The transmembrane and cytosolic domains of equine herpesvirus type 1 glycoprotein D determine Golgi retention by regulating vesicle formation.

Accumulating evidence underscores the pivotal role of envelope proteins in viral secondary envelopment. However, the intricate molecular mechanisms governing this phenomenon remain elusive. To shed light on these mechanisms, we investigated a Golgi-retained gD of EHV-1 (gDEHV-1), distinguishing it from its counterparts in Herpes Simplex Virus-1 (HSV-1) and Pseudorabies Virus (PRV). To unravel the specific sequences responsible for the Golgi retention phenotype, we employed a gene truncation and replacement strategy. The results suggested that Golgi retention signals in gDEHV-1 exhibiting a multi-domain character. The extracellular domain of gDEHV-1 was identified as an endoplasmic reticulum (ER)-resident domain, the transmembrane domain and cytoplasmic tail (TM-CT) of gDEHV-1 were integral in facilitating the protein's residence within the Golgi complex. Deletion or replacement of either of these dual domains consistently resulted in the mutant gDEHV-1 being retained in an ER-like structure. Moreover, (TM-CT)EHV-1 demonstrated a preference for binding to endomembranes, inducing the generation of a substantial number of vesicles, potentially originate from the Golgi complex or the ER-Golgi intermediate compartment. In conclusion, our findings provide insights into the intricate molecular mechanisms governing the Golgi retention of gDEHV-1, facilitating the comprehension of the processes underlying viral secondary envelopment.

Equine herpesvirus type 1 ORF51 encoding UL11 as an essential gene for replication in cultured cells.

Equine herpesvirus type 1 (EHV-1) UL11 is a 74-amino-acid tegument protein encoded by ORF51 of the EHV-1 genome. EHV-1 UL11 was previously reported by other researchers using the RacL22 and RacH strains to be nonessential for viral replication in cultured cells. Here, we constructed UL11 mutant viruses including a UL11 null mutant and three C-terminal truncated mutants, for further characterization of EHV-1 UL11 using bacterial artificial chromosome (BAC) technology based on the neuropathogenic strain Ab4p. EHV-1 Ab4p UL11 was localized to juxtanuclear and Golgi regions as reported by other researchers. We found that no progeny viruses were produced by transfection of fetal equine kidney cells and rabbit kidney (RK-13) cells with the UL11 null mutant and truncation mutant BAC DNAs. However, mutant viruses were generated after transfection of RK13-UL11 cells constitutively expressing EHV-1 UL11 with the mutant BAC DNAs. In conclusion, UL11 of EHV-1 Ab4p is essential for replication in cultured cells.

[Impact of fluorescent protein tag on gD envelope protein subcellular localization in BHK-21 cells].

Objective: The fluorescent protein and gD envelope protein of equine herpes virus type 1 (EHV-1) were used to study the impact of tags on gD protein subcellular localization in BHK-21 cells. Methods: With the EHV-1 genome as a template, the gD complete gene was amplified by PCR technique. The product of PCR was cloned to pAcGFP1-C1 and pDsRed2-N1 plasmids. The recombinant plasmids were designated as pAc-GFP-gD (GFP-gD) and pDs-gD-Red (gD-Red). The GFP gene was inserted into the posterior position of gD gene signal peptide sequence. The modified gD gene signal peptide sequence was cloned to pVAX-1 plasmid, so that pVAX-S-GFP-gD' (S-GFPgD') recombinant plasmid was constructed. Meanwhile, the flag tag was added to N-terminal of gD sequence and they were cloned to pVAX-1 expression vector for constructing pVAX-Flag-gD recombinant plasmid. The BHK-21 cells were transfected with the 4 different recombinant plasmids and the subcellular localizations of fusion proteins were determined by lasar confocal scan microscopy. Results: Four eukaryotic expression vectors were constructed successfully. In BHK-21 cells, the vast majority of gD envelope proteins was localized in Golgi, and a small amount of gD was localized in the nucleus. Conclusion: Our finding reveals that the fluorescent protein of different insertion sites has no significant effects on the subcellular localization of gD, and provides a useful reference for other researchers.

Equine herpesvirus type 1 tegument protein VP22 is not essential for pathogenicity in a hamster model, but is required for efficient viral growth in cultured cells.

