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.

Herpes simplex virus DNA packaging sequences adopt novel structures that are specifically recognized by a component of the cleavage and packaging machinery.

The product of the herpes simplex virus type 1 U(L)28 gene is essential for cleavage of concatemeric viral DNA into genome-length units and packaging of this DNA into viral procapsids. To address the role of U(L)28 in this process, purified U(L)28 protein was assayed for the ability to recognize conserved herpesvirus DNA packaging sequences. We report that DNA fragments containing the pac1 DNA packaging motif can be induced by heat treatment to adopt novel DNA conformations that migrate faster than the corresponding duplex in nondenaturing gels. Surprisingly, these novel DNA structures are high-affinity substrates for U(L)28 protein binding, whereas double-stranded DNA of identical sequence composition is not recognized by U(L)28 protein. We demonstrate that only one strand of the pac1 motif is responsible for the formation of novel DNA structures that are bound tightly and specifically by U(L)28 protein. To determine the relevance of the observed U(L)28 protein-pac1 interaction to the cleavage and packaging process, we have analyzed the binding affinity of U(L)28 protein for pac1 mutants previously shown to be deficient in cleavage and packaging in vivo. Each of the pac1 mutants exhibited a decrease in DNA binding by U(L)28 protein that correlated directly with the reported reduction in cleavage and packaging efficiency, thereby supporting a role for the U(L)28 protein-pac1 interaction in vivo. These data therefore suggest that the formation of novel DNA structures by the pac1 motif confers added specificity on recognition of DNA packaging sequences by the U(L)28-encoded component of the herpesvirus cleavage and packaging machinery.

Characterization of the U(L)33 gene product of herpes simplex virus 1.

The U(L)33 protein is one of six genes (including U(L)6, U(L)15, U(L)17, U(L)28, and U(L)32) required for cleavage of viral concatemeric DNA into unit-length genomes and packaging of the virus genomes into preformed capsids. The U(L)25 gene product is dispensable for cleavage of viral DNA but essential for packaging of DNA into capsids. A polyclonal antiserum was produced against an affinity-purified protein containing the full-length U(L)33 gene product of herpes simplex virus 1 fused to glutathione-S-transferase. A protein of approximate M(r) 19,000 that reacted with the antiserum was detected in immunoblots of herpes simplex virus 1-infected cellular lysates. This protein was not detected in lysates of mock-infected cells or cells infected with a mutant virus containing a stop codon in U(L)33, indicating that the 19,000 M(r) protein is the product of the U(L)33 open reading frame. The U(L)33 gene product was not detected in purified virions or capsids. Accumulation of the U(L)33 protein to detectable levels required viral DNA synthesis, indicating that the protein was regulated as a late gene. Indirect immunofluorescence analysis demonstrated that U(L)33 protein accumulated predominantly within replication compartments in the central domains of infected cell nuclei and within the cytoplasm. Localization of the U(L)33 gene product in replication compartments was maintained in cells infected with a variety of cleavage/packaging mutants.

Herpes simplex virus type 1 gene UL14: phenotype of a null mutant and identification of the encoded protein.

Herpes simplex virus type 1 (HSV-1) gene UL14 is located between divergently transcribed genes UL13 and UL15 and overlaps the promoters for both of these genes. UL14 also exhibits a substantial overlap of its coding region with that of UL13. It is one of the few HSV-1 genes for which a phenotype and protein product have not been described. Using mass spectrometric and immunological approaches, we demonstrated that the UL14 protein is a minor component of the virion tegument of 32 kDa which is expressed late in infection. In infected cells, the UL14 protein was detected in the nucleus at discrete sites within electron-dense nuclear bodies and in the cytoplasm initially in a diffuse distribution and then at discrete sites. Some of the UL14 protein was phosphorylated. A mutant with a 4-bp deletion in the central region of UL14 failed to produce the UL14 protein and generated small plaques. The mutant exhibited an extended growth cycle at low multiplicity of infection and appeared to be compromised in efficient transit of virus particles from the infected cell. In mice injected intracranially, the 50% lethal dose of the mutant was reduced more than 30,000-fold. Recovery of the mutant from the latently infected sacral ganglia of mice injected peripherally was significantly less than that of wild-type virus, suggesting a marked defect in the establishment of, or reactivation from, latent infection.

