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Recently, the product of equine herpesvirus type 1 (EHV-1) ORF1, a homolog to HSV-1 pUL56, was shown to modulate MHC-I expression and innate immunity. Here, we investigated modulation of respiratory epithelial immunity by EHV-1 pUL56 and compared responses to those of PBMCs, which are important target cells that allow cell-associated EHV-1 viremia. The salient observations are as follows: (i) EHV-1 significantly down-modulated MHC-I and MHC-II expression in equine respiratory epithelial cells (ERECs). MHC-I expression remained unaffected in PBMCs and MHC-II expression was increased. (ii) Infection with an EHV-1 ORF1 deletion mutant partially restored MHC-I and MHC-II expression and altered IFN-alpha and IL-10 mRNA expression. (iii) Deletion of EHV-1 ORF1 also significantly increased chemokine expression and chemotaxis of monocytes and neutrophils in ERECs. Collectively, these results suggest a role for EHV-1 pUL56 in modulation of antigen presentation, cytokine expression and chemotaxis at the respiratory epithelium, but not in PBMC., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Equine herpesvirus type 1 (EHV-1) ORF59 is predicted to encode a protein consisting of 180 amino acids. To determine whether ORF59 in fact encodes a protein, sequences encoding an HA epitope (YPYDVPDYA) was inserted at the carboxyterminus of the ORF59 protein in EHV-1 strain Ab4. Using anti-HA monoclonal antibodies, a 21-kDa band was specifically detected by western blot analysis in lysates of cells infected with a recombinant EHV-1 from strain Ab4 that carries the pORF59-HA but not in cells infected with parental Ab4. Further characterization of the protein using immunofluorescence and fractionation studies showed that pORF59 is an early protein that localizes to the cytosol in virus-infected cells. Recombinant EHV-1 lacking ORF59 (rAb4∆59) exhibited a small-plaque phenotype and could not be propagated. Our findings suggest that the ORF59 protein plays a major role in EHV-1 replication in vitro and likely in vivo., (Crown Copyright © 2013 Published by Elsevier Inc. All rights reserved.)

Transcriptome analysis of polar bears (Ursus maritimus) yielded sequences with highest similarity to the human endogenous retrovirus group HERV-K(HML-2). Further analysis of the polar bear draft genome identified an endogenous betaretrovirus group comprising 26 proviral copies and 231 solo LTRs. Molecular dating indicates the group originated before the divergence of bears from a common ancestor but is not present in all carnivores. Closely related sequences were identified in the giant panda (Ailuropoda melanoleuca) and characterized from its genome. We have designated the polar bear and giant panda sequences U. maritimus endogenous retrovirus (UmaERV) and A. melanoleuca endogenous retrovirus (AmeERV), respectively. Phylogenetic analysis demonstrated that the bear virus group is nested within the HERV-K supergroup among bovine and bat endogenous retroviruses suggesting a complex evolutionary history within the HERV-K group. All individual remnants of proviral sequences contain numerous frameshifts and stop codons and thus, the virus is likely non-infectious., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Marek's disease virus (MDV) encodes an ubiquitin-specific protease (USP) within its UL36 gene. USP is highly conserved among herpesviruses and was shown to be important for MDV replication and pathogenesis in MDV's natural host, the chicken. To further investigate the role of MDV USP, several recombinant (r) MDVs were generated and their in vitro phenotypes were evaluated using plaque size and growth kinetics assays. We discovered that the N-terminus of pUL36 is essential for MDV replication and could not be complemented by ectopic expression of MDV USP. In addition, we demonstrated that the region located between the conserved glutamine (Q85) and leucine (L106) residues comprising the active site cysteine (C98) is also essential for MDV replication. Based on the analyses of the rMDVs generated here, we concluded that MDV USP likely contributes to the structure and/or stability of pUL36 and affects replication and oncogenesis of MDV beyond its enzymatic activity., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Marek's disease is a devastating neoplastic disease of chickens caused by Marek's disease virus (MDV). MDV down-regulates surface expression of MHC class I molecules, although the mechanism has remained elusive. MDV harbors a UL49.5 homolog that has been shown to down-regulate MHC class I expression in other Varicelloviruses. Using in vitro assays, we showed that MDV pUL49.5 down-regulates MHC class I directly and identified its cytoplasmic tail as essential for this function. In vivo, viruses lacking the cytoplasmic tail of pUL49.5 showed no differences in MD pathogenesis compared to revertant viruses in highly susceptible chickens of the B(19)B(19) MHC class I haplotype, while there was a mild reduction in pathogenic potential of the deletion viruses in chickens more resistant to MD pathogenesis (MHC:B(21)B(21)). We concluded that the pathogenic effect of MHC class I down-regulation mediated by pUL49.5 is small because virus immune evasion possibly requires more than one viral protein., (Copyright 2010 Elsevier Inc. All rights reserved.)

