The expression of the proteins of equine herpesvirus 1 which share homology with herpes simplex virus 1 glycoproteins H and L.

Several expression systems were used in studies aimed at characterizing the equine herpesvirus 1 (EHV-1) glycoprotein H and L homologues of HSV-1 (EHV-1 gH and gL) and the products were compared to the authentic proteins synthesized in virus infected cells. Using an in vitro transcription/translation system two gH species were detected (an unprocessed 89 kDa and a processed 116 kDa product). Three low molecular weight proteins were found in the case of gL (21.8 kDa, 22.9 kDa and 26.9 kDa) and these showed a slight reduction in mobility on the addition of microsomal membranes to the reactions. A gL fusion protein was produced in pGEX-2T, expression being confirmed by Western blotting using a gL-specific antiserum raised against a peptide incorporating the 13 carboxyl terminal amino acids of the protein. A gH specific peptide antiserum precipitated both gH and two smaller proteins from EHV-1 infected cells thought to be two forms of gL. Insect cells infected with gH or gL baculovirus recombinants were used to vaccinate C3H (H-2k) mice. Some protection against EHV-1 infection was conferred to the gH inoculated mice. The results will enable further studies on the importance of the gH and gL interaction in the pathogenesis of EHV-1 to be evaluated and their potential in contributing to a subunit vaccine to be assessed.

Inter- and intra-strain genomic variation in equine herpesvirus type 1 isolates.

Restriction enzyme digests of DNA from 22 unselected isolates of EHV-1 were analysed by hybridization with cloned DNA fragments covering the genome. In addition to a small amount of inter-strain variation, heterogeneity within strains was observed, caused by loss of specific restriction endonuclease sites in the DNA of a proportion of the virus particles of any one stock. Fifteen strains demonstrated the same intra-strain variation involving loss of the BamHI L-M site which was shown to lie within coding sequence for the large subunit of ribonucleotide reductase. This particular mutation may therefore be selected for by passage in RK13 cells.

Glycoprotein 300 is encoded by gene 28 of equine herpesvirus type 1: a new family of herpesvirus membrane proteins?

A portion of equine herpesvirus type 1 (EHV-1) gene 28, which is homologous to herpes simplex virus type 1 gene UL32, was expressed using a prokaryotic system to yield a fusion protein which reacted on Western blots with P19, a monoclonal antibody (MAb) that reacts with EHV-1 glycoprotein 300 (gp300), confirming that this gene encodes gp300. Hydrophobicity analysis showed that gp300 is a glycoprotein with multiple hydrophobic domains that might interact with, or span, the membrane several times. As such, it may represent the first member of a new family of herpesvirus glycoproteins to be identified as a virus structural component. Gp300 was also shown to be modified by palmitic acid residues, and a second MAb (1G12) directed against gp300 inhibited fusion between EHV-1-infected cells.

Identification of the equine herpesvirus type 1 glycoprotein 17/18 as a homologue of herpes simplex virus glycoprotein D.

The DNA sequence of the equine herpesvirus type 1 (EHV-1) gD gene homologue has been determined for the strain Ab1 and compared with previously published sequences. A portion of the gene has been located to a region of the genome which also encodes homologues of the herpes simplex virus type 1 genes for gE and gI and is known to encode an epitope of the virion protein gp17/18. Analysis of the EHV-1 strain Kentucky A (KyA) by DNA hybridization showed the presence of a gD gene homologue and established the absence of genes for gI and gE. Western blot analysis, however, showed that KyA virus particles contain gp17/18, thus indicating that this protein is encoded by the gD gene homologue. The KyA gp17/18 was found to be smaller than that detected in other strains and this is accounted for by a frameshift mutation in the KyA sequence relative to Ab1. The mutation in the KyA strain results in an altered C-terminal sequence and could explain the apparent structural differences suggested by the reactivities with monoclonal antibodies (MAbs). We have also expressed part of the Ab1 gD gene as a fusion protein with glutathione S-transferase in Escherichia coli and shown that this reacts with the MAb 5H6 originally used to map gp17/18. These experiments establish that gp17/18 is encoded by the gD gene homologue.

Glycoprotein 60 of equine herpesvirus type 1 is a homologue of herpes simplex virus glycoprotein D and plays a major role in penetration of cells.

