Regulatory function of the equine herpesvirus 1 ICP27 gene product.

The UL3 protein of equine herpesvirus 1 (EHV-1) KyA strain is a homolog of the ICP27 alpha regulatory protein of herpes simplex virus type 1 (HSV-1) and the ORF 4 protein of varicella-zoster virus. To characterize the regulatory function of the UL3 gene product, a UL3 gene expression vector (pSVUL3) and a vector expressing a truncated version of the UL3 gene (pSVUL3P) were generated. These effector plasmids, in combination with an EHV-1 immediate-early (IE) gene expression vector (pSVIE) and chimeric EHV-1 promoter-chloramphenicol acetyltransferase (CAT) reporter constructs, were used in transient transfection assays. These assays demonstrated that the EHV-1 UL3 gene product is a regulatory protein that can independently trans activate the EHV-1 IE promoter; however, this effect can be inhibited by the repressive function of the IE gene product on the IE promoter (R. H. Smith, G. B. Caughman, and D. J. O'Callaghan, J. Virol. 66:936-945, 1992). In the presence of the IE gene product, the UL3 gene product significantly augments gene expression directed by the promoters of three EHV-1 early genes (thymidine kinase; IR4, which is the homolog of HSV-1 ICP22; and UL3 [ICP27]) and the promoter of the EHV-1 late gene IR5, which is the homolog of HSV-1 US10. Sequences located at nucleotides -123 to +20 of the UL3 promoter harbor a TATA box, SP1 binding site, CAAT box, and octamer binding site and, when linked to the CAT reporter gene, are trans activated to maximal levels by the pSVIE construct in transient expression assays. Results from CAT assays also suggest that the first 11 amino acids of the UL3 protein are not essential for the regulatory function of the UL3 gene product.     

The herpes simplex virus virion host shutoff function.

The virion host shutoff (vhs) function of herpes simplex virus (HSV) limits the expression of genes in the infected cells by destabilizing both host and viral mRNAs. vhs function mutants have been isolated which are defective in their ability to degrade host mRNA. Furthermore, the half-life of viral mRNAs is significantly longer in cells infected with the vhs-1 mutant virus than in cells infected with the wild-type (wt) virus. Recent data have shown that the vhs-1 mutation resides within the open reading frame UL41. We have analyzed the shutoff of host protein synthesis in cells infected with a mixture of the wt HSV-1 (KOS) and the vhs-1 mutant virus. The results of these experiments revealed that (i) the wt virus shutoff activity requires a threshold level of input virions per cell and (ii) the mutant vhs-1 virus protein can irreversibly block the wt virus shutoff activity. These results are consistent with a stoichiometric model in which the wt vhs protein interacts with a cellular factor which controls the half-life of cell mRNA. This wt virus interaction results in the destabilization of both host and viral mRNAs. In contrast, the mutant vhs function interacts with the cellular factor irreversibly, resulting in the increased half-life of both host and viral mRNAs.     

The equine herpesvirus 1 Us2 homolog encodes a nonessential membrane-associated virion component

Experiments were conducted to analyze the equine herpesvirus 1 (EHV-1) gene 68 product which is encoded by the EHV-1 Us2 homolog. An antiserum directed against the amino-terminal 206 amino acids of the EHV-1 Us2 protein specifically detected a protein with an Mr of 34,000 in cells infected with EHV-1 strain RacL11. EHV-1 strain Ab4 encodes a 44,000-Mr Us2 protein, whereas vaccine strain RacH, a high-passage derivative of RacL11, encodes a 31,000-Mr Us2 polypeptide. Irrespective of its size, the Us2 protein was incorporated into virions. The EHV-1 Us2 protein localized to membrane and nuclear fractions of RacL11-infected cells and to the envelope fraction of purified virions. To monitor intracellular trafficking of the protein, the green fluorescent protein (GFP) was fused to the carboxy terminus of the EHV-1 Us2 protein or to a truncated Us2 protein lacking a stretch of 16 hydrophobic amino acids at the extreme amino terminus. Both fusion proteins were detected at the plasma membrane and accumulated in the vicinity of nuclei of transfected cells. However, trafficking of either GFP fusion protein through the secretory pathway could not be demonstrated, and the EHV-1 Us2 protein lacked detectable N- and O-linked carbohydrates. Consistent with the presence of the Us2 protein in the viral envelope and plasma membrane of infected cells, a Us2-negative RacL11 mutant (L11DeltaUs2) exhibited delayed penetration kinetics and produced smaller plaques compared with either wild-type RacL11 or a Us2-repaired virus. After infection of BALB/c mice with L11DeltaUs2, reduced pathogenicity compared with the parental RacL11 virus and the repaired virus was observed. It is concluded that the EHV-1 Us2 protein modulates virus entry and cell-to-cell spread and appears to support sustained EHV-1 replication in vivo.     

