The nucleotide sequence of the equine herpesvirus 4 gC gene homologue

The genomic position of an equine herpesvirus 4 (EHV-4) gene homologue of the herpes simplex virus 1 (HSV-1) gC gene was determined by Southern analysis and DNA sequencing. The gene lies within a 2-kbp Bg/II-EcoRI fragment mapping between 0.15 and 0.17 within the long unique component of the EHV-4 genome and is transcribed from right to left. Putative promoter elements were identified upstream of the 1455-bp open reading frame which encodes a 485-amino-acid protein of unglycosylated molecular weight 52,513. Computer-assisted analysis of the primary sequence predicts the protein possesses a domain structure characteristic of a type 1 integral membrane glycoprotein. Four domains were distinguished--(i) an N-terminal signal sequence, (ii) a large extracellular domain containing 11 putative N-linked glycosylation sites, (iii) a hydrophobic transmembrane domain, and (iv) a C-terminal charged domain. Comparison of the predicted amino acid sequence to that of other herpesvirus glycoproteins indicated identities of between 22 and 29% with HSV-1 gC, HSV-2 gC, VZV gpV, PRV gIII, BHV-1 gIII, and MDV A antigen and of 79% with EHV-1 gp13. A gene with no apparent homologue in HSV-1 or VZV maps immediately downstream of the EHV-4 gC gene homologue.     

Open reading frames encoding a protein kinase, homolog of glycoprotein gX of pseudorabies virus, and a novel glycoprotein map within the unique short segment of equine herpesvirus type 1.

DNA sequence analysis of the unique short (Us) segment of the genome of equine herpesvirus type 1 Kentucky A strain (EHV-1) by our laboratory and strains Kentucky D and AB1 by other workers identifies a total of nine open reading frames (ORF). In this report, we present the DNA sequence of three of these newly identified ORFs, designated EUS 2, EUS 3, and EUS 4. The EUS 2 ORF is 1146 nucleotides (nt) in length and encodes a potential protein of 382 amino acids. Cis-regulatory sequences upstream of the putative ATG start codon include a G/C box 112 nt upstream and two potential TATA-like elements located between 15 and 90 nt before the ATG. The EUS 2 translation product exhibits significant homology to Ser/Thr protein kinases encoded within the Us segments of other herpesviruses, such as herpes simplex virus (26% homology) and pseudorabies virus (PRV), (45% homology), and possesses sequence domains conserved in protein kinases of cellular and viral origin. The EUS 3 ORF begins 127 nt downstream from the EUS 2 stop codon and ends at a stop codon 1119 nt further downstream. A single TATA-like element maps 61 nt upstream of the ORF. This ORF encodes a potential protein of 373 amino acids and is a homolog of glycoprotein gX of PRV, as judged by overall homology of amino acid residues, cysteine displacement, and presence of potential glycosylation sites and signal sequence. Interestingly, the EUS 4 ORF encodes a potential membrane glycoprotein that does not exhibit homology to any reported protein sequence. The EUS 4 ORF encodes a 383 amino acid polypeptide with a sequence indicative of a signal sequence at its amino terminal end, glycosylation sites for N-linked oligosaccharides, and a transmembrane domain near its carboxyl terminus. Several cis-acting regulatory sequences lie upstream of this ORF. These findings support the observation that the short region of alphaherpesviruses show considerable variation in their genetic content and gene organization.     

Molecular biological characterization of equine herpesvirus type 1 (EHV-1) isolates from ruminant hosts

Isolates of equine herpesvirus type 1 (EHV-1) originating from affected antelope and cattle were compared with reference EHV-1 and EHV-4 isolates and were characterized. Based on cross-neutralization, DNA restriction profiles and blot-hybridization data these isolates could be characterized as EHV-1. One isolate (from an antelope) with a different restriction profile showed significant DNA homology with EHV-1, partial homology with EHV-4, and little or no homology with EHV-2 and HSV-1 DNAs. Blot hybridization revealed differences in DNA restriction fragments located at the termini of two isolates and size heterogeneity in the unique long/internal repeat junction fragment (UL/IR) of one isolate.     

