Equine herpesvirus type 4 UL56 and UL49.5 proteins downregulate cell surface major histocompatibility complex class I expression independently of each other

Major histocompatibility complex class I (MHC-I) molecules are critically important in the host defense against various pathogens through presentation of viral peptides to cytotoxic T lymphocytes (CTLs), a process resulting in the destruction of virus-infected cells. Herpesviruses interfere with CTL-mediated elimination of infected cells by various mechanisms, including inhibition of peptide transport and loading, perturbation of MHC-I trafficking, and rerouting and proteolysis of cell surface MHC-I. In this study, we show that equine herpesvirus type 4 (EHV-4) modulates MHC-I cell surface expression through two different mechanisms. First, EHV-4 can lead to a significant downregulation of MHC-I expression at the cell surface through the product of ORF1, a protein expressed with early kinetics from a gene that is homologous to herpes simplex virus 1 UL56. The EHV-4 UL56 protein reduces cell surface MHC-I as early as 4 h after infection. Second, EHV-4 can interfere with MHC-I antigen presentation, starting at 6 h after infection, by inhibition of the transporter associated with antigen processing (TAP) through its UL49.5 protein. Although pUL49.5 has no immediate effect on overall surface MHC-I levels in infected cells, it blocks the supply of antigenic peptides to the endoplasmic reticulum (ER) and transport of peptide-loaded MHC-I to the cell surface. Taken together, our results show that EHV-4 encodes at least two viral immune evasion proteins: pUL56 reduces MHC-I molecules on the cell surface at early times after infection, and pUL49.5 interferes with MHC-I antigen presentation by blocking peptide transport in the ER.     

Equus caballus Major Histocompatibility Complex Class I Is an Entry Receptor for Equine Herpesvirus Type 1

In this study, Equus caballus major histocompatibility complex class I (MHC-I) was identified as a cellular entry receptor for the alphaherpesvirus equine herpesvirus type 1 (EHV-1). This novel EHV-1 receptor was discovered using a cDNA library from equine macrophages. cDNAs from this EHV-1-susceptible cell type were inserted into EHV-1-resistant B78H1 murine melanoma cells, these cells were infected with an EHV-1 lacZ reporter virus, and cells that supported virus infection were identified by X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) staining. Positive cells were subjected to several rounds of purification to obtain homogeneous cell populations that were shown to be uniformly infected with EHV-1. cDNAs from these cell populations were amplified by PCR and then sequenced. The sequence data revealed that the EHV-1-susceptible cells had acquired an E. caballus MHC-I cDNA. Cell surface expression of this receptor was verified by confocal immunofluorescence microscopy. The MHC-I cDNA was cloned into a mammalian expression vector, and stable B78H1 cell lines were generated that express this receptor. These cell lines were susceptible to EHV-1 infection while the parental B78H1 cells remained resistant to infection. In addition, EHV-1 infection of the B78H1 MHC-I-expressing cell lines was inhibited in a dose-dependent manner by an anti-MHC-I antibody.Equine herpesvirus type 1 (EHV-1) is a major pathogen affecting horses worldwide. Clinical signs of infection range from initial respiratory distress, fever, inappetance, and malaise to more serious secondary conditions including paralysis in some cases and abortigenic disease in pregnant mares (2). The virus is readily spread via direct transmission from horse to horse or via contact with contaminated surroundings. Due to the latent program of the virus, there is a constant reservoir of EHV-1 within the equine population, and frequent reactivation events trigger outbreaks and expose naïve horses to the virus (35).At the cellular level, EHV-1 initially attaches to cells via an interaction between two of its glycoproteins, gC and gB, and cell surface heparin sulfate (36, 41). While these electrostatic interactions mediate virus binding, they do not trigger the entry of the virus into cells. For entry to proceed, a secondary triggering event mediated by gD must occur (10, 14). After entry is initiated, EHV-1 enters cells either by directly fusing with the plasma membrane or via endocytosis (17). After fusion between the viral envelope and a cellular membrane, viral capsids are released into the cytoplasm and then actively transported to the nucleus along microtubules (18).Previous studies showed that EHV-1 utilizes a cell receptor that is distinct from any of the known alphaherpesvirus entry receptors (14). The goal of the present study was to identify a functional EHV-1 entry receptor by screening an equine macrophage cDNA library. To identify a receptor, we transferred equine cDNAs (48) from an equine macrophage library into cells that are highly resistant to EHV-1. These cDNA-transduced cells were then screened for their ability to mediate EHV-1 infection. Using this approach, we successfully converted a set of highly resistant cells to a state of complete susceptibility to EHV-1. From this converted set of cells, we amplified and sequenced the incorporated equine cDNA. The sequencing results revealed that the equine cDNA isolated from our screen codes for Equus caballus major histocompatibility complex class I (MHC-I) protein, and further assays confirmed that this receptor is utilized by EHV-1 for entry into cells.     