VP22 is a major tegument protein of Equine herpesvirus type 1 (EHV-1) that is a conserved protein among alphaherpesviruses. However, the roles of VP22 differ among each virus, and the roles of EHV-1 VP22 are still unclear. Here, we constructed an EHV-1 VP22 deletion mutant and a revertant virus to clarify the role of VP22. We found that EHV-1 VP22 was required for efficient viral growth in cultured cells, but not for virulence in a hamster model.

Equid herpesvirus type 1 activates platelets.

Equid herpesvirus type 1 (EHV-1) causes outbreaks of abortion and neurological disease in horses. One of the main causes of these clinical syndromes is thrombosis in placental and spinal cord vessels, however the mechanism for thrombus formation is unknown. Platelets form part of the thrombus and amplify and propagate thrombin generation. Here, we tested the hypothesis that EHV-1 activates platelets. We found that two EHV-1 strains, RacL11 and Ab4 at 0.5 or higher plaque forming unit/cell, activate platelets within 10 minutes, causing α-granule secretion (surface P-selectin expression) and platelet microvesiculation (increased small events double positive for CD41 and Annexin V). Microvesiculation was more pronounced with the RacL11 strain. Virus-induced P-selectin expression required plasma and 1.0 mM exogenous calcium. P-selectin expression was abolished and microvesiculation was significantly reduced in factor VII- or X-deficient human plasma. Both P-selectin expression and microvesiculation were re-established in factor VII-deficient human plasma with added purified human factor VIIa (1 nM). A glycoprotein C-deficient mutant of the Ab4 strain activated platelets as effectively as non-mutated Ab4. P-selectin expression was abolished and microvesiculation was significantly reduced by preincubation of virus with a goat polyclonal anti-rabbit tissue factor antibody. Infectious virus could be retrieved from washed EHV-1-exposed platelets, suggesting a direct platelet-virus interaction. Our results indicate that EHV-1 activates equine platelets and that α-granule secretion is a consequence of virus-associated tissue factor triggering factor X activation and thrombin generation. Microvesiculation was only partly tissue factor and thrombin-dependent, suggesting the virus causes microvesiculation through other mechanisms, potentially through direct binding. These findings suggest that EHV-1-induced platelet activation could contribute to the thrombosis that occurs in clinically infected horses and provides a new mechanism by which viruses activate hemostasis.

Ubiquitination and degradation of the ORF34 gene product of equine herpesvirus type 1 (EHV-1) at late times of infection.

The equine herpesvirus type 1 (EHV-1) open reading frame 34 (ORF34) is predicted to encode a polypeptide of 161 amino acids. We show that an ORF34 deletion mutant exhibited a significant growth defect in equine peripheral blood mononuclear cells taken directly ex vivo during early but not late times of infection. ORF34 protein (pORF34)-specific antibodies specifically reacted with a 28-kDa early polypeptide present in the cytosol of infected cells. From 10h post infection, multiple smaller pORF34-specific protein moieties were detected indicating that expression of a late viral gene product(s) caused pORF34 degradation. Proteasome inhibitors blocked pORF34 degradation as did treatment of infected cells with a ubiquitin-activating enzyme (E1) inhibitor. Finally, kinetic studies showed that pORF34 is modified by addition of multiple copies of ubiquitin. Taken together, our findings suggest that the ubiquitin proteasome pathway is required for pORF34 degradation that may modulate protein activity in the course of infection.

Equine herpesvirus type 1 (EHV-1) utilizes microtubules, dynein, and ROCK1 to productively infect cells.

To initiate infection, equine herpesvirus type 1 (EHV-1) attaches to heparan sulfate on cell surfaces and then interacts with a putative glycoprotein D receptor(s). After attachment, virus entry occurs either by direct fusion of the virus envelope with the plasma membrane or via endocytosis followed by fusion between the virus envelope and an endosomal membrane. Upon fusion, de-enveloped virus particles are deposited into the cytoplasm and travel to the nucleus for viral replication. In this report, we examined the mechanism of EHV-1 intracellular trafficking and investigated the ability of EHV-1 to utilize specific cellular components to efficiently travel to the nucleus post-entry. Using a panel of microtubule-depolymerizing drugs and inhibitors of microtubule motor proteins, we show that EHV-1 infection is dependent on both the integrity of the microtubule network and the minus-end microtubule motor protein, dynein. In addition, we show that EHV-1 actively induces the acetylation of tubulin, a marker of microtubule stabilization, as early as 15 min post-infection. Finally, our data support a role for the cellular kinase, ROCK1, in virus trafficking to the nucleus.