Monoclonal antibodies (MAbs) specific for equine herpesvirus type 1 (EHV-1) glycoprotein 60 (gp60) and gp 17/18 (F3132 and 5H6 respectively) were found to react with the same protein, which was identified as a homologue of herpes simplex virus type 1 gD. MAb F3132 strongly neutralized virus infectivity and inhibited the penetration of the virus into the cell. The effects on penetration were shared with three other MAbs against this protein (P68, F3116 and F3129), but no effect on virus penetration was found with any other anti-EHV-1 MAb tested. The level of glycosylation of gp60 was analysed using glycanase enzymes and glycosylation inhibitors, and consisted of mainly N-linked carbohydrate. The M(r) of non-N-glycosylated gp60 was 50K.

Post-translational modification of the tegument proteins (VP13 and VP14) of herpes simplex virus type 1 by glycosylation and phosphorylation.

VP13 and VP14, major tegument proteins of herpes simplex virus type 1 (HSV-1) and the products of the UL47 gene, have been shown by partial proteolytic mapping to have closely related protein sequences. These proteins are phosphorylated in virus-infected cells, but not in preparations of purified virus. They also contain O-linked oligosaccharide units which include beta-1,4-N-acetyl galactosamine residues, as demonstrated by the binding of Dolichos biflorus lectin. This modification was detected only in purified virus and appears to be restricted to VP13/14 and VP22, another HSV-1 tegument protein.

Location of open reading frames coding for equine herpesvirus type-1 glycoproteins with homology to gE and gI of herpes simplex virus.

The DNA fragments representing the entire short unique region and part of the repeat sequences of the equine herpesvirus type-1 genome were cloned into plasmid vectors. The approximate positions of the junctions between the short unique region and the inverted repeats were then located by restriction endonuclease mapping. Two open reading frames coding for potential glycoproteins have been identified within the short unique region, using DNA sequence analysis. The predicted amino acid sequences of these open reading frames had extensive homology to the herpes simplex virus glycoproteins gE and gI and the related glycoproteins of pseudorabies virus and varicella-zoster virus.

Antigenic and protein sequence homology between VP13/14, a herpes simplex virus type 1 tegument protein, and gp10, a glycoprotein of equine herpesvirus 1 and 4.

Monospecific polyclonal antisera raised against VP13/14, a major tegument protein of herpes simplex virus type 1 cross-reacted with structural equine herpesvirus 1 and 4 proteins of Mr 120,000 and 123,000, respectively; these proteins are identical in molecular weight to the corresponding glycoprotein 10 (gp10) of each virus. Using a combination of immune precipitation and Western immunoblotting techniques, we confirmed that anti-VP13/14 and a monoclonal antibody to gp10 reacted with the same protein. Sequence analysis of a lambda gt11 insert of equine herpesvirus 1 gp10 identified an open reading frame in equine herpesvirus 4 with which it showed strong homology; this open reading frame also shared homology with gene UL47 of herpes simplex virus type 1 and gene 11 of varicella-zoster virus. This showed that, in addition to immunological cross-reactivity, VP13/14 and gp10 have protein sequence homology; it also allowed identification of VP13/14 as the gene product of UL47.

Sequence analysis of the 4.7-kb BamHI-EcoRI fragment of the equine herpesvirus type-1 short unique region.

To localize gene that may encode immunogens potentially important for recombinant vaccine design, we have analysed a region of the equine herpesvirus type-1 (EHV-1) genome where a glycoprotein-encoding gene had previously been mapped. The 4707-bp BamHI-EcoRI fragment from the short unique region of the EHV-1 genome was sequenced. This sequence contains three entire open reading frames (ORFs), and portions of two more. ORF1 codes for 161 amino acids (aa), and represents the C terminus of a possible membrane-bound protein. ORF2 (424 aa) and ORF3 (550 aa) are potential glycoprotein-encoding genes; the predicted aa sequences contain possible signal sequences, N-linked glycosylation sites and transmembrane domains; they also show homology to the glycoproteins gI and gE of herpes simplex virus type-1 (HSV-1), and the related proteins of pseudorabies virus and varicella-zoster virus. The predicted aa sequence of ORF4 shares no homology with other known herpesvirus proteins, but the nucleotide sequence shows a high level of homology with the corresponding region of the EHV-4 genome. ORF5 may be related to US9 of HSV-1.