Glycoproteins D of equine herpesvirus type 1 (EHV-1) and EHV-4 determine cellular tropism independently of integrins

Equine herpesvirus type 1 (EHV-1) and EHV-4 are genetically and antigenically very similar, but their pathogenic potentials are strikingly different. The differences in pathogenicity between both viruses seem to be reflected in cellular host range: EHV-1 can readily be propagated in many cell types of multiple species, while EHV-4 entry and replication appear to be restricted mainly to equine cells. The clear difference in cellular tropism may well be associated with differences in the gene products involved in virus entry and/or spread from cell to cell. Here we show that (i) most of the EHV-1 permissive cell lines became resistant to EHV-1 expressing EHV-4 glycoprotein D (gD4) and the opposite was observed for EHV-4 harboring EHV-1 gD (gD1). (ii) The absence of integrins did not inhibit entry into and replication of EHV-1 in CHO-K1 or peripheral blood mononuclear cells (PBMC). Furthermore, integrin-negative K562 cells did not acquire the ability to bind to gD1 when αVβ3 integrin was overexpressed. (iii) PBMC could be infected with similar efficiencies by both EHV-1 and EHV-4 in vitro. (iv) In contrast to results for equine fibroblasts and cells of endothelial or epithelial origin, we were unable to block entry of EHV-1 or EHV-4 into PBMC with antibodies directed against major histocompatibility complex class I (MHC-I), a result that indicates that these viruses utilize a different receptor(s) to infect PBMC. Cumulatively, we provide evidence that efficient EHV-1 and EHV-4 entry is dependent mainly on gD, which can bind to multiple cell surface receptors, and that gD has a defining role with respect to cellular host range of EHV-1 and EHV-4.     

Identification and characterization of equine herpesvirus type 1 pUL56 and its role in virus-induced downregulation of major histocompatibility complex class I

Major histocompatibility complex class I (MHC-I) molecules play an important role in host immunity to infection by presenting antigenic peptides to cytotoxic T lymphocytes (CTLs), which recognize and destroy virus-infected cells. Members of the Herpesviridae have developed multiple mechanisms to avoid CTL recognition by virtue of downregulation of MHC-I on the cell surface. We report here on an immunomodulatory protein involved in this process, pUL56, which is encoded by ORF1 of equine herpesvirus type 1 (EHV-1), an alphaherpesvirus. We show that EHV-1 pUL56 is a phosphorylated early protein which is expressed as different forms and predominantly localizes to Golgi membranes. In addition, the transmembrane (TM) domain of the type II membrane protein was shown to be indispensable for correct subcellular localization and a proper function. pUL56 by itself is not functional with respect to interference with MHC-I and likely needs another unidentified viral protein(s) to perform this action. Surprisingly, pUL49.5, an inhibitor of the transporter associated with antigen processing (TAP) and encoded by EHV-1 and related viruses, appeared not to be required for pUL56-induced early MHC-I downmodulation in infected cells. In conclusion, our data identify a new immunomodulatory protein, pUL56, involved in MHC-I downregulation which is unable to perform its function outside the context of viral infection.     

Oncogenic transformation by by equine herpesviruses. II. Coestablishment of persistent infection and oncogenic transformation of hamster embryo cells by equine herpesvirus type 1 preparations enriched for defective interfering particles.