Marek's disease (MD) is a highly contagious, lymphoproliferative disease of chickens caused by the cell-associated MD virus (MDV), a member of the alphaherpesvirus subfamily. In a previous study we showed that the absence of the serine/threonine protein kinase (pU(S)3) encoded in the MDV unique-short region resulted in accumulation of primarily enveloped virions in the perinuclear space and significant impairment of virus growth in vitro. It was also shown that pU(S)3 is involved in actin stress fiber breakdown [Schumacher, D., Tischer, B. K., Trapp, S., and Osterrieder, N. (2005). Here, we constructed a recombinant virus to test the importance of pU(S)3 kinase activity for MDV replication and its functions in actin rearrangement. Disruption of the kinase active site was achieved by substituting a lysine at position 220 with an alanine (K220A). Titers of a kinase-negative MDV mutant, 20U(S)3()K220A, were reduced when compared to parental virus similar to those of the U(S)3 deletion mutant. We were also able to demonstrate complete absence of phosphorylation of MDV-specific phosphoprotein pp38 in cells infected with the kinase-deficient virus, indicating that pp38 phosphorylation depends entirely on the kinase activity of pU(S)3. Enzymatically inactive pU(S)3()K220A was, however, still capable of mediating breakdown of the actin cytoskeleton in transfection studies, and this activity was indistinguishable from that of wild-type pU(S)3(). Furthermore, we demonstrated that pU(S)3 possesses anti-apoptotic activity, which is dependent on its kinase activity. Taken together, our results demonstrate that pU(S)3 and MDV-specific phosphoprotein pp38 represent a kinase-substrate pair and that growth impairment in the absence of pU(S)3 is caused by the absence of kinase activity. The unaltered disruption of F-actin by the K220A pU(S)3 mutant suggests that F-actin disassembly is unrelated to MDV growth restrictions in the absence of the unique-short protein kinase.

Glycoprotein G (gG) of equine herpesvirus type 1 (EHV-1), a structural component of virions and secreted from virus-infected cells, was shown to bind to a variety of different chemokines and as such might be involved in immune modulation. Little is known, however, about its role in the replication cycle and infection of EHV-1 in vivo. Here we report on the function of gG in context of virus infection in vitro and in vivo. A gG deletion mutant of pathogenic EHV-1 strain RacL11 (vL11DeltagG) was constructed and analyzed. Deletion of gG had virtually no effect on the growth properties of vL11DeltagG in cell culture when compared to parental virus or a rescuant virus vL11DeltagGR, respectively, and virus titers and plaque formation were unaffected in the absence of the glycoprotein. Similarly, in the murine model of EHV-1 infection, no significant differences in virulence between the gG deletion mutant and RacL11 or vL11DeltagGR were found at high doses of infection. However, infection of mice at lower doses revealed that the gG deletion mutant was able to replicate to higher titers in lungs of infected mice. Additionally, these mice lost significantly more weight than those infected with RacL11 and a more pronounced inflammatory response in lungs was observed. Therefore we concluded that deletion of gG in EHV-1 seems to lead to an exacerbation of respiratory disease in the mouse.

