Crystal structure of the oligomerization domain of NSP4 from rotavirus reveals a core metal-binding site.

During the maturation of rotaviral particles, non-structural protein 4 (NSP4) plays a critical role in the translocation of the immature capsid into the lumen of the endoplasmic reticulum. Full-length NSP4 and a 22 amino acid peptide (NSP4(114-135)) derived from this protein have been shown to induce diarrhea in young mice in an age-dependent manner, and may therefore be the agent responsible for rotavirally-induced symptoms. We have determined the crystal structure of the oligomerization domain of NSP4 which spans residues 95 to 137 (NSP4(95-137)). NSP4(95-137) self-associates into a parallel, tetrameric coiled-coil, with the hydrophobic core interrupted by three polar layers occupying a and d-heptad positions. Side-chains from two consecutive polar layers, consisting of four Gln123 and two of the four Glu120 residues, coordinate a divalent cation. Two independent structures built from MAD-phased data indicated the presence of a strontium and calcium ion bound at this site, respectively. This metal-binding site appears to play an important role in stabilizing the homo-tetramer, which has implications for the engagement of NSP4 as an enterotoxin.

A subunit gIV vaccine, produced by transfected mammalian cells in culture, induces mucosal immunity against bovine herpesvirus-1 in cattle.

A truncated version of bovine herpesvirus-1 (BHV-1) glycoprotein IV (tgIV) was produced in a novel, non-destructive expression system based upon regulation of gene expression by the bovine heat-shock protein 70A (hsp70) gene promoter in Madin Darby bovine kidney (MDBK) cells. In this system, up to 20 micrograms ml-1 of secreted tgIV, which is equivalent to the yield from 4 x 10(6) cells, was produced daily over a period of up to 18 days. Different doses of tgIV were injected intramuscularly into seronegative calves. Virus-neutralizing antibodies were induced by all doses of tgIV, both in the serum and in the nasal superficial mucosa. However, the low dose (2.3 micrograms) induced significantly (p < 0.05) lower antibody titres than the medium (7 micrograms) and high (21 micrograms) doses. The medium and high doses of tgIV conferred protection from BHV-1 infection, as demonstrated by a significant (p < 0.05) reduction in clinical signs of respiratory disease and virus shedding in the nasal secretions postchallenge. However, the 2.3 micrograms group, although partially protected, was not significantly (p > 0.05) different from the placebo group. This study demonstrated the potential of an intramuscularly administered tgIV subunit vaccine to induce mucosal immunity to BHV-1 using an economic protein production system and an acceptable vaccine formulation. In addition, a strong correlation was observed between neutralizing antibodies in the serum and nasal superficial mucosa, virus shedding and clinical disease. Thus, serum neutralizing antibody levels in tgIV-immunized animals may be a good prognosticator of protection from BHV-1 infection and disease.

Identification of a putative cellular receptor for bovine herpesvirus 1.

Recognition of a host cell receptor by a virus is the first and perhaps the most crucial step in initiating the disease process. This study was undertaken to identify the cellular receptor(s) for bovine herpesvirus 1 (BHV-1). Previously, we reported the development and characterization of bovine anti-idiotypic antibodies (anti-ids) that induce neutralizing antibodies to BHV-1. These anti-ids inhibit BHV-1 penetration of permissive cells. We have used these anti-ids, which mimic an epitope on the virus glycoprotein IV (gIV), and gradient-purified virus in immunoprecipitation (IP) as well as photoaffinity labelling (PAL) assays. In the IP assays, both bovine anti-ids and BHV-1 virions coupled to Sepharose precipitated a 60K protein from 125I-labelled BHV-1 permissive cell membrane extracts. Normal bovine IgG or an irrelevant virus, transmissible gastroenteritis virus (TGEV), used as negative controls failed to precipitate this protein. Similarly, in the PAL assays, the 60K cell surface protein was identified on cells permissive for BHV-1 infection, but not on non-permissive cells when 125I-labelled ligands, the anti-ids or BHV-1 were used as probes. The iodinated ligands failed to identify the 60K protein if they had been pretreated with the antibody 1. Pretreatment of the iodinated ligands with an isotype-matched control antibody had no effect on the identification of the 60K protein present on cells permissive for BHV-1 infection. The negative controls, i.e. normal bovine IgG and TGEV, failed to identify this 60K protein on permissive or non-permissive cells. These results suggest that the 60K protein is a cellular receptor recognized by BHV-1 during the infection process.

Bovine herpesvirus 1: immune responses in mice and cattle injected with plasmid DNA.