Infection of permissive hamster embryo cells with virus preparations enriched for defective interfering (DI) particles of equine herpesvirus type 1 (EHV-1) resulted in persistent infection and oncogenic transformation. Six cell lines, designated DI-5 to -10, exhibited biological properties (immortality, increased saturation density, growth in soft agar, etc.) inherent to transformed cells, but 2 to 18% of the total cells in these cell lines were shown to release virus as judged by electron microscope studies and infectious center assays. The released virus was shown to be standard EHV-1 and not to contain DI particles as determined by density measurements of the viral DNA in the analytical ultracentrifuge and by interference assays using the released virus. Tumorigenicity studies revealed that inoculation of these persistently infected cells into newborn LSH inbred hamsters resulted in a lethal, fulminating hepatitis, whereas inoculation into older immunocompetent hamsters (+4 weeks) led to the development of metastatic fibrous sarcomas. Tumor cell lines (DI-5T to -10T) established from these sarcomas were shown to be transplantable and virus nonproducers. Hybridization analyses of cellular DNAs from DI transformed and tumor cell lines using 32P-labeled genomic EHV-1 DNA as probes indicated that the whole virus genome was detectable in multiple copies (23 to 45) in the transformed cells and that DNA sequences representing only 43.5 to 56.6% of the virus genome were present in amounts of 2 to 4 copies per cell in the DI tumor cells. Expression of these viral DNA sequences as demonstrated by the detection of virus-neutralizing antibodies, 50% neutralizing dose titers ranging from 1:50 to 1:1,000, in the sera of animals inoculated with either the virus-producing transformed cells or the virus-nonproducing tumor cells. Further, EHV-1-specific proteins were detected in the membrane and the perinuclear region of bothDI transformed and tumor cells by indirect immunofluorescent assays using antisera against EHV-1 structural antigens, EHV-1 nonstructural antigens, or preparations of EHV-1 DI particles. The roles of DI particles in mediating persistent infection and cellular transformation are discussed.     

Down-modulation of MHC-I in a CD4+ T cell line, CEM-E5, after HIV-1 infection

HIV-1 is capable of infecting many different cell types that express the CD4 molecule. In vivo and in vitro this infection is associated with profound immunologic defects. We have examined the effect of HIV-1 infection on the expression of MHC class I (MHC-I) molecules to explore the possibility that this important immune system molecule is perturbed after HIV-1 infection. Our data show that in vitro, HIV-1 infection of CD4+ PBL, and the CD4+ cell lines, CEM-E5, HT, and U937, results in decreased expression of MHC-I molecules on the cell surface. This down-modulation is transient, occurring 18 h after HIV-1 infection of CD4+ PBL and returning to normal expression by 24 h. In CEM-E5, MHC-I down-modulation occurs over the course of days, reaching its greatest decrease (40%) about the time the cells are producing the most virus. Reversal of MHC-I expression to normal levels occurs as viral production decreases. Down-regulation during the time periods examined appear to be specific for MHC-I and does not occur with other cell-surface Ag nor is it caused by selection of a preexisting cell population with low MHC-I expression. Radioimmunoprecipitation of MHC-I protein from CEM-E5 indicated that the decrease of surface MHC-I is caused by decreased total protein secondary to a decrease in the level of mRNA for MHC-I. These decreased levels of MHC-I are biologically relevant because HIV-1 infected CEM-E5 cells are less susceptible to CTL lysis determined by the use of MHC-I cytolytic T cell clones and with the use of cold target-inhibition assay.     

A specific viral DNA sequence is stably integrated in herpesvirus oncogenically transformed cells

The integration patterns of viral DNA sequences in three hamster embryo cell lines independently derived by transformation with equine herpesvirus type 1 (EHV-1) have been investigated by DNA blot hybridization analyses for the restriction enzymes Eco RI, Bgl II, Xba I and Bam HI with ³²P-labeled selected DNAs from a collection of cloned EHV-1 restriction enzyme fragments as probes. These EHV-1-transformed cell lines contained subgenomic portions of the viral genome in an integrated state at multiple sites in the host genome. At least one copy of a viral DNA sequence mapping colinearly from 0.32 to 0.38 map units within the EHV-1 genome was common among these three EHV-1 transformed cell lines. The 0.32–0.38 viral DNA sequence was maintained stably even after 125 cell passages, whereas sequences from other positions in the EHV-1 genome were lost progressively during continued cell passage. The significance of the findings that these oncogenically transformed cell lines harbor a specific region of the EHV-1 genome is discussed with regard to stable maintenance of the oncogenically transformed state.     