Proteolytic cleavage of the amino terminus of the U(L)15 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.

Three closely related herpesviruses are associated with fibropapillomatosis in marine turtles.

Green turtle fibropapillomatosis is a neoplastic disease of increasingly significant threat to the survivability of this species. Degenerate PCR primers that target highly conserved regions of genes encoding herpesvirus DNA polymerases were used to amplify a DNA sequence from fibropapillomas and fibromas from Hawaiian and Florida green turtles. All of the tumors tested (n = 23) were found to harbor viral DNA, whereas no viral DNA was detected in skin biopsies from tumor-negative turtles. The tissue distribution of the green turtle herpesvirus appears to be generally limited to tumors where viral DNA was found to accumulate at approximately two to five copies per cell and is occasionally detected, only by PCR, in some tissues normally associated with tumor development. In addition, herpesviral DNA was detected in fibropapillomas from two loggerhead and four olive ridley turtles. Nucleotide sequencing of a 483-bp fragment of the turtle herpesvirus DNA polymerase gene determined that the Florida green turtle and loggerhead turtle sequences are identical and differ from the Hawaiian green turtle sequence by five nucleotide changes, which results in two amino acid substitutions. The olive ridley sequence differs from the Florida and Hawaiian green turtle sequences by 15 and 16 nucleotide changes, respectively, resulting in four amino acid substitutions, three of which are unique to the olive ridley sequence. Our data suggest that these closely related turtle herpesviruses are intimately involved in the genesis of fibropapillomatosis.

Detection of a novel bovine lymphotropic herpesvirus.

Degenerate PCR primers which amplify a conserved region of the DNA polymerase genes of the herpesvirus family were used to provide sequence evidence for a new bovine herpesvirus in bovine B-lymphoma cells and peripheral blood mononuclear cells (PBMC). The sequence of the resultant amplicon was found to be distinct from those of known herpesvirus isolates. Alignment of amino acid sequences demonstrated 70% identity with ovine herpesvirus 2, 69% with alcelaphine herpesvirus 1, 65% with bovine herpesvirus 4, and 42% with bovine herpesvirus 1. Phylogenetic analysis placed this putative virus within the tumorigenic Gammaherpesvirinae subfamily, and it is tentatively identified as bovine lymphotropic herpesvirus. This novel agent was expressed in vitro from infected PBMC, and cell-free supernatants were used to transfer infection to a bovine B-cell line, BL3. Analysis, with specific PCR primers, of DNA from bovine PBMC and lymphoma cells identified infection in blood of 91% of adult animals (n = 101), 63% of lymphomas (n = 32), and 38% of juveniles (n = 13). Of the adults, herpesvirus infection was present in 94% of animals that were seropositive for bovine leukemia virus (BLV) (n = 63) and in 87% of BLV-seronegative animals (n = 38). Of the seropositive group, 17 animals exhibited persistent lymphocytosis, and 100% of these were herpesvirus positive by PCR. A role for bovine lymphotropic herpesvirus as a cofactor in BLV pathogenesis is considered.

Herpes simplex virus DNA cleavage and packaging: association of multiple forms of U(L)15-encoded proteins with B capsids requires at least the U(L)6, U(L)17, and U(L)28 genes.