Studies on glycoprotein 13 (gp13) of equid herpesvirus 1 using affinity-purified gp13, glycoprotein-specific monoclonal antibodies and synthetic peptides in a hamster model.

Hamsters were immunized with either an affinity-purified preparation of equid herpesvirus 1 (EHV-1) glycoprotein 13 (gp13) or synthetic peptides representing three sequences within the homologous glycoprotein of EHV-4, resulting in the production of anti-peptide (in the case of peptide-immunized animals) or antivirus antibodies. The sera from gp13-immunized hamsters contained antibodies which showed virus-neutralizing activity and complement-mediated antibody lysis of EHV-1-infected target cells. These hamsters were protected from EHV-1 challenge. The characteristics of a panel of anti-gp13 monoclonal antibodies (P28, P17, 14H7, 16E4 and 16H9) were assessed both in vivo and in vitro. 16E4 and P28 showed high levels of complement-mediated neutralization of virus, complement-mediated lysis of virus-infected target cells and passive protection of hamsters. Furthermore, epitope mapping studies demonstrated that this glycoprotein contains a neutralizing epitope recognized by EHV-1-immune horse serum. The data imply that gp13 has potential as a candidate antigen for a molecular vaccine.

Characterization of the high Mr glycoprotein (gP300) of equine herpesvirus type 1 as a novel glycoprotein with extensive O-linked carbohydrate.

The high Mr glycoprotein (gp300) of equine herpesvirus type 1 was found to have an Mr, estimated by SDS-PAGE, of over 400,000 and was confirmed as being a surface glycoprotein by 125I-labelling. In contrast to [3H]glucosamine, gp300 showed very low levels of [3H]glucosamine, gp300 showed very low levels of [3H]mannose incorporation. The Mr of gp300 showed no detectable change upon treatment of purified virus with N-glycanase, and showed only a small change in virus-infected cells treated with tunicamycin. In addition, gp300 failed to bind the lectin concanavalin A. Taken together, these results indicate a lack of N-linked carbohydrate on gp300. The major carbohydrate species were found to be composed primarily of O-linked chains, as indicated by the sensitivity of the protein to monensin, to exoglycanase enzymes specific for sugars present in O-linked chains and to mild alkaline borohydride treatment, which revealed three species of carbohydrate of Mr of greater than 10,000, 2400 and 1100, respectively. Neuraminidase treatment and binding of Helix pomatia lectin indicated the presence of alpha-N-acetylglucosamine and sialic acid as terminal sugars. Immunological cross-reactivity of gp300 with a high Mr protein of equine herpesvirus type 4 was shown and it also exhibited a marked Mr variation in the vaccine strain Rhinomune.

Identification of the gB homologues of equine herpesvirus types 1 and 4 as disulphide-linked heterodimers and their characterization using monoclonal antibodies.

Equine herpesvirus types 1 and 4 (EHV-1 and EHV-4) labelled with [14C]glucosamine were purified from infected cell culture medium and profiles of their structural proteins were obtained that enabled identification of the major glycoproteins. Nine glycosylated polypeptides were identified for each virus. Preparations of the purified viruses each contained a glycoprotein which was linked by disulphide bonds, as determined by diagonal gel electrophoresis under reducing/non-reducing conditions. High Mr forms of this glycoprotein were detected for EHV-1 when the sample was not heated. The EHV-1 protein consisted of three polypeptides of Mr 108K, 76K and 58K and the EHV-4 protein consisted of three polypeptides of Mr 112K, 74K and 61K. Western blotting and immunoprecipitation with monoclonal antibodies confirmed that the EHV-1 gB homologue migrates with an apparent Mr of 108K (140K under non-reducing conditions) but is cleaved to give glycoproteins of 76K and 58K which are held together by disulphide bonds. The EHV-4 gB homologue consists of a 112K glycoprotein which is cleaved to give glycoproteins of 74K and 61K which are also linked by disulphide bonds.

Immunological conservation between Epstein-Barr virus and herpes simplex virus.