Potential of equine herpesvirus 1 as a vector for immunization

Key problems using viral vectors for vaccination and gene therapy are antivector immunity, low transduction efficiencies, acute toxicity, and limited capacity to package foreign genetic information. It could be demonstrated that animal and human cells were efficiently transduced with equine herpesvirus 1 (EHV-1) reconstituted from viral DNA maintained and manipulated in Escherichia coli. Between 13 and 23% of primary human CD3+, CD4+, CD8+, CD11b+, and CD19+ cells and more than 70% of CD4+ MT4 cells or various human tumor cell lines (MeWo, Huh7, HeLa, 293T, or H1299) could be transduced with one infectious unit of EHV-1 per cell. After intranasal instillation of EHV-1 into mice, efficient transgene expression in lungs was detectable. Successful immunization using EHV-1 was shown after delivery of the human immunodeficiency virus type 1 Pr55gag precursor by the induction of a Gag-specific CD8+ immune response in mice. Because EHV-1 was not neutralized by human sera containing high titers of antibodies directed against human herpesviruses 1 to 5, it is concluded that this animal herpesvirus has enormous potential as a vaccine vector, because it is able to efficiently transduce a variety of animal and human cells, has high DNA packaging capacity, and can conveniently be maintained and manipulated in prokaryotic cells.     

Establishment of a cottontail rabbit papillomavirus/HLA-A2.1 transgenic rabbit model

Three transgenic rabbit lines that express a well-characterized human major histocompatibility complex class I (MHC-I) gene (HLA-A2.1) have been established. All three lines carry the HLA-A2.1 heavy chain and are able to pass the transgene to their offspring with both the outbred and the inbred EIII/JC genetic background. HLA-A2.1 colocalizes exclusively with rabbit MHC-I on the cell surfaces. These HLA-A2.1 transgenic rabbits demonstrated infection patterns similar to those found after cottontail rabbit papillomavirus (CRPV) challenge when compared with results in normal rabbits, although higher regression rates were found in HLA-A2.1 transgenic rabbits. Because the CRPV genome can accommodate significant modifications, the CRPV/HLA-A2.1 rabbit model has the potential to be used to screen HLA-A2.1-restricted immunogenic epitopes from human papillomaviruses in the context of in vivo papillomavirus infection.     

Expression of superoxide dismutases, catalase, and glutathione peroxidase in glioma cells

Four primary antioxidant enzymes were measured in both human and rat glioma cells. Both manganese-containing superoxide dismutase (MnSOD) and copper-zinc-containing superoxide dismutase (CuZnSOD) activities varied greatly among the different glioma cell lines. MnSOD was generally higher in human glioma cells than in rat glioma cells and relatively higher than in other tumor types. High levels of MnSOD in human glioma cells were due to the high levels of expression of MnSOD mRNA and protein. Heterogeneous expression of MnSOD was present in individual glioma cell lines and may be due to subpopulations or cells at different differentiation stages. Less difference in CuZnSOD, catalase, or glutathione peroxide was found between human and rat glioma cells. The human glioma cell lines showed large differences in sensitivity to the glutathione modulating drugs 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) and buthionine sulfoximine (BSO). A good correlation was found between sensitivity to BCNU and the activities of catalase in these cell lines. Only one cell line was sensitive to BSO and this line had low CuZnSOD activity.     

Investigation of apoptosis in cultured cells infected with equine herpesvirus 1

Many viruses alter different stages of apoptosis of infected cells as a strategy for successful infection. Few studies have addressed mechanisms of equine herpesvirus 1 (EHV-1) strain-induced cell death. We investigated the effect of an abortigenic strain (AR8 strain) on heterologous Madin–Darby bovine kidney cells and homologous equine dermis (ED) cells cell lines. We compared morphologic and biochemical features of early and late apoptosis at different postinfection times. We investigated translocation of phosphatidylserine to the cell surface, nuclear fragmentation and changes in the cytoskeleton using flow cytometry and annexin V/propidium iodide staining, DNA laddering, terminal deoxynucleotidyl transferase UTP nick-end labeling assay and immunofluorescence staining of cytokeratin 18 cleavage. AR8 EVH-1 strain interfered with apoptosis in both cell lines, particularly during the middle stage of the replication cycle; this was more evident in ED cells. Although this antiapoptotic effect has been reported for other alpha herpesviruses, our findings may help elucidate how EHV-1 improves its infectivity during its cycle.     