The function of the equine herpesvirus type 1 (EHV-1) glycoprotein K (gK) homologue was investigated. Deletion of 88% of the UL53-homologous open reading frame in EHV-1 strain RacH resulted in a severe growth defect of the gK-negative virus (HDeltagK) as reflected by a significant decrease in the production of infectious virus progeny on RK13 cells. The HDeltagK virus induced only minute plaques, was unable to form syncytia, and its penetration efficiency into RK13 cells was reduced by approximately 40%. To further analyze gK function and intracellular trafficking, gK of strain RacH was replaced by a C-terminally truncated gK-green fluorescent protein fusion protein (gK-GFP). The generated recombinant virus was shown to replicate well on non-complementing cells, and virus penetration and syncytium formation were comparable to parental RacH. A reduction in plaque size and slightly decreased intra- and extracellular virus titers, however, were observed. The gK-GFP fusion protein was expressed with early-late kinetics, and multiple forms of the protein exhibiting M(r)s between 50,000 and 85,000 were detected by Western blot analysis. The various gK-GFP forms were shown to be N-glycosylated, associated with membranes of the Golgi apparatus, and were incorporated into extracellular virions. Complete processing of gK-GFP was only observed within the context of viral infection. From the results, we concluded that EHV-1 gK is required for efficient virus growth in vitro and that the carboxy-terminal amino acids are not required for its function, because the gK-GFP fusion protein was able to complement for EHV-1 growth in the absence of authentic gK.

The structure and function of the equine herpesvirus type 1 (EHV-1) UL34 homologous protein were characterized. A UL34 protein-specific antiserum reacted with an M(r)28,000 protein that could not be detected in purified extracellular virions. Confocal laser scanning microscopy demonstrated that UL34 reactivity mainly concentrated at the nuclear rim, which changed into a punctuate and filamentous pattern at late times after infection. These changes in UL34 distribution were especially prominent when analyzing the distribution of a GFP-UL34 fusion protein. A UL34-negative EHV-1 was generated by mutagenesis of a recently established BAC clone of EHV-1 strain RacH (pRacH). Release of extracellular infectious virus was severely impaired after infection of Rk13 cells with HDelta34. Electron microscopy revealed a virtual absence of virus particles in the cytoplasm of infected cells, whereas nucleocapsid formation and maturation within the nucleus appeared unaffected. A UL34-GFP fusion protein with GFP linked to the C-terminus of UL34 was able to complement for the UL34 deletion in trans, while a GFP-UL34-fusion protein with GFP linked to the N-terminus of UL34 was able to only partially restore virus growth. It was concluded that the EHV-1 UL34 product is essential for an early step in virus egress, i.e., release of capsids from infected-cell nuclei.

Experiments were conducted to analyze the effects of a simultaneous deletion of glycoprotein M (gM) and glycoprotein 2 (gp2) of equine herpesvirus type 1 (EHV-1). EHV-1 strain RacH was cloned as a bacterial artificial chromosome (pRacH) by homologous recombination of a mini F plasmid into the unique short region of the genome, thereby deleting gene 71 encoding gp2. Upon transfection of the pRacH DNA into rabbit kidney RK13 cells, virus plaques were visible from day 1 after transfection. The mutant RacH virus (H Delta gp2) reconstituted from pRacH lacked gene 71 and did not express gp2 as assayed by indirect immunofluorescence analysis using gp2-specific monoclonal antibodies. The H Delta gp2 virus exhibited 10-fold reduced extracellular titers and an approximately 10% reduction in mean plaque diameters when compared to parental or gp2-revertant virus. The gM open reading frame was deleted from pRacH by recE/T mediated mutagenesis in Escherichia coli. The gM-gp2 double negative virus mutant (H Delta gp2gM) did not express either of the deleted glycoproteins as demonstrated by indirect immunofluorescence analysis. The H Delta gp2gM virus exhibited a 200-fold reduction of end-point extracellular titers when compared to parental RacH virus, which could not be compensated for by growth of the mutant virus on gM-expressing cells. After restoration of the gM open reading frame, however, growth of the mutant virus was comparable to the H Delta gp2 virus. Plaque diameters of the gM-gp2 double-negative mutant were reduced by only 16% when compared to that of parental RacH virus. From the results it was concluded that the simultaneous absence of gM and gp2 had an additive effect on egress but not secondary envelopment or cell-to-cell spread of EHV-1.