Mice and cattle injected with plasmids encoding bovine herpesvirus 1 (BHV-1) glycoproteins developed gene-specific antibody responses capable of neutralizing BHV-1. The ability of animals to respond serologically to DNA injections was in part dependent on the quantity of DNA injected and was also negatively affected by carrier DNA. Calves injected with a plasmid encoding BHV-1 gIV developed significant antibody titers to gIV and shed less virus than did the control calf after challenge. This report indicates the potential of DNA injection as a method of vaccination.

Expression of glycoprotein gIII-human decay-accelerating factor chimera on the bovine herpesvirus 1 virion via a glycosyl phosphatidylinositol-based membrane anchor.

Mutants of bovine herpesvirus 1 that express a truncated envelope glycoprotein gIII or a gIII-human decay-accelerating factor (hDAF) chimeric protein (gIII.hDAF) were employed to evaluate the function of the transmembrane and cytoplasmic domains of the gIII molecule. Truncated gIII (i.e., lacking the transmembrane and cytoplasmic region) was readily released from infected cells and was not detected on mature virus particles. In contrast, replacement of the transmembrane and cytoplasmic domains with the carboxyl-terminal portion of hDAF restored the expression of gIII on the membranes of infected cells as well as on virion surfaces. The presence of the gIII.hDAF chimera on virus particles was also associated with normal gIII function, i.e., the mediation of virus attachment and penetration. The gIII-hDAF chimera, which is present on both infected cell surfaces and virions, could be cleaved by a phosphatidylinositol-specific phospholipase C, indicating that it was anchored in the membrane via glycosyl phosphatidylinositol. Our results from this study suggest that the transmembrane and cytoplasmic regions of the gIII molecule serve as a general membrane anchor, but they do not contain structural signals required for the specific assembly of envelope proteins into mature virions.

Identification and deletion mutagenesis of the bovine herpesvirus 1 dUTPase gene and a gene homologous to herpes simplex virus UL49.5.

The gene encoding bovine herpesvirus 1 (BHV 1) deoxyuridine triphosphatase (dUTPase) was isolated by a PCR procedure using degenerate oligonucleotide primers whose sequences were based upon conserved motifs commonly present in dUTPase genes. This gene was found to reside between 0.059 and 0.066 map units in the BHV 1 Cooper strain. DNA sequence analysis of this region revealed an open reading frame of 975 base pairs capable of encoding 325 amino acids. The deduced amino acid sequence of the open reading frame exhibits significant homology with dUTPases of other herpesviruses (including human herpes simplex virus, varicella-zoster virus, and Epstein-Barr virus), and it contains five conserved amino acid motifs characteristics of all dUTPases identified to date. A mutant virus carrying a partial deletion of the putative dUTPase gene was made and was found to lack virus-encoded dUTPase activity. This further confirmed that we have identified the BHV 1 dUTPase gene. In addition, a further analysis of the genomic fragment which contains the dUTPase coding sequence revealed an additional 288-base-pair open reading frame which appears to be colinear with the HSV 1 UL49.5 gene. The deduced amino acid sequence of this open reading frame is significantly homologous to the HSV 1 UL49.5 gene product, and as with UL49.5, it contains a potential signal sequence and transmembrane domain characteristic of membrane-associated proteins. These results suggest that this open reading frame represents the BHV 1 homolog of the HSV 1 UL49.5 gene. Since our dUTPase negative mutant was fully viable and since the mutant was constructed such that the UL49.5 gene was also deleted, both the dUTPase and the UL49.5 gene homolog are not required for virus growth in cell culture.

Mapping of heparin-binding structures on bovine herpesvirus 1 and pseudorabies virus gIII glycoproteins.

The gIII glycoproteins of both bovine herpesvirus 1 (BHV 1) and pseudorabies virus (PrV) mediate the initial and dominant interactions between virus and permissive host cells. By studying virus binding to wild-type and heparin-deficient CHO cells, we demonstrated that the cellular heparin-like moieties play an essential role in BHV 1 and PrV gIII-mediated virus attachment. Subsequent studies were carried out to map the gIII structures that are responsible for heparin binding. First, based on the observation that BHV 1 and PrV are differentially sensitive to heparin inhibition of gIII-mediated attachment to cells, we conducted a gIII domain shuffling experiment. This involved the construction of a set of recombinant BHV 1 expressing BHV 1 and PrV gIII chimeras and then using the sensitivity to heparin inhibition as a means of mapping the potential heparin-binding regions on the gIII molecules. