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.     

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.     

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.     

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 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.     

DNA cleavage and packaging proteins encoded by genes U(L)28, U(L)15, and U(L)33 of herpes simplex virus type 1 form a complex in infected cells

Previous studies have indicated that the U(L)6, U(L)15, U(L)17, U(L)28, U(L)32, and U(L)33 genes are required for the cleavage and packaging of herpes simplex viral DNA. To identify proteins that interact with the U(L)28-encoded DNA binding protein of herpes simplex virus type 1 (HSV-1), a previously undescribed rabbit polyclonal antibody directed against the U(L)28 protein fused to glutathione S-transferase was used to immunopurify U(L)28 and the proteins with which it associated. It was found that the antibody specifically coimmunoprecipitated proteins encoded by the genes U(L)28, U(L)15, and U(L)33 from lysates of both HEp-2 cells infected with HSV-1(F) and insect cells infected with recombinant baculoviruses expressing these three proteins. In reciprocal reactions, antibodies directed against the U(L)15- or U(L)33-encoded proteins also coimmunoprecipitated the U(L)28 protein. The coimmunoprecipitation of the three proteins from HSV-infected cells confirms earlier reports of an association between the U(L)28 and U(L)15 proteins and represents the first evidence of the involvement of the U(L)33 protein in this complex.     

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.     

Effects of charged cluster mutations on the function of herpes simplex virus type 1 UL34 protein

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 (DeltaTM) were generated. Mutants in this library were analyzed in a complementation assay for their ability to function in the production of infectious virus. Seven of the mutants failed to complement a U(L)34-null virus. The remainder of the mutants complemented at or near wild-type U(L)34 levels. Failure of a mutant protein to function might be the result of incorrect subcellular localization. To address this possibility, confocal microscopy was used to determine the localization of the U(L)34 protein in charged cluster mutants and DeltaTM. In transfection-infection experiments, all of the functional U(L)34 mutants and four of the six noncomplementing mutants localized to the inner nuclear envelope in a manner indistinguishable from that of wild-type U(L)34. All of the noncomplementing U(L)34 mutants mediated proper localization of U(L)31. Charged clusters critical for U(L)34 function are dispersed throughout the protein sequence and do not correlate well with highly conserved regions of the protein. These data suggest that U(L)34 has at least one function in addition to mediating proper localization of U(L)31 in infected cells and provide further support for the role of U(L)34 in mediating proper localization of U(L)31 in infected cells.     

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.     

Small dense nuclear bodies are the site of localization of herpes simplex virus 1 U(L)3 and U(L)4 proteins and of ICP22 only when the latter protein is present

The herpes simplex virus 1 U(L)3 and U(L)4 open reading frames are expressed late in infection and are not essential for viral replication in cultured cells in vitro. An earlier report showed that the U(L)4 protein colocalizes with the products of the alpha22/U(S)1.5 genes in small nuclear dense bodies. Here we report that the U(L)3 protein also colocalized in these small nuclear dense bodies and the localization of U(L)3 and U(L)4 proteins in these bodies required the presence of alpha22/U(S)1.5 genes. In cells infected with a mutant lacking intact alpha22/U(S)1.5 genes, U(L)3 was diffused throughout the nucleus even though the overall accumulation of the gamma2 U(L)3 protein was decreased. The results suggest that ICP22 acts both as a regulator of U(L)3 accumulation and as the structural component and anchor of these small dense nuclear bodies.     

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.     

UL34, the target of the herpes simplex virus U(S)3 protein kinase, is a membrane protein which in its unphosphorylated state associates with novel phosphoproteins.