Equine herpesvirus 1 (EHV-1) glycoprotein M: effect of deletions of transmembrane domains

Equine herpesvirus 1 (EHV-1) recombinants that carry either a deletion of glycoprotein M (gM) or express mutant forms of gM were constructed. The recombinants were derived from strain Kentucky A (KyA), which also lacks genes encoding gE and gI. Plaques on RK13 cells induced by the gM-negative KyA were reduced in size by 80%, but plaque sizes were restored to wild-type levels on gM-expressing cells. Electron microscopic studies revealed a massive defect in virus release after the deletion of gM in the gE- and gI-negative KyA, which was caused by a block in secondary envelopment of virions at Golgi vesicles. Recombinant KyA expressing mutant gM with deletions of predicted transmembrane domains was generated and characterized. It was shown that mutant gM was expressed and formed dimeric and oligomeric structures. However, subcellular localization of mutant gM proteins differed from that of wild-type gM. Mutant glycoproteins were not transported to the Golgi network and consequently were not incorporated into the envelope of extracellular virions. Also, a small plaque phenotype of mutant viruses that was indistinguishable from that of the gM-negative KyA was observed. Plaque sizes of mutant viruses were restored to wild-type levels by plating onto RK13 cells constitutively expressing full-length EHV-1 gM, indicating that mutant proteins did not exert a transdominant negative effect on wild-type gM.     

Contribution of gene products encoded within the unique short segment of equine herpesvirus 1 to virulence in a murine model

The pathogenesis of three equine herpesvirus 1 (EHV-1) recombinants was assessed in a CBA mouse model. Sequences encoding the majority of glycoproteins I (gI) and E (gE) were deleted from the pathogenic EHV-1 strain RacL11 (L11ΔgIΔgE), and sequences comprising the 3859 bp deletion within the strain KyA U_S segment, which includes genes 73 (gI), 74 (gE), and 75 (putative 10 kDa protein 75), were re-inserted into attenuated KyA (KgI/gE/75). In addition, genes gE and 75 were inserted into KyA to generate the EHV-1 recombinant KgE/75. The insertion of the 3859 bp U_S segment was sufficient to confer virulence to KyA, as indicated by pronounced signs of clinical disease including substantial weight loss. A large plaque morphology was observed in cells infected with KgI/gE/75 compared with KyA, and a small plaque phenotype was observed in cells infected with L11ΔgIΔgE compared with RacL11. These data indicate that gI and/or gI and gE contribute to the ability of EHV-1 to spread directly from cell-to-cell. The deletion of both gI and gE from the pathogenic RacL11 strain did not reduce clinical signs of disease in infected mice, but did decrease mortality compared with RacL11. Furthermore, the insertion of genes 74 (gE) and 75 into the vaccine strain KyA did not alter the attenuated phenotype of this virus. Finally, KgI/gE/75 and RacL11 elicited the production of the proinflammatory chemokines MIP-1, MIP-1β, and MIP-2 in the lungs of infected mice, while KyA did not, suggesting that gI and/or gI and gE contribute to the up-regulation of these mediators of inflammation. These findings show that gI, and/or gI and gE restore a virulent phenotype to the EHV-1 KyA strain, and indicate that virulence factors, in addition to gI and gE, contribute to the pathogenesis of the RacL11 strain.     

Equine herpes virus 1 and pseudorabies virus resistance to 2'-fluoropyrimidine analogs and to bromovinyldeoxyuridine: implications for dTMP kinase activity

The 2'-fluoropyrimidine nucleoside analogs 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC). 1(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-methyluracil (FMAU), and 1(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU) showed higher in vitro activity against herpes simplex virus type 1 (HSV-1), than equine herpesvirus 1 (EHV-1) or pseudorabies virus (PRV). Comparison of the 50% plaque inhibitory doses for HSV-1 and its mutant MMdUr-20 in cell cultures with inhibition constants (Ki's) for the viral deoxythymidine kinases (dTKs) suggests that in the infected cell FMAU is phosphorylated by host enzymes. As compared to HSV-1, EHV-1 and PRV were more resistant to E-5-(2-bromovinyl-2'-deoxyuridine (BVdU) and to the 2'-fluoropyrimidine analogs, as are HSV-2 and the HSV-1 mutants MMdUr-20 and S1. Because the dTKs of the latter lack deoxythymidylate kinase (dTMPK) activity, there appears to be a correlation between resistance to these analogs and BVdU on the one hand, and lack of dTMPK activity on the other. We predict that EHV-1 and PRV dTKs will be shown to lack significant dTMPK activity.     