The U(L)15 gene of herpes simplex virus (HSV) is one of several genes required for the packaging of viral DNA into intranuclear B capsids to produce C capsids that become enveloped at the inner nuclear membrane. A rabbit antiserum directed against U(L)15-encoded protein recognized three proteins with apparent Mrs of 79,000, 80,000, and 83,000 in highly purified B capsids. The 83,000-Mr protein was detected in type C capsids and comigrated with the product of a U(L)15 cDNA transcribed and translated in vitro. The 83,000- and 80,000-Mr proteins were readily detected in purified virions. Inasmuch as (i) none of these proteins were detectable in capsids purified from cells infected with HSV-1(deltaU(L)15), a virus lacking an intact U(L)15 gene, and (ii) corresponding proteins in capsids purified from cells infected with a recombinant virus [HSV-1(R7244), containing a 20-codon tag at the 3' end of U(L)15] were decreased in electrophoretic mobility relative to the wild-type proteins, we conclude that the proteins with apparent Mrs of 83,000, 80,000, and 79,000 are products of U(L)15 with identical C termini. The 79,000-, 80,000-, and 83,000-Mr proteins remained associated with B capsids in the presence of 0.5 M guanidine HCl and remained detectable in capsids treated with 2.0 M guanidine HCl and lacking proteins associated with the capsid core. These data, therefore, indicate that U(L)15-encoded proteins are integral components of B capsids. Only the 83,000-Mr protein was detected in B capsids purified from cells infected with viruses lacking the U(L)6, U(L)17, or U(L)28 genes, which are required for DNA cleavage and packaging, suggesting that capsid association of the 80,000- and 79,000-Mr proteins requires intact cleavage and packaging machinery. These data, therefore, indicate that capsid association of the 80,000- and 79,000-Mr U(L)15-encoded proteins reflects a previously unrecognized step in the DNA cleavage and packaging reaction.

The herpes simplex virus type 1 U(L)17 gene encodes virion tegument proteins that are required for cleavage and packaging of viral DNA.

Previous studies have suggested that the U(L)17 gene of herpes simplex virus type 1 (HSV-1) is essential for virus replication. In this study, viral mutants incorporating either a lacZ expression cassette in place of 1,490 bp of the 2,109-bp U(L)17 open reading frame [HSV-1(deltaU(L)17)] or a DNA oligomer containing an in-frame stop codon inserted 778 bp from the 5' end of the U(L)17 open reading frame [HSV-1(U(L)17-stop)] were plaque purified on engineered cell lines containing the U(L)17 gene. A virus derived from HSV-1(U(L)17-stop) but containing a restored U(L)17 gene was also constructed and was designated HSV-1(U(L)17-restored). The latter virus formed plaques and cleaved genomic viral DNA in a manner indistinguishable from wild-type virus. Neither HSV-1(deltaU(L)17) nor HSV-1(U(L)17-stop) formed plaques or produced infectious progeny when propagated on noncomplementing Vero cells. Furthermore, genomic end-specific restriction fragments were not detected in DNA purified from noncomplementing cells infected with HSV-1(deltaU(L)17) or HSV-1(U(L)17-stop), whereas end-specific fragments were readily detected when the viruses were propagated on complementing cells. Electron micrographs of thin sections of cells infected with HSV-1(deltaU(L)17) or HSV-1(U(L)17-stop) illustrated that empty capsids accumulated in the nuclei of Vero cells, whereas DNA-containing capsids accumulated in the nuclei of complementing cells and enveloped virions were found in the cytoplasm and extracellular space. Additionally, protein profiles of capsids purified from cells infected with HSV-1(deltaU(L)17) compared to wild-type virus show no detectable differences. These data indicate that the U(L)17 gene is essential for virus replication and is required for cleavage and packaging of viral DNA. To characterize the U(L)17 gene product, an anti-U(L)17 rabbit polyclonal antiserum was produced. The antiserum reacted strongly with a major protein of apparent Mr 77,000 and weakly with a protein of apparent Mr 72,000 in wild-type infected cell lysates and in virions. Bands of similar sizes were also detected in electrophoretically separated tegument fractions of virions and light particles and yielded tryptic peptides of masses characteristic of the predicted U(L)17 protein. We therefore conclude that the U(L)17 gene products are associated with the virion tegument and note that they are the first tegument-associated proteins shown to be required for cleavage and packaging of viral DNA.