Previous studies (F. C. Purves, D. Spector, and B. Roizman, J. Virol. 65:5757-5764, 1991) have shown that the protein kinase encoded by the U(S)3 gene mediates posttranslational modification of a viral phosphoprotein with an apparent M(r) of 30,000 encoded by the UL34 gene. Here we report the following. (i) UL34 protein is not phosphorylated in cells infected with recombinant viruses deleted in the U(S)3 gene. (ii) Several new phosphoproteins (apparent M(r)s, 25,000 to 35,000) are present in cells infected with recombinant viruses deleted in the U(S)3 gene or with viruses carrying a mutation in the UL34 gene that precluded phosphorylation of the UL34 gene product by the U(S)3 protein kinase, but not in cells infected under conditions which permit phosphorylation of the UL34 protein. These proteins are genetically unrelated to the product of the UL34 gene. (iii) Polyclonal rabbit anti-UL34 protein serum precipitated not only the UL34 protein but also the other (25,000- to 35,000-M(r)) phosphoproteins from lysates of cells infected with U(S)3- virus. (iv) The UL34 gene product is a membrane protein inasmuch as the polyclonal anti-UL34 serum reacted with surfaces of intact, unfixed, infected cells and the antigen-antibody complex formed in this reaction contained the UL34 protein. (v) Small amounts of the UL34 protein were present in virions of infected cells. We conclude that the UL34 gene product is a membrane protein exclusively phosphorylated by the U(S)3 protein kinase which can either directly or indirectly form complexes with several other phosphoproteins. Experiments done thus far suggest that these phosphoproteins are present only under conditions in which the UL34 protein is not phosphorylated.     

Cell surface major histocompatibility complex class II proteins are regulated by the products of the gamma(1)34.5 and U(L)41 genes of herpes simplex virus 1

Modulation of host immune responses has emerged as a common strategy employed by herpesviruses both to establish life-long infections and to affect recovery from infection. Herpes simplex virus 1 (HSV-1) blocks the major histocompatibility complex (MHC) class I antigen presentation pathway by inhibiting peptide transport into the endoplasmic reticulum. The interaction of viral gene products with the MHC class II pathway, however, has not been thoroughly investigated, although CD4(+) T cells play an important role in human recovery from infection. We have investigated the stability, distribution, and state of MHC class II proteins in glioblastoma cells infected with wild-type HSV-1 or mutants lacking specific genes. We report the following findings. (i) Wild-type virus infection caused a decrease in the accumulation of class II protein on the surface of cells and a decrease in the endocytosis of lucifer yellow or dextran conjugated to fluorescein isothiocyanate but no decrease in the total amount of MHC class II proteins relative to the levels seen in mock-infected cells. (ii) Although the total amount of MHC class II protein remained unchanged, the amounts of cell surface MHC class II proteins were higher in cells infected with the U(L)41-negative mutant, which lacks the virion host shutoff protein, and especially high in cells infected with the gamma(1)34.5-negative mutant. We conclude that infected cells attempt to respond to infection by increased acquisition of antigens and transport of MHC class II proteins to the cell surface and that these responses are blocked in part by the virion host shutoff protein encoded by the U(L)41 gene and in large measure by the direct or indirect action of the infected cell protein 34.5, the product of the gamma(1)34.5 gene.     

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.     

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.     

US3 Protein Kinase of Herpes Simplex Virus 1 Blocks Caspase 3 Activation Induced by the Products of US1.5 and UL13 Genes and Modulates Expression of Transduced US1.5 Open Reading Frame in a Cell Type-Specific Manner

The coding domain of the herpes simplex virus type 1 (HSV-1) alpha22 gene encodes two proteins, the 420-amino-acid infected-cell protein 22 (ICP22) and U(S)1.5, a protein colinear with the carboxyl-terminal domain of ICP22. In HSV-1-infected cells, ICP22 and U(S)1.5 are extensively modified by the U(L)13 and U(S)3 viral protein kinases. In this report, we show that in contrast to other viral proteins defined by their properties as alpha proteins, U(S)1.5 becomes detectable and accumulated only at late times after infection. Moreover, significantly more U(S)1.5 protein accumulated in cells infected with a mutant lacking the U(L)13 gene than in cells infected with wild-type virus. To define the role of viral protein kinases on the accumulation of U(S)1.5 protein, rabbit skin cells or Vero cells were exposed to recombinant baculoviruses that expressed U(S)1.5, U(L)13, or U(S)3 proteins under a human cytomegalovirus immediate-early promoter. The results were as follows. (i) Accumulation of the U(S)1.5 protein was reduced by concurrent expression of the U(L)13 protein kinase and augmented by concurrent expression of the U(S)3 protein kinase. The magnitude of the reduction or increase in the accumulation of the U(S)1.5 protein was cell type dependent. The effect of U(L)13 kinase appears to be specific inasmuch as it did not affect the accumulation of glycoprotein D in cells doubly infected by recombinant baculoviruses expressing these genes. (ii) The reduction in accumulation of the U(S)1.5 protein was partially due to proteasome-dependent degradation. (iii) Both U(S)1.5 and U(L)13 proteins activated caspase 3, indicative of programmed cell death. (iv) Concurrent expression of the U(S)3 protein kinase blocked activation of caspase 3. The results are concordant with those published elsewhere (J. Munger and B. Roizman, Proc. Natl. Acad. Sci. USA 98:10410-10415, 2001) that the U(S)3 protein kinase can block apoptosis by degradation or posttranslational modification of BAD.     