Genomic termini of equine herpesvirus 1.

After cell infection with the equine herpesvirus 1 (EHV-1), the termini of the linear double-stranded DNA genome fuse to form circular forms. To investigate the mechanisms in the generation and cleavage of such replicative-form DNAs, the genomic termini, the fusion of termini from replicative-form molecules, and the junction between the short and long genome segments have been analyzed by restriction mapping, blot hybridizations, cloning, and sequencing. The data suggest that the genome ends are not redundant and that the genomic termini are fused in replicative intermediates via 3' single-base extensions at the termini of the unique long segment (UL) and terminal repeat (TR). Adjacent to the EHV-1 termini are AT and gamma sequence elements highly conserved among different herpesviruses. We propose that both of these sequence elements are important for the cleavage of EHV-1 replicative forms.     

Expression of equine herpesvirus 1 glycoprotein D by using a recombinant baculovirus.

Glycoprotein D (gD) of equine herpesvirus 1 (EHV-1) was expressed at the surface of insect cells infected by a recombinant baculovirus. EHV-1 gD was detected as multiple forms (56, 52, and 48 kDa) from 18 to 96 h postinfection. Laboratory animals inoculated with the recombinant EHV-1 gD developed neutralizing antibody responses against both EHV-1 and EHV-4.     

Role of the virion host shutoff protein in neurovirulence of monkey B virus (Macacine herpesvirus 1)

Monkey B virus(Macacine herpesvirus 1; BV) is noted for its extreme neurovirulence in humans. Since the vhs protein encoded by the UL41 gene has been shown to be a neurovirulence factor in the related human herpes simplex viruses, the role of the UL41 gene in BV neurovirulence was investigated. BV mutants were constructed that lacked the entire UL41 ORF(Δ41) or had the RNase active site mutated(Δ41A). Neither mutant shut off host protein synthesis, degraded β-actin mRNA, or prevented an IFN-β response, indicating that the vhs protein and its RNase activity are both necessary for these activities. Replication of both mutants in primary mouse cells was impaired and they exhibited a prolonged disease course in mice. Whereas Δ41 infected mice were euthanized for symptoms related to central nervous system(CNS) infection, Δ41A infected mice were euthanized primarily for symptoms of autonomic nervous system dysfunction. While neuroinvasiveness was not affected, lesions in the CNS were more limited in size, anatomical distribution, and severity than for wild-type virus. These results indicate that the vhs protein affects the general replicative efficiency of BV in vivo rather than being a specific neurovirulence factor critical for invasion of or preferential replication in the CNS.     

Role of the virion host shutoff protein in neurovirulence of monkey B virus(Macacine herpesvirus 1)

Monkey B virus(Macacine herpesvirus 1; BV) is noted for its extreme neurovirulence in humans. Since the vhs protein encoded by the UL41 gene has been shown to be a neurovirulence factor in the related human herpes simplex viruses, the role of the UL41 gene in BV neurovirulence was investigated. BV mutants were constructed that lacked the entire UL41 ORF(Δ41) or had the RNase active site mutated(Δ41A). Neither mutant shut off host protein synthesis, degraded β-actin mRNA, or prevented an IFN-β response, indicating that the vhs protein and its RNase activity are both necessary for these activities. Replication of both mutants in primary mouse cells was impaired and they exhibited a prolonged disease course in mice. Whereas Δ41 infected mice were euthanized for symptoms related to central nervous system(CNS) infection, Δ41A infected mice were euthanized primarily for symptoms of autonomic nervous system dysfunction. While neuroinvasiveness was not affected, lesions in the CNS were more limited in size, anatomical distribution, and severity than for wild-type virus. These results indicate that the vhs protein affects the general replicative efficiency of BV in vivo rather than being a specific neurovirulence factor critical for invasion of or preferential replication in the CNS.     

Detection of equine herpesvirus and differentiation of equine herpesvirus type 1 from type 4 by the polymerase chain reaction.