Herpes simplex virus 1 DNA cleavage/packaging: the UL28 gene encodes a minor component of B capsids.

An antiserum directed against a bacterial fusion protein containing UL28 protein sequences specifically recognized an 86,000 apparent Mr protein in immunoblots of wild-type capsids. This protein was not detected in immunoblots of capsids purified from cells infected with a UL28 deletion virus, indicating that the protein was a product of UL28. The 86,000 Mr protein was also detected in capsids purified from cells infected with mutant viruses lacking the UL6, UL15, and UL25 genes, indicating that the UL28 protein can associate with capsids independently of successful DNA packaging and other minor capsid components. The UL6 protein, full-length UL15 protein, and UL25-encoded proteins were also detected in capsids purified from cells infected with the UL28 deletion virus. The UL28 and UL6 proteins remained associated with capsids treated with 1.0 M guanidine-HCl, indicating that, like the UL6 protein, the UL28 protein was an integral component of capsids. Amounts of UL28 protein were reduced in DNA-containing capsids and UL28 protein was not detected in virions, suggesting that some UL28 protein is lost during the cleavage-packaging reaction.

The herpes simplex virus 1 UL 17 gene is required for localization of capsids and major and minor capsid proteins to intranuclear sites where viral DNA is cleaved and packaged.

In nuclei of cells infected with herpes simplex virus (HSV), synthesized viral DNA accumulates as concatamers that are cleaved into genomic lengths and inserted into preformed capsids. Whereas newly replicated DNA and enzymes required for DNA synthesis accumulate in sites of infected cell nuclei termed replication compartments, the intranuclear site of DNA cleavage and packaging is currently controversial. DNA packaging requires the UL6, UL15, UL17, UL25, UL28, UL32, and UL33 genes in addition to the major capsid proteins. Using confocal immunofluorescence microscopy, it was observed that in > 95% of HEp-2 cells fixed at late times after infection with wild-type HSV-1, capsids, major capsid proteins ICP5 and ICP35, and the UL6-encoded minor capsid protein localized in DNA replication compartments. These data support the hypothesis that capsid assembly and DNA cleavage/packaging normally occur in HEp-2 cell replication compartments. In contrast, cells infected with a viral mutant lacking functional UL17 contained antigenically dense nuclear aggregates that stained with ICP35, ICP5, and capsid specific antibodies. Cells infected with the UL17 mutant virus also displayed UL6-specific fluorescence in a diffuse pattern at the nuclear periphery in regions not containing ICP35 and ICP5. Displacement of ICP35 from replication compartments was not observed in cells infected with cleavage/packaging mutants lacking UL28 and UL33. We conclude that the UL17 gene is required for correct targeting of capsids and major and minor capsid proteins to the DNA replication compartment of HEp-2 cells and deduce that this targeting reflects one functional role of UL17 in viral DNA cleavage and packaging.

Synthesis and processing of the equine herpesvirus 1 glycoprotein M.

In a previous report, the function of the equine herpesvirus 1 (EHV-1) glycoprotein M (gM) homolog was investigated. It was shown that EHV-1 gM is involved in both virus entry and direct cell-to-cell spread of infection (N. Osterrieder et al., J. Virol. 70, 4110-4115, 1996). In this study, experiments were conducted to analyze the synthesis, posttranslational processing, and the putative ion channel function of EHV-1 gM. It was demonstrated that EHV-1 gM is synthesized as an Mr 44,000 polypeptide, which is cotranslationally N-glycosylated to an Mr 46,000-48,000 glycoprotein. The Mr 46,000-48,000 gM moiety is processed to an Mr 50,000-55,000 glycoprotein, which is resistant to treatment with endoglycosidase H, indicating that processing occurs in the Golgi network. EHV-1 gM forms a dimer in infected cells and the virion, as was demonstrated by the presence of an Mr 105,000-110,000 gM-containing band in electrophoretically separated lysates of infected cells and purified extracellular virions. The Mr 105,000-110,000 protein band containing gM was also observed in lysates of cells that had been transfected with EHV-1 gM DNA. The translation of EHV-1 gM is initiated at the first in-frame methionine of the gM open reading frame as shown by transient transfection experiments of full-length gM and a truncated gM lacking the aminoterminal 83 amino acids. Functional expression of EHV-1 gM in Xenopus laevis oocytes together with voltage-clamp analyses demonstrated that gM per se does not exhibit ion channel activity as had been speculated from the predicted structure of the polypeptide.