Conservation of a gene cluster including glycoprotein B in bovine herpesvirus type 2 (BHV-2) and herpes simplex virus type 1 (HSV-1)

A library of subgenomic fragments of bovine herpesvirus type 2 (BHV-2) DNA was constructed in the expression cloning vector lambda gt11 and screened with monoclonal antibodies to the glycoprotein gb BHV-2, which is homologous to glycoprotein gB (gB-1) of herpes simplex virus type 1 (HSV-1). Lambda gt11 clones containing gB BHV-2-specific sequences were used to identify lambda EMBL3 vectors with DNA inserts which contained the complete gB BHV-2 gene. Nucleotide sequencing revealed that the gB BHV-2 gene is highly conserved compared to gB-1. The amino acid sequences and the predicted secondary structures of both glycoproteins are very similar. Two further open reading frames (ORF) in close vicinity to the gene encoding gB BHV-2 showed considerable homology to HSV-1 genes. They code for the major DNA-binding protein (dbp) of BHV-2 and a putative 72-kDa polypeptide. The gene of the latter protein corresponding to ICP18.5 of HSV-1 is interspersed between the ORFs of gB BHV-2 and the dbp of BHV-2. All three genes map in the unique long region of the genome. Their homology and the colinear arrangement compared to HSV-1 indicate a close relationship between the two viruses.     

Localization of the UsProtein Kinase of Equine Herpesvirus Type 1 Is Affected by the Cytoplasmic Structures Formed by the Novel IR6 Protein

Previous work revealed that the Us(unique short) segment of equine herpesvirus type-1 (EHV-1), like that of other alphaherpesviruses, encodes a serine/threonine protein kinase (PK). Experiments were carried out to identify the PK encoded by the EHV-1 EUS2 gene (ORF 69) and to ascertain its time course of synthesis and cellular localization. Western blot and immunoprecipitation analyses of EHV-1-infected cell extracts using a PK-specific polyclonal antibody generated against a bacterially expressed TrpE/PK fusion protein identified the UsPK as a 42- to 45-kDa phosphoprotein. The PK protein is first synthesized at 3 hr postinfection, is produced throughout the infection cycle, and is incorporated into EHV-1 virions. Interestingly, immunoprecipitation analyses revealed that the PK protein within the cytoplasm is associated with the 33-kDa IR6 novel protein of EHV-1, is expressed abundantly as an early protein, and is present in the large rod-like structures formed by the IR6 protein (ORF67 protein) within the cytoplasm of infected cells. Confocal microscopic examination of cells stained with fluorescein-labeled antibody clearly showed that the PK protein colocalized with the cytoplasmic IR6 rod-like structures and remained associated with these unique structures during infection. In contrast, in cells infected with the EHV-1 RacM strain in which the IR6 protein harbors four amino acid substitutions that prevent formation of the rod-like structures (Osterriederet al.,1996,Virology217, 442–451), the PK protein localized predominantly to the nucleus. The possible significance of the association of the IR6 and PK proteins in EHV-1 replication is discussed.     

A genomic map of infectious laryngotracheitis virus and the sequence and organization of genes present in the unique short and flanking regions

We present a genomic map of infectious laryngotracheitis virus (ILT) and an 18,912 bp sequence containing the entire unique short region and a portion of the flanking short repeats. In determining the genomic map, an 856 bp region repeated as many as 13 times was identified within the short repeats. The unique short sequence contains nine potential open reading frames (ORFs). Six of these ORFs show homology to other known herpesvirus unique short genes. Using the herpes simplex virus nomenclature, these genes are the US2, protein kinase, and glycoproteins G, D, I, and E (ORF 1, 2, 4, 6, 7, and 8, respectively). Interestingly, an open reading frame with homology to HSV-1 UL47 (ORF 3) is found in the unique short. One very large open reading frame (ORF 5) is present and contains a threonine-rich, degenerate repeat sequence. This gene appears to be unique to ILT among sequenced herpesviruses. Two ORFs were identified within the short repeat (SR) region. SRORF 1 is homologous to a gene (SORF3) found in the unique short region in both MDV and HVT, and appears to be specific to avian herpesviruses. SRORF 2 has homology to HSV US10.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence data-base and have been assigned the accession number U28832.     