Equine herpesvirus 1 enters cells by two different pathways, and infection requires the activation of the cellular kinase ROCK1

Equine herpesvirus type 1 (EHV-1), a member of the Alphaherpesviridae, displays a broad host range in vitro, allowing for detailed study of the mechanisms of productive infection, including attachment and entry, in various cell culture systems. Previously, we showed that EHV-1 infects Chinese hamster ovary (CHO-K1) cells even though these cells do not express a known alphaherpesvirus entry receptor. In this report, we show by electron microscopy and an infectious recovery assay that entry into CHO-K1 cells occurs via an endocytic or phagocytic mechanism, while entry into equine dermal (ED) or rabbit kidney (RK13) cells occurs by direct fusion at the cell surface. In both cases (endocytic/phagocytic or direct fusion), entry leads to productive infection. Using drugs that inhibit clathrin-dependent or caveola-dependent endocytosis, we showed that EHV-1 entry into CHO-K1 cells does not require clathrin or caveolae. We also show that EHV-1 infection requires the activation of cell signaling molecules. In particular, we demonstrate that activation of the serine/threonine Rho kinase ROCK1 is critical for infection. Inhibition of this kinase by drugs or overexpression of a negative regulator of ROCK1 significantly blocked EHV-1 infection. These results show that EHV-1 can enter disparate cell types by at least two distinct mechanisms and that productive infection is dependent upon the activation of ROCK1.     

Comprehensive Serology Based on a Peptide ELISA to Assess the Prevalence of Closely Related Equine Herpesviruses in Zoo and Wild Animals

Equine herpesvirus type 1 (EHV-1) causes respiratory disorders and abortion in equids while EHV-1 regularly causes equine herpesvirus myeloencephalopathy (EHM), a stroke-like syndrome following endothelial cell infection in horses. Both EHV-1 and EHV-9 infections of non-definitive hosts often result in neuronal infection and high case fatality rates. Hence, EHV-1 and EHV-9 are somewhat unusual herpesviruses and lack strict host specificity, and the true extent of their host ranges have remained unclear. In order to determine the seroprevalence of EHV-1 and EHV-9, a sensitive and specific peptide-based ELISA was developed and applied to 428 sera from captive and wild animals representing 30 species in 12 families and five orders. Members of the Equidae, Rhinocerotidae and Bovidae were serologically positive for EHV-1 and EHV-9. The prevalence of EHV-1 in the sampled wild zebra populations was significantly higher than in zoos suggesting captivity may reduce exposure to EHV-1. Furthermore, the seroprevalence for EHV-1 was significantly higher than for EHV-9 in zebras. In contrast, EHV-9 antibody prevalence was high in captive and wild African rhinoceros species suggesting that they may serve as a reservoir or natural host for EHV-9. Thus, EHV-1 and EHV-9 have a broad host range favoring African herbivores and may have acquired novel natural hosts in ecosystems where wild equids are common and are in close contact with other perissodactyls.     

Influence of long-term equine herpesvirus type 1 (EHV-1) infection on primary murine neurons—the possible effects of the multiple passages of EHV-1 on its neurovirulence

Equine herpesvirus 1 (EHV-1), like other members of the Alphaherpesvirinae subfamily, is a neurotropic virus causing latent infections in the nervous system of the natural host. In the present study, we have investigated EHV-1 replication (wild-type Jan-E strain and Rac-H laboratory strain) during long-term infection and during the passages of the virus in cultured neurons. The studies were performed on primary murine neurons, which are an excellent in vitro model for studying neurotropism and neurovirulence of EHV-1. Using real-time cell growth analysis, we have demonstrated for the first time that primary murine neurons are able to survive long-term EHV-1 infection. Positive results of real-time PCR test indicated a high level of virus DNA in cultured neurons, and during long-term infection, these neurons were still able to transmit the virus to the other cells. We also compared the neurovirulence of Rac-H and Jan-E EHV-1 strains after multiple passages of these strains in neuron cell culture. The results showed that multiple passages of EHV-1 in neurons lead to the inhibition of viral replication as early as in the third passage. Interestingly, the inhibition of the EHV-1 replication occurred exclusively in neurons, because the equine dermal (ED) cells co-cultivated with neuroculture medium from the third passage showed the presence of large amount of viral DNA. In conclusion, our results showed that certain balance between EHV-1 and neurons has been established during in vitro infection allowing neurons to survive long-term infection.     

Adhesion of Human Glioma Cell Lines to Fibronectin, Laminin, Vitronectin and Collagen I Is Modulated by Gangliosides in vitro