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.

The UL 16 gene product of herpes simplex virus 1 is a virion protein that colocalizes with intranuclear capsid proteins.

The UL16 gene of herpes simplex virus maps within the intron of the UL 15 gene. This report shows the following: (i) A polyclonal antiserum directed against a bacterial fusion protein containing glutathione S-transferase fused to the C-terminus of the UL 16 gene reacted with an apparent M(r) 40,000 protein in HSV-1 infected cell lysates. (ii) The protein encoded by UL 16 was dependent on viral DNA synthesis for accumulation to detectable levels. (iii) In immunofluorescence studies, the polyclonal UL 16/GST-specific antiserum was shown to stain the nucleus of infected cells at 18 hr after infection in areas containing high concentrations of HSV capsid proteins. These nuclear compartments have been described previously as viral assemblons (Ward et al., J. Virol. 70, 4623-4631, 1996) and are distinct from compartments containing replicating DNA. Localization within assemblons argues for a role of UL 16 encoded protein in capsid assembly or maturation. (iv) At 22 hr after infection, UL 16-specific immunofluorescence was present in both the nucleus and the cytoplasm. (v) Consistent with the change in localization at late times after infection, the UL 16 protein was found to be a component of purified virions.

The equine herpesvirus 1 glycoprotein gp21/22a, the herpes simplex virus type 1 gM homolog, is involved in virus penetration and cell-to-cell spread of virions.

Experiments to analyze the function of the equine herpesvirus 1 (EHV-1) glycoprotein gM homolog were conducted. To this end, an Rk13 cell line (TCgM) that stably expressed EHV-1 gM was constructed. Proteins with apparent M(r)s of 46,000 to 48,000 and 50,000 to 55,000 were detected in TCgM cells with specific anti-gM antibodies, and the gM protein pattern was indistinguishable from that in cells infected with EHV-1 strain RacL11. A viral mutant (L11deltagM) bearing an Escherichia coli lacZ gene inserted into the EHV-1 strain RacL11 gM gene (open reading frame 52) was purified, and cells infected with L11deltagM did not contain detectable gM. L11deltagM exhibited approximately 100-fold lower titers and a more than 2-fold reduction in plaque size relative to wild-type EHV-1 when grown and titrated on noncomplementing cells. Viral titers were reduced only 10-fold when L11deltagM was grown on the complementing cell line TCgM and titrated on noncomplementing cells. L11deltagM also exhibited slower penetration kinetics compared with those of the parental EHV-1 RacL11. It is concluded that EHV-1 gM plays important roles in the penetration of virus into the target cell and in spread of EHV-1 from cell to cell.

The herpes simplex virus 1 UL11 proteins are associated with cytoplasmic and nuclear membranes and with nuclear bodies of infected cells.