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., (Copyright 2000 Academic Press.)

Equine herpesvirus 1 (EHV-1) mutants devoid of the open reading frames (ORFs) of either glycoprotein (g) B or M were constructed and tested for their immunogenic potential in a murine model of EHV-1 infection. The mutant viruses were engineered using the virulent EHV-1 strain RacL11 or the modified live vaccine strain RacH by inserting the Escherichia coli LacZ gene into the viral ORFs. RacL11-infected mice showed signs typical of an EHV-1 infection, whereas mice infected with the EHV-1 gB- or gM-negative mutants or with RacH did not develop disease. No difference in the pathogenic potential of RacL11 gB- and gM-negative viruses was observed after application of either phenotypically completed or negative viruses. However, revertant RacL11 viruses in which the gB or gM gene had been restored caused EHV-1-related symptoms that were indistinguishable from those induced by RacL11. Mice that had been immunized with phenotypically negative gB- and gM-deficient EHV-1 were challenged with the RacL11 virus 25 days after immunization. Mock-immunized mice developed EHV-1 disease and high virus loads in their lungs were observed. In contrast, mice developed not exhibit EHV-1-caused disease. It was concluded (i) that deletion of either gB or gM abolished the virulence of strain RacL11 and (ii) that immunization with gB- or gM-negative EHV-1 elicited a protective immunity that was reflected by both virus-neutralizing antibodies and EHV-1-specific T-cells in spleens of immunized mice.

Noninfectious Pr55gag virus-like particles containing high quantities of oligomeric human immunodeficiency virus type 1 (HIV-1) envelope (Env) proteins represent potential candidate immunogens for a vaccine against HIV-1 infection. Thus, chimeric env genes were constructed encoding the HIV-1 exterior glycoprotein gp120 which was covalently linked at different C-terminal positions to a transmembrane domain (TM) from the Epstein-Barr virus (EBV) major Env glycoprotein gp220/ 350. All chimeric Env-TM polypeptides as well as the wild-type HIV Env proteins were equally produced and incorporated at the outer surface of insect cells using the baculovirus expression system. In the presence of coexpressed HIV Pr55gag polyproteins significantly decreased amounts of wild-type Env proteins were presented at the cell surface, whereas the membrane incorporation of the Env-TM chimeras was not affected. Biochemical and immunoelectron microscopical analysis of particles that were efficiently released from these cells displayed the incorporation of both wild-type Env and chimeric Env-TM proteins on the surface of VLPs. However, the quantities of particle-associated chimeric Env-TM proteins exceeded those of incorporated wild-type Env proteins by a factor of 5-10. Chemical cross-linking and subsequent polyacrylamide gel electrophoresis of VLP-entrapped Env proteins revealed that the chimeric Env-TM proteins form homodimers and a higher-order oligomer, similar to that observed for wild-type Env proteins. Thus, the results of this study clearly demonstrate that the replacement of the gp41 transmembrane protein of gp160 by a heterologous, EBV gp220/350-derived membrane anchor provides an effective strategy to incorporate high quantities of oligomeric HIV gp120 proteins on the surface of Pr55gag virus-like particles.

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.