Meningoencephalitis in mice infected with an equine herpesvirus 1 strain KyA recombinant expressing glycoprotein I and glycoprotein E.

One of the consequences of equine herpesvirus 1 (EHV-1) infection in the natural host is a neurological disease that can lead to paralysis. The pathology associated with EHV-1-induced neurological disease includes vasculitis of the small blood vessels within the central nervous system and subsequent damage to the surrounding neural tissue. In a previous study, an EHV-1 recombinant KyA virus (KgI/gE/75) was generated in which the sequences encoding glycoprotein I (gI) and glycoprotein E (gE) were repaired [Frampton et al. 2002 (Virus Research 90: 287-301)] using genes of the pathogenic EHV-1 strain 89c25. In contrast to the parental KyA virus that lacks gI and gE, the recombinant KgI/gE/75 was able to spread to the brains of CBA mice after intranasal infection. Infection resulted in a meningoencephalitis characterized by lymphocytic cuffing of small blood vessels within the brain, consistent with that observed in EHV-1-infected horses exhibiting neurological signs. KgI/gE/75 was able to elicit cytopathology in the lung prior to spread to the brain. However, like the attenuated KyA strain, KgI/gE/75 did not persist in the lung and was completely cleared from lung tissue by day 5 postinfection. We propose that gI and gE are neurovirulence factors for EHV-1, and that the CBA mouse model can be extended to study neurologic sequelae resulting after EHV-1 infection.

The equine herpesvirus 1 UL11 gene product localizes to the trans-golgi network and is involved in cell-to-cell spread.

Experiments were conducted to identify and characterize the equine herpesvirus type 1 (EHV-1) UL11 homologous protein. At early-late times after EHV-1 infection of Rk13 cells several proteins at an M(r) of 8000 to 12,000 were detected using a UL11 protein-specific antiserum. Particularly, an M(r) of 11,000 protein was found abundantly in purified virions and could be assigned to the tegument fraction. As demonstrated by confocal laser scanning microscopy, UL11 reactivity localized predominantly to the trans-Golgi network of infected cells, but was also noted at the plasma membrane, specifically of transfected cells. Deletion of UL11 sequences in EHV-1 vaccine strain RacH (Hdelta11) and in the virulent isolate RacL22 (Ldelta11) resulted in viruses that were able to replicate on noncomplementing cells. It was shown in one-step growth kinetics on Rk13 cells that the reduction of intracellular and of extracellular virus titers caused by the absence of UL11 expression in either virus was somewhat variable, but approximately 10- to 20-fold. In contrast, a marked influence on the plaque phenotype was noted, as mean maximal diameters of plaques were reduced to 23.2% (RacL22) or 34.7% (RacH) of parental virus plaques and as an effect on the ability of RacH to cause syncytia upon infection was noted. It was therefore concluded that the EHV-1 UL11 product is not essential for virus replication in Rk13 cells but is involved in cell-to-cell spread.

Glycoprotein G isoforms from some alphaherpesviruses function as broad-spectrum chemokine binding proteins.

Mimicry of host chemokines and chemokine receptors to modulate chemokine activity is a strategy encoded by beta- and gammaherpesviruses, but very limited information is available on the anti-chemokine strategies encoded by alphaherpesviruses. The secretion of chemokine binding proteins (vCKBPs) has hitherto been considered a unique strategy encoded by poxviruses and gammaherpesviruses. We describe a family of novel vCKBPs in equine herpesvirus 1, bovine herpesvirus 1 and 5, and related alphaherpesviruses with no sequence similarity to chemokine receptors or other vCKBPs. We show that glycoprotein G (gG) is secreted from infected cells, binds a broad range of chemokines with high affinity and blocks chemokine activity by preventing their interaction with specific receptors. Moreover, gG also blocks chemokine binding to glycosaminoglycans, an interaction required for the correct presentation and function of chemokines in vivo. In contrast to other vCKBPs, gG may also be membrane anchored and, consistently, we show chemokine binding activity at the surface of cells expressing full-length protein. These alphaherpesvirus vCKBPs represent a novel family of proteins that bind chemokines both at the membrane and in solution.

