids bud through the inner nuclear membrane. Underlying this membrane is the nuclear lamina, a meshwork of intermediate filaments with which it is tightly associated. Details of alterations to the lamina and the inner nuclear membrane during infection and the mechanisms involved in capsid transport across these structures remain unclear. Here we describe the fate of key protein components of the nuclear envelope and lamina during herpes simplex virus type 1 (HSV-1) infection. We followed the distribution of the inner nuclear membrane protein lamin B receptor (LBR) and lamins A and B(2) tagged with green fluorescent protein (GFP) in live infected cells. Together with additional results from indirect immunofluorescence, our studies reveal major morphologic distortion of nuclear-rim LBR and lamins A/C, B(1), and B(2). By 8 h p.i., we also observed a significant redistribution of LBR-GFP to the endoplasmic reticulum, where it colocalized with a subpopulation of cytoplasmic glycoprotein B by immunofluorescence. In addition, analysis by fluorescence recovery after photobleaching reveals that LBR-GFP exhibited increased diffusional mobility within the nuclear membrane of infected cells. This is consistent with the disruption of interactions between LBR and the underlying lamina. In addition to studying stably expressed GFP-lamins by fluorescence microscopy, we studied endogenous A- and B-type lamins in infected cells by Western blotting. Both approaches reveal a loss of lamins associated with virus infection. These data indicate major disruption of the nuclear envelope and lamina of HSV-1-infected cells and are consistent with a virus-induced dismantling of the nuclear lamina, possibly in order to gain access to the inner nuclear membrane.     

Structural determinants for nuclear envelope localization and function of pseudorabies virus pUL34

Herpesvirus proteins pUL34 and pUL31 form a complex at the inner nuclear membrane (INM) which is necessary for efficient nuclear egress. Pseudorabies virus (PrV) pUL34 is a type II membrane protein of 262 amino acids (aa). The transmembrane region (TM) is predicted to be located between aa 245 and 261, leaving only one amino acid in the C terminus that probably extends into the perinuclear space. It is targeted to the nuclear envelope in the absence of other viral proteins, pointing to intrinsic localization motifs, and shows structural similarity to cellular INM proteins like lamina-associated polypeptide (Lap) 2ß and Emerin. To investigate which domains of pUL34 are relevant for localization and function, we constructed chimeric proteins by replacing parts of pUL34 with regions of cellular INM proteins. First the 18 C-terminal amino acids encompassing the TM were exchanged with TM regions and C-terminal domains of Lap2ß and Emerin or with the first TM region of the polytopic lamin B receptor (LBR), including the nine following amino acids. All resulting chimeric proteins complemented the replication defect of PrV-ΔUL34, demonstrating that the substitution of the TM and the extension of the C-terminal domain does not interfere with the function of pUL34. Complementation was reduced but not abolished when the C-terminal 50 aa were replaced by corresponding Lap2ß sequences (pUL34-LapCT50). However, replacing the C-terminal 100 aa (pUL34-LapCT100) resulted in a nonfunctional protein despite continuing pUL31 binding, pointing to an important functional role of this region. The replacement of the N-terminal 100 aa (pUL34-LapNT100) had no effect on nuclear envelope localization but abrogated pUL31 binding and function.     

Functional domains of murine cytomegalovirus nuclear egress protein M53/p38

Two conserved herpes simplex virus 1 proteins, UL31 and UL34, form a complex at the inner nuclear membrane which governs primary envelopment and nuclear egress of the herpesvirus nucleocapsids. In mouse cytomegalovirus, a member of the betaherpesvirus subfamily, the homologous proteins M53/p38 and M50/p35 form the nuclear egress complex (NEC). Since the interaction of these proteins is essential for functionality, the definition of the mutual binding sites is a prerequisite for further analysis. Using a comprehensive random mutagenesis procedure, we have mapped the M53/p38 binding site of M50/p35 (A. Bubeck, M. Wagner, Z. Ruzsics, M. L?tzerich, M. Iglesias, I. R. Singh, and U. H. Koszinowski, J. Virol. 78:8026-8035). Here we describe a corresponding analysis for the UL31 homolog M53/p38. A total of 72 individual mutants were reinserted into the genome to test the complementation of the lethal M53 null phenotype. The mutants were also studied for colocalization and for coprecipitation with M50/p35. The analysis revealed that the nonconserved N-terminal one-third of M53/p38 provides the nuclear localization signal as an essential function. The collective results for many mutants localized the binding site for M50/p35 to amino acids (aa) 112 to 137. No single aa exchange for alanine could destroy NEC formation, but virus attenuation revealed a major role for aa K128, Y129, and L130. The lethal phenotype of several insertion and stop mutants indicated the functional importance of the C terminus of the protein.     

Herpes simplex virus type 1 U(L)34 gene product is required for viral envelopment

The herpes simplex virus type 1 U(L)34 gene encodes a protein that is conserved in all human herpesviruses. The association of the U(L)34 protein with membranes in the infected cell and its expression as a gamma-1 gene suggest a role in maturation or egress of the virus particle from the cell. To determine the function of this gene product, we have constructed a recombinant virus that fails to express the U(L)34 protein. This recombinant virus, in which the U(L)34 protein coding sequence has been replaced by green fluorescent protein, forms minute plaques and replicates in single-step growth experiments to titers 3 to 5 log orders of magnitude lower than wild-type or repair viruses. On Vero cells, the deletion virus synthesizes proteins of all kinetic classes in normal amounts. Electron microscopic and biochemical analyses show that morphogenesis of the deletion virus proceeds normally to the point of formation of DNA-containing nuclear capsids, but electron micrographs show no enveloped virus particles in the cytoplasm or at the surface of infected cells, suggesting that the U(L)34 protein is essential for efficient envelopment of capsids.