Experiments to analyze the functions of the equine herpesvirus 1 (EHV-1) glycoprotein gB were performed. Cell lines which stably expressed either the full-length EHV-1 gB or only the extracellular portion of gB (amino acids 1 to 844) were constructed and were termed TCgBf and TCgB delta, respectively. Using the cell line TCgBf, a gB-negative viral mutant, L11delta gB, was generated by replacing a 2.1-kb BglII-NruI fragment in the EHV-1 strain RacL11 gB with the Escherichia coli LacZ gene. EHV-1 strain RacL11, the modified live vaccine strain RacH, and L11delta gB were used for functional studies. It was shown that: (i) EHV-1 gB is essential for virus growth in vitro since gB-negative L11delta gB exhibited titers of <10 PFU/ml when grown and titrated on noncomplementing cells. (ii) The cell line expressing truncated gB (TCgB delta) did not complement for the growth of L11delta gB, but the RacH virus grew to titers comparable to those of RacL11 in all cell lines tested. Since RacH had amino acids 944-980 of gB replaced by 7 missense amino acids as determined by nucleotide sequence analysis, the extreme carboxyterminus but not a domain between amino acid residues 845 and 943, probably the transmembrane domain, of EHV-1 gB is dispensable for virus growth in cultured cells. (iii) Single infected cells but no plaque formation were observed after infection of noncomplementing cells with L11delta gB, demonstrating the requirement of EHV-1 gB for direct cell-to-cell spread of infection. (iv) The attachment of gB-negative L11delta gB virions to target cells was similar to both phenotypically complemented L11delta gB and parent RacL11 virus. (v) L11delta gB viral titers could be enhanced by using the fusogen polyethylene glycol (PEG). The increase of L11delta gB titers by PEG treatment, however, was considerably lower compared to gB-negative pseudorabies virus, suggesting that EHV-1 gB might not be as stringently required for virus penetration as are its homologs in other Alphaherpesvirinae.

The diploid IR6 gene (ORF 67) of equine herpesvirus type 1 (EHV-1) is absent in the modified live EHV-1 vaccine strain RacH and is present in a mutated form in the avirulent EHV-1 strains RacM24 and RacM36, such that the IR6 protein fails to form the typical rod-like structures observed for wild-type EHV-1 RacL11. To assess the role of the IR6 protein in EHV-1 replication and virulence, two recombinant RacH viruses, HIR6-1 and HIR6-2, that harbor a single copy of the wild-type IR6 gene were engineered and characterized. It was shown that: (i) HIR6-1 or HIR6-2 virus encoded for an IR6 protein that was capable of forming the rod-like structures typical of cells infected with the wild-type virulent virus strain RacL11. (ii) Whereas the avirulent EHV-1 strains RacH and RacM36 are temperature-sensitive (in that virus replication at 40 degrees versus that at 37 degrees was reduced by as much as 7,500-fold), the HIR6-1 and HIR6-2 viruses, like RacL11 virus, were capable of significant replication at the elevated temperature. (iii) Electron microscopic analyses revealed that cells infected with the HIR6-1 or HIR6-2 virus, like those infected with virulent RacL11 virus, produced and released comparable numbers of virus particles at both 37 and 40 degrees. In cells infected with the RacH virus at 40 degrees, however, release of extracellular particles was inhibited by greater than 90% and relatively few of the particles were enveloped. (iv) Infections of BALB/cA mice revealed that both the HIR6-1 and HIR6-2 viruses, unlike the parent RacH virus, were as virulent as the wild-type RacL11 strain as judged by the criteria of body weight loss, development of clinical signs of EHV-1 infection, virus titers in the lung, and ability to cause viremia. These findings and those of our recent studies indicate that the IR6 protein is a major determinant of EHV-1 virulence and that the IR6 protein may play a role in virus maturation and/or egress.