Interaction of the equine herpesvirus 1 EICP0 protein with the immediate-early (IE) protein, TFIIB, and TBP may mediate the antagonism between the IE and EICP0 proteins.

The equine herpesvirus 1 (EHV-1) immediate-early (IE) and EICP0 proteins are potent trans-activators of EHV-1 promoters; however, in transient-transfection assays, the IE protein inhibits the trans-activation function of the EICP0 protein. Assays with IE mutant proteins revealed that its DNA-binding domain, TFIIB-binding domain, and nuclear localization signal may be important for the antagonism between the IE and EICP0 proteins. In vitro interaction assays with the purified IE and EICP0 proteins indicated that these proteins interact directly. At late times postinfection, the IE and EICP0 proteins colocalized in the nuclei of infected equine cells. Transient-transfection assays showed that the EICP0 protein trans-activated EHV-1 promoters harboring only a minimal promoter region (TATA box and cap site), suggesting that the EICP0 protein trans-activates EHV-1 promoters by interactions with general transcription factor(s). In vitro interaction assays revealed that the EICP0 protein interacted directly with the basal transcription factors TFIIB and TBP and that the EICP0 protein (amino acids [aa] 143 to 278) mediated the interaction with aa 125 to 174 of TFIIB. Our unpublished data showed that the IE protein interacts with the same domain (aa 125 to 174) of TFIIB and with TBP. Taken together, these results suggested that interaction of the EICP0 protein with the IE protein, TFIIB, and TBP may mediate the antagonism between the IE and EICP0 proteins.

Egress of alphaherpesviruses: comparative ultrastructural study.

Egress of four important alphaherpesviruses, equine herpesvirus 1 (EHV-1), herpes simplex virus type 1 (HSV-1), infectious laryngotracheitis virus (ILTV), and pseudorabies virus (PrV), was investigated by electron microscopy of infected cell lines of different origins. In all virus-cell systems analyzed, similar observations were made concerning the different stages of virion morphogenesis. After intranuclear assembly, nucleocapsids bud at the inner leaflet of the nuclear membrane, resulting in enveloped particles in the perinuclear space that contain a sharply bordered rim of tegument and a smooth envelope surface. Egress from the perinuclear cisterna primarily occurs by fusion of the primary envelope with the outer leaflet of the nuclear membrane, which has been visualized for HSV-1 and EHV-1 for the first time. The resulting intracytoplasmic naked nucleocapsids are enveloped at membranes of the trans-Golgi network (TGN), as shown by immunogold labeling with a TGN-specific antiserum. Virions containing their final envelope differ in morphology from particles within the perinuclear cisterna by visible surface projections and a diffuse tegument. Particularly striking was the addition of a large amount of tegument material to ILTV capsids in the cytoplasm. Extracellular virions were morphologically identical to virions within Golgi-derived vesicles, but distinct from virions in the perinuclear space. Studies with gB- and gH-deleted PrV mutants indicated that these two glycoproteins, which are essential for virus entry and direct cell-to-cell spread, are dispensable for egress. Taken together, our studies indicate that the deenvelopment-reenvelopment process of herpesvirus maturation also occurs in EHV-1, HSV-1, and ILTV and that membrane fusion processes occurring during egress are substantially different from those during entry and direct viral cell-to-cell spread.

The equine herpesvirus 1 UL45 homolog encodes a glycosylated type II transmembrane protein and is involved in virus egress.

Experiments to analyze the product of the equine herpesvirus type 1 (EHV-1) UL45 homolog were conducted. Using an antiserum generated against the carboxylterminal 114 amino acids of the EHV-1 UL45 protein, proteins of M(r) 32,000, 40,000, and 43,000 were detected specifically in EHV-1-infected cells. Neither form of the protein was located in purified virions of EHV-1 wild-type strain RacL22 or the modified live vaccine strain RacH, but UL45 was demonstrated to be expressed as a late (gamma-2) protein. Fractionation of infected cells and deglycosylation experiments demonstrated that the EHV-1 UL45 protein represents a type II membrane glycoprotein. Deletion of the UL45 gene in RacL22 and RacH (LDelta45 and HDelta45) showed that UL45 is nonessential for EHV-1 growth in vitro, but that deletion reduced the viruses' replication efficiency. A marked reduction of virus release was observed although no significant influence was noticed either on plaque size or on the syncytial phenotype of the EHV-1 strain RacH.