Earlier studies have shown that the UL11 gene of herpes simplex virus encodes a myristylated virion protein and that the UL11 gene enables efficient virion envelopment and export from infected cells. A rabbit polyclonal antibody directed against an affinity-purified UL11-glutathione-S-transferase fusion protein was made and used to study the properties of the UL11 protein and its distribution in infected cells. We report the following: (i) UL11 protein formed up to five bands (apparent M(r)s, 17,000 to 22,000) in denaturing polyacrylamide gels; (ii) fluorescent-antibody studies revealed the presence of UL11 protein in the perinuclear space and in sites within the nucleus; (iii) immune electron microscopic studies indicated that the UL11 gene products were associated with the inner nuclear membrane, with cytoplasmic membranes and ribbon-like cytoplasmic structures resembling membranous organelles, with nuclear bodies shown by fluorescence microscopy to be different from nucleoli in which US11 protein accumulates, and with enveloped virions but not with nuclear capsids; and (iv) the nuclear bodies containing UL11 protein were reminiscent both of type IV morphotypes consisting of an electron-dense core containing the UL11 proteins surrounded by a more electron-transluscent core and of type V morphotypes consisting of material homogenous in electron opacity. We conclude that (i) the UL11 protein is processed after synthesis; (ii) the localization of UL11 protein with virions and membranes is consistent with the hypothesis that UL11 plays a role in the transport of virions to the extracellular space; and (iii) although the significance of the association of UL11 proteins with nuclear bodies is unknown, the results indicate that nuclear bodies differ with respect to their morphologies and contents of viral protein and suggest that UL11 protein may have more than one function in the infected cell.

The herpes simplex virus 1 UL15 gene encodes two proteins and is required for cleavage of genomic viral DNA.

Previous studies have shown that a ts mutant [herpes simplex virus 1 (mP)ts66.4] in the UL15 gene fails to package viral DNA into capsids (A. P. W. Poon and B. Roizman, J. Virol. 67:4497-4503, 1993) and that although the intron separating the first and second exons of the UL15 gene contains UL16 and UL17 open reading frames, replacement of the first exon with a cDNA copy of the entire gene does not affect viral replication (J.D. Baines, and B. Roizman, J. Virol. 66:5621-5626, 1992). We report that (i) a polyclonal rabbit antiserum generated against a chimeric protein consisting of the bacterial maltose-binding protein fused in frame to the majority of sequences contained in the second exon of the UL15 gene reacted with two proteins with M(r) of 35,000 and 75,000, respectively, in cells infected with a virus containing the authentic gene yielding a spliced mRNA or with a virus in which the authentic UL15 gene was replaced with a cDNA copy. (ii) Insertion of 20 additional codons into the C terminus of UL15 exon II caused a reduction in the electrophoretic mobility of both the apparently 35,000- and 75,000-M(r) proteins, unambiguously demonstrating that both share the carboxyl terminus of the UL15 exon II. (iii) Accumulation of the 35,000-M(r) protein was reduced in cells infected and maintained in the presence of phosphonoacetate, an inhibitor of viral DNA synthesis. (iv) The UL15 proteins were localized in the perinuclear space at 6 h after infection and largely in the nucleus at 12 h after infection. (v) Viral DNA accumulating in cells infected with herpes simplex virus 1(mP)ts66.4 and maintained at the nonpermissive temperature was in an endless (concatemeric) form, and therefore UL15 is required for the cleavage of mature, unit-length molecules for packaging into capsids.

The UL21 gene products of herpes simplex virus 1 are dispensable for growth in cultured cells.

A viral deletion mutant (delta UL21) that lacked the sequences encoding 484 of the predicted first 535 amino acids of the UL21 open reading frame was genetically engineered and studied with respect to its phenotype in cells in culture. We report the following. (i) The replication of delta UL21 was identical to that of the parent herpes simplex virus 1 (HSV-1) strain F in Vero cells, but the yields were three- to fivefold lower than those of the parent virus in human embryonic lung cells. (ii) To characterize the UL21 protein, we immunized rabbits against a purified bacterial fusion protein consisting of glutathione S-transferase fused to the majority of the coding domain of the UL21 gene. Rabbit antiserum directed against the fusion protein recognized a broad band with an apparent M(r) of 62,000 to 64,000 in lysates of cells infected with HSV-1 strain F and in virions purified from the infected cell cytoplasm. This band was absent from lysates of mock-infected cells or cells infected with the delta UL21 virus. The band was significantly reduced in intensity in lysates of cells infected in the presence of phosphonoacetic acid, indicating that it is expressed as a late (gamma 1) gene. (iii) Immunofluorescence studies localized the UL21 antigen primarily in brightly staining granules in the cytoplasms of infected cells. Taken together, the data indicate that the UL21 protein is a virion component dispensable for all aspects of replication of HSV-1 in the cells tested. The electrophoretic mobility of the UL21 protein suggests that it is extensively modified posttranslationally.