The IR6 protein of different plaque isolates from three passages of the equine herpesvirus 1 strain Rac was investigated. Southern blot and DNA sequence analyses revealed that plaque isolates from the 12th passage (RacL11 and RacL22) retained both copies of the IR6 gene, whereas two different genotypes were observed by the 185th passage: RacM24 still harbored both copies of the IR6 gene, whereas RacM36 deleted one of the two copies. In the 256th passage (RacH), both copies of the IR6 gene were absent. As compared to the wild-type IR6 protein, both the RacM24 and RacM36 IR6 protein displayed amino acid exchanges at positions 34, 42, 110, and 134 of the 272 amino acid polypeptide. It is shown that (i) the IR6 protein is nonessential for virus growth in vitro. (ii) In RacL11-infected equine and rodent cells, the typical rod-like appearance of the IR6 protein could be detected from 6 hr p.i., whereas in RacM24- and RacM36-infected cells formation of these structures was not observed. (iii) The RacL11 IR6 gene product was present in both the nuclei and cytoplasmic fraction of infected cells. In contrast, the IR6 protein of both RacM24 and RacM36 colocalized with cytoplasmic membrane vesicles. (iv) The RacL11 and RacL22 IR6 protein is present in viral nucleocapsids, whereas that of RacM24 and RacM36 is not incorporated into virions. (v) The RacL11 IR6 gene product aggregated to disulfide-linked oligomers, whereas the RacM24 and RacM36 IR6 protein showed only marginal oligomerization. (vi) In COS7 cells transfected with constructs expressing either the full-length RacL11-IR6 protein or a truncated form lacking the 81 carboxyterminal amino acids, the formation of rod-like structures was observed, indicating that another viral protein is not necessary for aggregation of the IR6 protein. In contrast, the IR6 protein expressed from constructs derived from either RacM24 or RacM36 failed to form these structures. (vii) Analyses of chimeric RacL11-RacM24 IR6 proteins suggest a crucial role for amino acid Leu-134 in the ability of the IR6 protein to form the rod-like structures.

Four formulations of the equine herpesvirus type 1 (EHV-1) glycoprotein gp 14 (gB), were tested for their ability to protect mice against intranasal (inas) EHV-1 challenge infection. The preparations tested included (i) a truncated gp14 produced in Escherichia coli or (ii) a truncated gp14 expressed in insect cells by a recombinant baculovirus, (iii) truncated gp14 coexpressed with human immunodeficiency virus type 1 (HIV-1) gag virus-like particles (VLP) in insect cells, and (iv) a gp14-DNA vaccine under the control of the cytomegalovirus immediate early promoter. All antigen preparations and the DNA vaccine elicited a humoral and delayed-type hypersensitivity (DTH) immune response to EHV-1 after intramuscular (im) immunization. After inas immunization, only the VLP-gp14 preparation produced both a good humoral and a prominent DTH immune response; gp14 expressed by insect cells elicited high titers of EHV-1-specific antibodies, whereas gp14 produced in E. coli and the DNA vaccine elicited only low antibody titers. Glycoprotein gp14 expressed by bacteria, however, induced a strong DTH reaction after inas application. Mice were completely protected against EHV-1 challenge infection after both the im and the inas immunization with the VLP-gp14 preparation. Protection was less efficient after immunization with the E. coli and insect cell gp14s as well as after DNA vaccination. Although the transmembrane domain of EHV-1 gp14 was deleted, recombinant gp14 could be demonstrated in insect cell membranes at late times postinfection and aggregated with the VLPs. It is suggested that the transmembrane domain is not required for gp14-association with membranes in that system.

A 14-kDa gene-specific probe of vaccinia virus Western Reserve (WR) hybridized to homologous sequences in the genomes of the orthopox virus species cowpox, camelpox, mousepox, and monkeypox virus. The corresponding genes were mapped and sequenced. Homologies of more than 95% were found when compared to the 14-kDa gene of vaccinia virus WR. However, point mutations which led to alterations in the amino acid sequences were mainly located between residues 26 and 40. By use of synthetic peptides, this part of the 14-kDa fusion protein could be identified as the binding site for four different neutralizing monoclonal antibodies.