Pseudorabies virus glycoprotein M inhibits membrane fusion.

A transient transfection-fusion assay was established to investigate membrane fusion mediated by pseudorabies virus (PrV) glycoproteins. Plasmids expressing PrV glycoproteins under control of the immediate-early 1 promoter-enhancer of human cytomegalovirus were transfected into rabbit kidney cells, and the extent of cell fusion was quantitated 27 to 42 h after transfection. Cotransfection of plasmids encoding PrV glycoproteins B (gB), gD, gH, and gL resulted in formation of polykaryocytes, as has been shown for homologous proteins of herpes simplex virus type 1 (HSV-1) (A. Turner, B. Bruun, T. Minson, and H. Browne, J. Virol. 72:873-875, 1998). However, in contrast to HSV-1, fusion was also observed when the gD-encoding plasmid was omitted, which indicates that PrV gB, gH, and gL are sufficient to mediate fusion. Fusogenic activity was enhanced when a carboxy-terminally truncated version of gB (gB-008) lacking the C-terminal 29 amino acids was used instead of wild-type gB. With gB-008, only gH was required in addition for fusion. A very rapid and extended fusion was observed after cotransfection of plasmids encoding gB-008 and gDH, a hybrid protein consisting of the N-terminal 271 amino acids of gD fused to the 590 C-terminal amino acids of gH. This protein has been shown to substitute for gH, gD, and gL function in the respective viral mutants (B. G. Klupp and T. C. Mettenleiter, J. Virol. 73:3014-3022, 1999). Cotransfection of plasmids encoding PrV gC, gE, gI, gK, and UL20 with gB-008 and gDH had no effect on fusion. However, inclusion of a gM-expressing plasmid strongly reduced the extent of fusion. An inhibitory effect was also observed after inclusion of plasmids encoding gM homologs of equine herpesvirus 1 or infectious laryngotracheitis virus but only in conjunction with expression of the gM complex partner, the gN homolog. Inhibition by PrV gM was not limited to PrV glycoprotein-mediated fusion but also affected fusion induced by the F protein of bovine respiratory syncytial virus, indicating a general mechanism of fusion inhibition by gM.

EHV-1 glycoprotein D (EHV-1 gD) is required for virus entry and cell-cell fusion, and an EHV-1 gD deletion mutant induces a protective immune response in mice.

Insertional mutagenesis was used to construct an equine herpesvirus 1 (EHV-1) mutant in which the open reading frame for glycoprotein D was replaced by a lacZ cassette. This gD deletion mutant (delta gD EHV-1) was unable to infect normally permissive RK cells in culture, but could be propagated in an EHV-1 gD-expressing cell line (RK/gD). Phenotypically complemented delta gD EHV-1 was able to infect RK cells, but did not spread to form syncytial plaques as seen with wild type EHV-1 or with delta gD EHV-1 infection of RK/gD cell cultures. Therefore EHV-1 gD is required for virus entry and for cell-cell fusion. The phenotypically complemented delta gD EHV-1 had very low pathogenicity in a mouse model of EHV-1 respiratory disease, compared to a fully replication-competent EHV-1 reporter virus (lacZ62/63 EHV-1). Intranasal or intramuscular inoculation of mice with delta gD EHV-1 induced protective immune responses that were similar to those elicited in mice inoculated with lacZ62/63 EHV-1 and greater than those following inoculation with UV-inactivated virus.

Construction and characterization of an equine herpesvirus 1 glycoprotein C negative mutant.