The UL10 gene of herpes simplex virus 1 encodes a novel viral glycoprotein, gM, which is present in the virion and in the plasma membrane of infected cells.

The herpes simplex virus 1 UL10 gene encodes a hydrophobic membrane protein dispensable for viral replication in cell culture (J.D. Baines and B. Roizman, J. Virol. 65:938-944, 1991). We report the following. (i) A fusion protein consisting of glutathione S-transferase fused to the C-terminal 93 amino acids of the UL10 protein was used to produce a rabbit polyclonal antiserum. The antiserum reacted with infected-cell proteins which formed in denaturing polyacrylamide gels a sharp band (apparent M(r) of 50,000) and a very broad band (M(r) of 53,000 to 63,000). These bands were not formed by lysates of UL10- virus or by lysates of infected cells boiled in the presence of sodium dodecyl sulfate before electrophoresis. (ii) The proteins forming both bands were labeled by [3H]glucosamine, indicating that they were glycosylated. (iii) The UL10 protein in cells treated with tunicamycin formed a single band (apparent M(r) of 47,000) reactive with the anti-UL10 antibody, indicating that the 47,000-M(r) protein was a precursor of N-glycosylated, more slowly migrating forms of UL10. Treatment of the immunoprecipitate with endoglycosidase H increased the electrophoretic mobility of the 50,000-M(r) species to that of the 47,000-M(r) species, indicating that the 50,000-M(r) species contained high-mannose polysaccharide chains, whereas the proteins forming the 53,000- to 63,000-M(r) bands contained mature chains inasmuch as they were resistant to digestion by the enzyme. (iv) The UL10 protein of R7221 carrying a 20-amino-acid epitope formed only one band with an M(r) of 53,000. This band was sensitive to endoglycosidase H, suggesting that the epitope inserted in the R7221 UL10 protein may have interfered with glycosylation. (v) The UL10 protein does not contain a cleavable signal sequence inasmuch as the first UL10 methionine codon was reflected in the 50,000-M(r) protein. (vi) The UL10 protein is present in virions and plasma membranes of unfixed cells that were reacted with the polyclonal rabbit antibody. In accordance with the current nomenclature, the UL10 protein is designated glycoprotein M.

The cDNA of UL15, a highly conserved herpes simplex virus 1 gene, effectively replaces the two exons of the wild-type virus.

The UL15 gene of herpes simplex virus 1 (HSV-1) is encoded by two or more exons in all herpesvirus genomes sequenced to date. The UL15 coding region is highly conserved, and the intron invariably encodes other genes transcribed antisense to the UL15 coding region. Previously we reported that we deleted the intron domain encoding UL16 but were unable to delete UL15 (J. D. Baines and B. Roizman, J. Virol. 65:938-944, 1991). Here we report that we replaced exon I of UL15 with an unspliced cDNA copy of UL15 in HSV-1 DNA and deleted 58% of the carboxyl-terminal sequences of the natural copy of exon II, including the polyadenylation signal. The yields of infectious virus obtained upon infection with viruses containing the cDNA copy of UL15 were similar to those of an isogenic virus with a wild-type UL15 gene. We therefore conclude that the separation of the two exons of UL15 by an intron encoding two genes is not essential for the replication of HSV, at least in cell culture.