Although both equine herpesvirus type 1 (EHV-1) and equine herpesvirus type 4 (EHV-4) can be associated with respiratory disease, epizootics caused by EHV-1 are much more serious because the virus can cause abortions and paralysis. It is, therefore, important to identify the type of EHV involved in an outbreak by a test that is quick, sensitive, and reliable. We have adapted the polymerase chain reaction (PCR) to detect and distinguish between EHV-1 and EHV-4 in the same reaction. Primers for PCR were designed from the sequences of the glycoprotein B genes of EHV-1 and EHV-4. The PCR products derived from EHV-1 and EHV-4 were 135 and 326 base pairs, respectively, and could be readily separated by electrophoresis. The identity of the PCR products was confirmed by determining their nucleotide sequence, which agreed with the published sequence of the gB genes. The test could be performed directly on virus pelleted from small volumes (300 microL) of medium in which nasal swabs were transported and did not rely on the presence of infectious virus. The PCR was unaffected by conditions that reduced the infectivity of a virus preparation by 99%. The PCR detected EHV-4 in 5 of 10 nasal mucous samples taken from an outbreak of respiratory disease in race horses. Virus isolation in indicator cells was successful in detecting virus in four of the five samples positive by PCR.     

Genetic relatedness of equine herpesvirus types 1 and 3.

The genetic relatedness of two types of equine herpesviruses (EHVs), 1 (EHV-1) and 3 (EHV-3), was determined by DNA-DNA reassociation kinetics. Denatured, labeled viral DNA probe was allowed to reassociate in the presence or absence of the second unlabeled viral DNA. The initial rate of reassociation of either labeled viral DNA was increased by the presence of the heterologous viral DNA to an extent indicating only 2 to 5% homology between the two EHV genomes. Moreover, labeled RNA extracted from EHV-3-infected cells hybridized to filter-immobilized EHV-1 DNA only 2 to 3 percent as efficiently as to the homologous EHV-3 DNA. These results demonstrate that the genital (EHV-3) and nongenital (EHV-1) types of EHVs exhibit very little genetic homology.     

The exonuclease and host shutoff functions of the SOX protein of Kaposi's sarcoma-associated herpesvirus are genetically separable

The Kaposi's sarcoma-associated herpesvirus (KSHV) SOX protein, encoded by ORF37, promotes shutoff of host cell gene expression during lytic viral replication by dramatically impairing mRNA accumulation. SOX is the KSHV homolog of the alkaline exonuclease of other herpesviruses, which has been shown to function as a DNase involved in processing and packaging the viral genome. Although the exonuclease activity of these proteins is widely conserved across all herpesviruses, the host shutoff activity observed for KSHV SOX is not. We show here that SOX expression sharply reduces the half-life of target mRNAs. Extensive mutational analysis reveals that the DNase and host shutoff activities of SOX are genetically separable. Lesions affecting the DNase activity cluster in conserved regions of the protein, but residues critical for mRNA degradation are not conserved across the viral family. Additionally, we present evidence suggesting that the two different functions of SOX occur within distinct cellular compartments-DNase activity in the nucleus and host shutoff activity in the cytoplasm.     

Experimental studies on equine herpesvirus type 1 infections.

The EHV-1 viruses of fetal origin grew better and had a wider tissue culture host range than those isolated from horses with respiratory diseases. Comparisons of a fetal isolate (F/304) and a respiratory disease isolate (R/NM-3) in partly immune horses showed that the F/304 virus infected horses more readily, grew better in the nasopharynx, was more likely to cause abortion, and was excreted to a greater extent into the environment.     

Electron microscopic study of equine herpesvirus type 1 DNA.

Electron microscopic studies of equine herpesvirus DNA revealed that single strands that were allowed to reanneal formed single-stranded loops with double-stranded stems only at one end of the molecule. These observations support restriction enzyme analyses which indicate that the 92-megadalton DNA molecule exists as a long region of unique sequences covalently linked to a short region. The short region is comprised of an internal unique sequence, which forms the loop during reannealing of single strands, and two terminal inverted repeat sequences that bracket the unique sequence and form the double-stranded stem structure observed upon reannealing of single strands. Measurements of the unique sequence and terminal inverted repeat subgenomic sequences indicate a size of 6.4 megadaltons for each and thus fix the size of the short region at approximately 19.2 megadaltons.     

Identification and characterization of the ICP22 protein of equine herpesvirus 1.