We have analysed Epstein-Barr virus (EBV)- and herpes simplex virus (HSV)-infected cells for evidence of antigenic conservation of virus-coded proteins. Immunofluorescence and Western blot analyses of EBV-transformed cell lines demonstrated the presence of proteins that are antigenically related to the HSV alkaline DNase, infected cell-specific protein 34/35, glycoprotein B, thymidine kinase and the major DNA-binding protein. These proteins were characterized on the basis of Mr and possible kinetic class.

Virulence is not conserved in recombinants between herpes simplex virus types 1 and 2.

The virulence of 31 herpes simplex virus type 1 X herpes simplex virus type 2 intertypic recombinants was determined following intraperitoneal inoculation into CBA mice. Only eight of the recombinants killed any of the mice and of these, only one recombinant was as virulent as its type 2 parent, the other seven recombinants being intermediate between their type 1 and type 2 parental viruses in virulence. These results indicate that most heterotypic combinations are attenuated independently of the virulence of the parental viruses and therefore that the virulence of HSV is controlled multigenically.

A comparison of the acid-soluble polypeptides of five herpesviruses.

The polypeptides soluble in 0.25 M-HCl were extracted from the nuclei of BHK cells infected with herpes simplex virus type 1 or type 2 and separated by SDS-PAGE. Seventeen polypeptides were detectable in each extract of which 10 type 1 and nine type 2 polypeptides were reproducibly effectively extracted. In cells infected with bovine mammillitis virus, pseudorabies virus or equine herpesvirus type 1, at least 12, 13 and eight polypeptides respectively were acid-soluble. In addition to histones, three other cellular polypeptides were present in sizeable quantities in the acid extracts and could obscure other acid-soluble viral polypeptides. Possible relationships between some polypeptides of the five herpesviruses are discussed.

Identification of cross-reacting glycoproteins of four herpesviruses by Western blotting.

Monospecific rabbit antisera against purified herpes simplex virus type 1 (HSV-1) gB, gC or gD antigens or polyvalent rabbit antiserum against equine herpesvirus type 1 (EHV-1)-infected cells was used in Western blotting to identify antigenically related proteins in cells infected with HSV-1, HSV-2, bovine mammillitis virus or EHV-1. Monomeric and oligomeric polypeptides related to HSV-1 gB were found in cells infected with each of the four herpesviruses. The gC antiserum was specific for HSV-1 and the gD antiserum reacted only with polypeptides in HSV-1- or HSV-2-infected cells. Neither gC nor gD antiserum revealed the existence of oligomeric forms of the glycoproteins with the single exception of HSV-1 strain KOS which had a heat-unstable high molecular weight form gD. The EHV-1 antiserum reacted with high molecular weight polypeptides of the same mobility as the oligomeric form of gB in cells infected with each of the four herpesviruses.

Herpes simplex virus 1 mutant deleted in the alpha 22 gene: growth and gene expression in permissive and restrictive cells and establishment of latency in mice.

R325-beta TK+, a herpes simplex virus 1 mutant carrying a 500-base-pair deletion in the alpha 22 gene and the wild-type (beta) thymidine kinase (TK) gene, was previously shown to grow efficiently in HEp-2 and Vero cell lines. We report that in rodent cell lines exemplified by the Rat-1 line, plating efficiency was reduced and growth was multiplicity dependent. A similar multiplicity dependence for growth and lack of virus spread at low multiplicity was seen in resting, confluent human embryonic lung (HEL) cells. The shutoff of synthesis of beta proteins was delayed and the duration of synthesis of gamma proteins was extended in R325-beta TK+-infected HEL cells relative to cells infected with the wild-type parent, but no significant differences were seen in the total accumulation of viral DNA. To quantify the effect on late (gamma 2) gene expression, a recombinant carrying the deletion in the alpha 22 gene and a gamma 2-TK gene (R325-gamma 2 TK) was constructed and compared with a wild-type virus (R3112) carrying a chimeric gamma 2-TK gene. In Vero cells, the gamma 2-TK gene of R325-gamma 2TK was expressed earlier than and at the same level as the gamma 2-TK gene of R3112. In the confluent resting HEL cells, the expression of the gamma 2-TK gene of the alpha 22- virus was grossly reduced relative to that of the alpha 22+ virus. Electron microscopic studies indicated that the number of intranuclear capsids of R325-beta TK+ virus was reduced relative to that of the parent virus in resting confluent HEL cells, but the number of DNA-containing capsids was higher. Notwithstanding the grossly reduced neurovirulence on intracerebral inoculation in mice, R325-beta TK+ virus was able to establish latency in mice. We conclude that (i) the alpha 22 gene affects late (gamma 2) gene expression, and (ii) a host cell factor complements that function of the alpha 22 gene to a greater extent in HEp-2 and Vero cells than in confluent, resting HEL cells.