Generation and characterization of an EICP0 null mutant of equine herpesvirus 1

The EICP0 gene (gene 63) of equine herpesvirus 1 (EHV-1) encodes an early regulatory protein that is a promiscuous trans-activator of all classes of viral genes. Bacterial artificial chromosome (BAC) technology and RecE/T cloning were employed to delete the EICP0 gene from EHV-1 strain KyA. Polymerase chain reaction, Southern blot analysis, and DNA sequencing confirmed the deletion of the EICP0 gene and its replacement with a kanamycin resistance gene in mutant KyA. Transfection of rabbit kidney cells with the EICP0 mutant genome produced infectious virus, indicating that the EICP0 gene is not essential for KyA replication in cell culture. Experiments to assess the effect of the EICP0 deletion on EHV-1 gene programming revealed that mRNA expression of the immediate-early gene and representative early and late genes as well as the synthesis of these viral proteins were reduced as compared to the kinetics of viral mRNA and protein synthesis observed for the wild type virus. However, the transition from early to late viral gene expression was not prevented or delayed, suggesting that the absence of the EICP0 gene did not disrupt the temporal aspects of EHV-1 gene regulation. The extracellular virus titer and plaque areas of the EICP0 mutant virus KyADeltaEICP0, in which the gp2-encoding gene 71 gene that is absent in the KyA BAC was restored, were reduced by 10-fold and 19%, respectively, when compared to parental KyA virus; while the titer and plaque areas of mutant KyADeltaEICP0Deltagp2 that lacks both the EICP0 gene and gene 71 were reduced more than 50-fold and 67%, respectively. The above results show that the EICP0 gene is dispensable for EHV-1 replication in cell culture, and that the switch from early to late viral gene expression for the representative genes examined does not require the EICP0 protein, but that the EICP0 protein may be structurally required for virus egress and cell-to-cell spread.     

Properties of an equine herpesvirus 1 mutant devoid of the internal inverted repeat sequence of the genomic short region

The 150 kbp genome of equine herpesvirus-1 (EHV-1) is composed of a unique long (UL) region and a unique short (Us) segment, which is flanked by identical internal and terminal repeat (IR and TR) sequences of 12.7 kbp. We constructed an EHV-1 lacking the entire IR (vL11ΔIR) and showed that the IR is dispensable for EHV-1 replication but that the vL11ΔIR exhibits a smaller plaque size and delayed growth kinetics. Western blot analyses of cells infected with vL11ΔIR showed that the synthesis of viral proteins encoded by the immediate-early, early, and late genes was reduced at immediate-early and early times, but by late stages of replication reached wild type levels. Intranasal infection of CBA mice revealed that the vL11ΔIR was significantly attenuated as mice infected with the vL11ΔIR showed a reduced lung viral titer and greater ability to survive infection compared to mice infected with parental or revertant virus.     

Identification of the envelope surface glycoproteins of equine herpesvirus type 1

The structural polypeptides of purified enveloped virions of the Army 183 strain of equine herpesvirus type 1 (EHV-1) were examined by different analytical techniques to identify the envelope glycoproteins. Glycoproteins were identified by electrophoretic analysis in polyacrylamide slab gels of virus labelled in vivo with [3H]glucosamine or labelled enzymically in vitro with either UDP-[14C]galactose or sodium [3H]borohydride. Fluorograms revealed eleven glycoproteins (mol. wt. 260000, 150000, 138000, 90000, 87000, 65000, 62000, 60000, 50000, 46000, and 24000). These glycoproteins probably correspond to virion protein (VP) 1-2, 9b, 10, 13, 14, 16, 17, 18, 21, 22a and 25 respectively, as designated in two other EHV-1 strains. In addition, a poorly resolved glucosamine-rich region (mol. wt. 250000 to 200000) corresponded to VP 3 to 8. The two isotopic surface labelling methods revealed that all the virus glycoproteins were exposed on the envelope surface.