An equine herpesvirus 1 (EHV-1) strain RacL 11 mutant was constructed that carries the Escherichia coli LacZ gene instead of the open reading frame encoding glycoprotein C (gC). The engineered virus mutant (L11(delta)gC) lacked codons 46-440 of the 1404 bp gene. On rabbit kidney cell line Rk13 and equine dermal cell line Edmin337, the L11(delta)gC virus grew to titers which were reduced by approximately 5- to 10-fold compared with wild-type RacL11 virus or a repaired virus (R-L11(delta)gC). However, when L11(delta)gC growth properties were analyzed on primary equine cells a decrease of viral titers was observed such that extracellular L11(delta)gC titers were reduced by 48- to 210-fold compared with those of wild-type or repaired virus. Heparin sensitive and heparin resistant attachment was assessed by binding studies using radiolabeled virion preparations. These studies revealed that EHV-1 gC is important for heparin sensitive attachment to the target cell. Similar results were obtained when cellular glycosaminoglycan (GAG) synthesis was inhibited by chlorate treatment or when cells defective in GAG synthesis were used. L11(delta)gC also exhibited significantly delayed penetration kinetics on Rk13 and primary equine cells. Infection of mice with L11(delta)gC did not cause EHV-1-related disease, whereas mice infected with either RacL11 or R-L11(delta)gC exhibited massive bodyweight losses, high virus titers in the lungs, and viremia. Taken together, EHV-1 gC was shown to play important roles in the early steps of infection and in release of virions, especially in primary equine cells, and contributes to EHV-1 virulence.

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.

Phosphorylation of structural components promotes dissociation of the herpes simplex virus type 1 tegument.

The role of phosphorylation in the dissociation of structural components of the herpes simplex virus type 1 (HSV-1) tegument was investigated, using an in vitro assay. Addition of physiological concentrations of ATP and magnesium to wild-type virions in the presence of detergent promoted the release of VP13/14 and VP22. VP1/2 and the UL13 protein kinase were not significantly solubilized. However, using a virus with an inactivated UL13 protein, we found that the release of VP22 was severely impaired. Addition of casein kinase II (CKII) to UL13 mutant virions promoted VP22 release. Heat inactivation of virions or addition of phosphatase inhibited the release of both proteins. Incorporation of radiolabeled ATP into the assay demonstrated the phosphorylation of VP1/2, VP13/14, VP16, and VP22. Incubation of detergent-purified, heat-inactivated capsid-tegument with recombinant kinases showed VP1/2 phosphorylation by CKII, VP13/14 phosphorylation by CKII, protein kinase A (PKA), and PKC, VP16 phosphorylation by PKA, and VP22 phosphorylation by CKII and PKC. Proteolytic mapping and phosphoamino acid analysis of phosphorylated VP22 correlated with previously published work. The phosphorylation of virion-associated VP13/14, VP16, and VP22 was demonstrated in cells infected in the presence of cycloheximide. Use of equine herpesvirus 1 in the in vitro release assay resulted in the enhanced release of VP10, the homolog of HSV-1 VP13/14. These results suggest that the dissociation of major tegument proteins from alphaherpesvirus virions in infected cells may be initiated by phosphorylation events mediated by both virion-associated and cellular kinases.

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.

N-terminal sequence analysis of equine herpesvirus 1 glycoproteins D and B and evidence for internal cleavage of the gene 71 product.

Signal cleavage sites of equine herpesvirus 1 (EHV-1) glycoproteins D and B (gD and gB) and an endoproteolytic cleavage site of EHV-1 gB were determined by N-terminal amino acid sequencing and compared with known cleavage sites of homologues in other herpesvirus. Signal cleavage of EHV-1 gD occurred between Arg35 and Ala36 in a region of basic amino acids resembling the endoproteolytic cleavage sites of viral glycoproteins, nine amino acids downstream of the predicted site, while EHV-1 gB was cleaved as predicted between Ala85 and Val86. Endoproteolytic cleavage of EHV-1 gB occurred between Arg548 and Ala549, 28 amino acids downstream of the cleavage site predicted from conserved sequences of other herpesvirus gB homologous. One interpretation of these data is that EHV-1 gB is cleaved internally at both sites, a possibility which was supported by the apparent molecular masses of the unglycosylated gB subunits produced in the presence of tunicamycin. This double cleavage would release a stretch of amino acids which is not present in sequenced gB molecules of other herpesviruses. Experiments with glycosylation inhibitors indicated that cleavage of EHV-1 gB can occur in the absence of glycosylation. N-terminal sequencing also determined that a 42 kDa EHV-1 glycoprotein was a product of internal cleavage of the protein encoded by gene 71. Staggered endoproteolytic cleavage after adjacent arginine residues 506 and 507 separates the 42 kDa C-terminal subunit containing all the cysteine residues from the serine and threonine rich N-terminal region.