Antigenic and biochemical analysis of gB of herpes simplex virus type 1 and type 2 and of cross-reacting glycoproteins induced by bovine mammillitis virus and equine herpesvirus type 1.

An antiserum was produced to the oligomeric form of glycoprotein B (gB) induced by herpes simplex virus type 1 (HSV-1) strain 17. This antiserum gave a single common precipitin line in agar gel immunodiffusion with HSV-1, HSV-2, bovine mammillitis virus (BMV) and equine herpesvirus type 1 (EHV-1). It also neutralized HSV-1, HSV-2 and BMV but not EHV-1. Absorption of the antiserum with excess HSV-2 or BMV antigen resulted in an HSV-1-specific neutralizing antiserum. In immunoprecipitation, two proteins, gB and pgB, were precipitated from HSV-1- and HSV-2-infected cells and at least three from BMV- and EHV-1-infected cells. Glycoprotein B and pgB of three HSV-1 and three HSV-2 strains and the corresponding antigenically related glycoproteins of BMV- and EHV-1-infected cells were labelled with 125I, digested with trypsin and the resulting peptides separated by two-dimensional thin-layer chromatography or high-pressure liquid chromatography. The resulting profiles were found to be almost identical, suggesting considerable structural conservation of the peptide backbone of the antigenically related glycoproteins of these four viruses.

Studies on the herpes simplex virus alkaline nuclease: detection of type-common and type-specific epitopes on the enzyme.

Five monoclonal antibodies to the alkaline nuclease of herpes simplex virus (HSV) types 1 and 2 have been used in immunoperoxidase tests to demonstrate the nuclear localization of the enzyme within HSV-1- and HSV-2-infected cells and to purify the enzyme from cells infected with either virus by immunoadsorbant chromatography. Affinity chromatography with a 32P-labelled extract of HSV-2-infected cells has enabled us to demonstrate that the nuclease eluting from the immunoadsorbant is a phosphoprotein, hence confirming the nuclease to be identical to the phosphorylated polypeptide previously referred to as ICSP 22 (HSV-2) or ICP 19 (HSV-1). In addition, the results clearly demonstrate that monoclonal antibodies Q1, CC1 and CH2 are directed against HSV type-common epitopes while V1 and T2T1 antibodies are against HSV-2-specific epitopes on the enzyme. Using the type-specific monoclonal antibodies in an immunoperoxidase test, the enzyme specified in cells infected with intertypic recombinants has been typed; correlation of these data with restriction endonuclease maps of the recombinants has enabled us to map the position of the active site of the nuclease gene to map units 0.168 to 0.184 on the genomes of both HSV-1 and HSV-2. Taken with the data mapping the mRNA encoding this enzyme, the nuclease active site can be mapped to 0.168 to 0.175 on the genome. Finally, the use of monoclonal antibodies in immunofluorescence tests on infected cells has demonstrated that the nuclease is synthesized within 2 h post-infection.

Efficiency of the use of pock size on the chorioallantoic membrane of fertile hen's eggs as a method of typing herpes simplex viruses.

One hundred and eighteen herpes simplex virus isolates were typed in a diagnostic virology laboratory using their standard procedure by pock size on the chorioallantoic membranes (CAMs) of fertile hen's eggs. Forty-three were typed as type 1 and 75 as type 2. The isolates were then sent to a research laboratory in which they were typed blind, with or without subsequent passage in tissue culture, by neutralization with type-specific antisera. Discrepant results were found with only two isolates. The isolates were then typed by the more time-consuming but unambiguous method of restriction endonuclease analysis of their DNAs. Typing by this method confirmed the typing by neutralization and established that typing by pock size on CAMs was correct in about 98% of cases.