Proteolytic Cleavage of the Amino Terminus of the UL15 Gene Product of Herpes Simplex Virus Type 1 Is Coupled with Maturation of Viral DNA into Unit-Length Genomes

The U(L)15 gene of herpes simplex virus type 1 (HSV-1), like U(L)6, U(L)17, U(L)28, U(L)32, and U(L)33, is required for cleavage of concatameric DNA into genomic lengths and for packaging of cleaved genomes into preformed capsids. A previous study indicated that the U(L)15 gene encodes minor capsid proteins. In the present study, we have shown that the amino-terminal 509 amino acids of the U(L)15-encoded protein are sufficient to confer capsid association inasmuch as a carboxyl-terminally truncated form of the U(L)15-encoded protein with an M(r) of approximately 55,000 readily associated with capsids. This and previous studies have shown that, whereas three U(L)15-encoded proteins with apparent M(r)s of 83,000, 80,000, and 79,000 associated with wild-type B capsids, only the full-length 83,000-M(r) protein associated with B capsids purified from cells infected with viruses lacking functional U(L)6, U(L)17, U(L)28, U(L)32, and U(L)33 genes (B. Salmon and J. D. Baines, J. Virol. 72:3045-3050, 1998). Thus, all viral mutants that fail to cleave viral DNA into genomic-length molecules also fail to produce capsid-associated U(L)15 80,000- and 79,000-M(r) proteins. In contrast, the 80,000- and 79,000-M(r) proteins were readily detected in capsids purified from cells infected with a U(L)25 null virus that cleaves, but does not package, DNA. The conclusion that the amino terminus of the 83,000-M(r) protein is truncated to produce the 80,000- and/or 79,000-M(r) protein was supported by the following observations. (i) Whereas the C termini of the 83,000-, 80, 000-, and 79,000-M(r) proteins are identical, immunoreactivity dependent on the first 35 amino acids of the U(L)15 83,000-M(r) protein was absent from the 80,000- and 79,000-M(r) proteins. (ii) The 79,000- and 80,000-M(r) proteins were detected in capsids from cells infected with HSV-1(U(L)15M36V), an engineered virus encoding valine rather than methionine at codon 36. Thus, initiation at codon 36 is unlikely to account for production of the 80,000- and/or 79, 000-M(r) protein. Taken together, these data strongly suggest that capsid-associated U(L)15-encoded protein is proteolytically cleaved near the N terminus and indicate that this modification is tightly linked to maturation of genomic DNA.     

Quantification of the DNA cleavage and packaging proteins U(L)15 and U(L)28 in A and B capsids of herpes simplex virus type 1

The proteins produced by the herpes simplex virus type 1 (HSV-1) genes U(L)15 and U(L)28 are believed to form part of the terminase enzyme, a protein complex essential for the cleavage of newly synthesized, concatameric herpesvirus DNA and the packaging of the resultant genome lengths into preformed capsids. This work describes the purification of recombinant forms of pU(L)15 and pU(L)28, which allowed the calculation of the average number of copies of each protein in A and B capsids and in capsids lacking the putative portal encoded by U(L)6. On average, 1.0 (+/-0.29 [standard deviation]) copies of pU(L)15 and 2.4 (+/-0.97) copies of pU(L)28 were present in B capsids, 1.2 (+/-0.72) copies of pU(L)15 and 1.5 (+/-0.86) copies of pU(L)28 were found in mutant capsids lacking the putative portal protein pU(L)6, and approximately 12.0 (+/-5.63) copies of pU(L)15 and 0.6 (+/-0.32) copies of pU(L)28 were present in each A capsid. These results suggest that the packaging machine is partly comprised of approximately 12 copies of pU(L)15, as found in A capsids, with wild-type B and mutant U(L)6(-) capsids containing an incomplete complement of cleavage and packaging proteins. These results are consistent with observations that B capsids form by default in the absence of packaging machinery in vitro and in vivo. In contrast, A capsids may be the result of initiated but aborted attempts at DNA packaging, resulting in the retention of at least part of the DNA packaging machinery.