The equine herpesvirus 1 (EHV-1) homolog of herpes simplex virus type 1 ICP22 is differently expressed from the fourth open reading frame of the inverted repeat (IR4) as a 1.4-kb early mRNA and a 1.7-kb late mRNA which are 3' coterminal (V. R. Holden, R. R. Yalamanchili, R. N. Harty, and D. J. O'Callaghan, J. Virol. 66:664-673, 1992). To extend the characterization of IR4 at the protein level, the synthesis and intracellular localization of the IR4 protein were investigated. Antiserum raised against either a synthetic peptide corresponding to amino acids 270 to 286 or against a TrpE-IR4 fusion protein (IR4 residues 13 to 150) was used to identify the IR4 protein. Western immunoblot analysis revealed that IR4 is expressed abundantly from an open reading frame composed of 293 codons as a family of proteins that migrate between 42 to 47 kDa. The intracellular localization of IR4 was examined by cell fractionation, indirect immunofluorescence, and laser-scanning confocal microscopy. These studies revealed that IR4 is localized predominantly in the nucleus and is dispersed uniformly throughout the nucleus. Interestingly, when IR4 is expressed transiently in COS-1 or LTK- cells, a punctate staining pattern within the nucleus is observed by indirect immunofluorescence. Cells transfected with an IR4 mutant construct that encodes a C-terminal truncated (19 amino acids) IR4 protein exhibited greatly reduced intranuclear accumulation of the IR4 protein, indicating that this domain possesses an important intranuclear localization signal. Western blot analysis of EHV-1 virion proteins revealed that IR4 proteins are structural components of the virions. Surprisingly, the 42-kDa species, which is the least abundant and the least modified form of the IR4 protein family in infected cell extracts, was the most abundant IR4 protein present in purified virions.     

The extreme carboxyl terminus of the equine herpesvirus 1 homolog of herpes simplex virus VP16 is essential for immediate-early gene activation.

Gene 12 of equine herpesvirus 1 (EHV-1), the homolog of herpes simplex virus (HSV) VP16 (alpha TIF, Vmw65), was cloned into a eukaryotic expression vector by PCR and used in transactivation studies of both the EHV-1 and HSV-1 IE1 promoters. Results demonstrated that the product of gene 12 is a potent transactivator of immediate-early gene expression of both viruses, which requires sequences in the upstream HSV-1 promoter for activity. Mutational analysis of the gene 12 open reading frame indicated that removal of the C-terminal 7 amino acids, which contain a short region of homology with the extreme C terminus of VP16, inactivated the protein. Within this region, only a single methionine residue appeared to be essential for activity, implying that gene 12 may have a modular array of organization similar to that of VP16. However, fusion of the gene 12 C terminus to a truncated form of VP16, which contained the complex formation domain, did not restore activity to the HSV-1 protein. These data demonstrate that the EHV-1 immediate-early transactivator may not be functionally colinear with VP16, with transactivation requiring both the C terminus and another region(s) present within the N-terminal portion.     

Identification and initial characterization of the IR6 protein of equine herpesvirus 1.

The IR6 gene of equine herpesvirus 1 (EHV-1) is a novel gene that maps within each inverted repeat (IR), encodes a potential protein of 272 amino acids, and is expressed as a 1.2-kb RNA whose synthesis begins at very early times (1.5 h) after infection and continues throughout the infection cycle (C. A. Breeden, R. R. Yalamanchili, C.F. Colle, and D.J. O'Callaghan, Virology 191:649-660,1992). To identify the IR6 protein and ascertain its properties, we generated an IR6-specific polyclonal antiserum to a TrpE/IR6 fusion protein containing 129 amino acids (residues 134 to 262) of the IR6 protein. This antiserum immunoprecipitated a 33-kDa protein generated by in vitro translation of mRNA transcribed from a pGEM construct (IR6/pGEM-3Z) that contains the entire IR6 open reading frame. The anti-IR6 antibody also recognized an infected-cell protein of approximately 33 kDa that was expressed as early as 1 to 2 h postinfection and was synthesized throughout the infection cycle. A variety of biochemical analyses including radiolabeling the IR6 protein with oligosaccharide precursors, translation of IR6 mRNA in the presence of canine pancreatic microsomes, radiolabeling the IR6 protein in the presence of tunicamycin, and pulse-chase labeling experiments indicated that the two potential sites for N-linked glycosylation were not used and that the IR6 protein does not enter the secretory pathway. To address the possibility that the unique IR6 gene encodes a novel regulatory protein, we transiently transfected an IR6 expression construct into L-M fibroblasts alone or with an immediate-early gene expression construct along with a representative EHV-1 immediate-early, early, or late promoter-chloramphenicol acetyltransferase reporter construct. The results indicated that the IR6 protein does not affect the expression of these representative promoter constructs. Interestingly, the IR6 protein was shown to be phosphorylated and to associate with purified EHV-1 virions and nucleocapsids. Lastly, immunofluorescence and laser-scanning confocal microscopic analyses revealed that the IR6 protein is distributed throughout the cytoplasm at early times postinfection and that by 4 to 6 h it appears as "dash-shaped" structures that localize to the perinuclear region. At late times after infection (8 to 12 h), these structures assemble around the nucleus, and three-dimensional image analyses reveal that the IR6 protein forms a crown-like structure that surrounds the nucleus as a perinuclear network.