Adhesion of eight cell lines, derived from human gliomas of different histological types, to fibronectin, collagen I, vitronectin, and laminin was investigated in vitro. The glioma cell lines were found to attach to these substrates to different extents. Interestingly, all cell lines strongly attached to laminin. In addition, glioma cell adhesion was found to be dose dependent. Moreover, adhesion of three cell lines to fibronectin and collagen I was partially inhibited and to vitronectin completely prevented by GRGDTP peptide, indicating the involvement of integrin receptors in glioma cell adhesion. We have demonstrated, recently, that gangliosides play an important role in promoting glioma cell invasion of the reconstituted basement membrane, Matrigel, in vitro. In order to study the mechanism of action of gangliosides in this process, the role of six gangliosides (GM1, GM3, GD3, GD1a, GD1b, and GT1b) in cell adhesion to the four proteins was investigated in three cell lines. Although all gangliosides, with the exception of GM3, were found to enhance cell adhesion to these proteins to different extents, GD3 proved to be the most effective adhesion-promoting ganglioside in all three cell lines. GM3 was found to inhibit cell adhesion to the four proteins in one cell line but enhanced cell adhesion in two other cell lines. The three cell lines were found to express both GD3 and gangliosides recognised by the A2B5 antibody. Furthermore, adhesion of the three cell lines to fibronectin, vitronectin, laminin, and collagen I was inhibited by incubation with A2B5, demonstrating the involvement of intrinsic cell membrane gangliosides in adhesion of glioma cells to these proteins. Taken together with the observation that gangliosides modulate integrin receptor function, these data suggest that gangliosides may play a central role in the control of the adhesive and invasive properties of human glioma cells.     

Early endosomal rerouting of major histocompatibility class I conformers

Major histocompatibility class I (MHC-I) molecules are present at the cell surface both as fully conformed trimolecular complexes composed of heavy chain (HC), beta-2-microglobulin (β2m) and peptide, and various open forms, devoid of peptide and/or β2m (open MHC-I conformers). Fully conformed MHC-I complexes and open MHC-I conformers can be distinguished by well characterized monoclonal antibody reagents that recognize their conformational difference in the extracellular domain. In the present study, we used these tools in order to test whether conformational difference in the extracellular domain determines endocytic and endosomal route of plasma membrane (PM) proteins. We analyzed PM localization, internalization, endosomal trafficking, and recycling of human and murine MHC-I proteins on various cell lines. We have shown that fully conformed MHC-I and open MHC-I conformers segregate at the PM and during endosomal trafficking resulting in the exclusion of open MHC-I conformers from the recycling route. This segregation is associated with their partitioning into the membranes of different compositions. As a result, the open MHC-I conformers internalized with higher rate than fully conformed counterparts. Thus, our data suggest the existence of conformation-based protein sorting mechanism in the endosomal system.     

Expression and function of the equine herpesvirus 1 virion-associated host shutoff homolog.

The ability of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2, respectively) to repress host cell protein synthesis early in infection has been studied extensively and found to involve the activities of the UL41 gene product, the virion-associated host shutoff (vhs) protein. To date, UL41 homologs have been identified in the genomes of three other alphaherpesviruses: equine herpesvirus 1 (EHV-1), varicella-zoster virus, and pseudorabies virus, but very little is known about the putative products of these homologous genes. Our earlier observations that no rapid early host protein shutoff occurred in EHV-1-infected cells led us to test EHV-1 vhs activity more thoroughly and to examine the expression and function of the EHV-1 UL41 homolog, ORF19. In the present study, the effects of EHV-1 and HSV-1 infections on cellular protein synthesis and mRNA degradation were compared at various multiplicities of infection in several cell types under an actinomycin D block. No virion-associated inhibition of cellular protein synthesis or vhs-induced cellular mRNA degradation was detected in cells infected with any of three EHV-1 strains (Ab4, KyA, and KyD) at multiplicities of infection at which HSV-1 strain F exhibited maximal vhs activity. However, further analyses revealed that (i) the EHV-1 vhs homolog gene, ORF19, was transcribed and translated into a 58-kDa protein in infected cells; (ii) the ORF19 protein was packaged into viral particles in amounts detectable in Western blots (immunoblots) with monoclonal antibodies; (iii) in cotransfection vhs activity assays, transiently-expressed ORF19 protein had intrinsic vhs activity comparable to that of wild-type HSV-1 vhs; and (iv) this intrinsic vhs activity was ablated by in vitro site-directed mutations in which either the functionally inactive HSV-1 vhs1 UL41 mutation (Thr at position 214 replaced by Ile [Thr-214-->Ile]) was recreated within ORF19 or two conserved residues within the putative poly(A) binding region of the ORF19 sequence were altered (Tyr-190, 192-->Phe). From these results we conclude that EHV-1's low vhs activity in infected cells is not a reflection of the ORF19 protein's intrinsic vhs activity but may be due instead to the amount of ORF19 protein associated with viral particles or to modulation of ORF19 protein's intrinsic activity by another viral component(s).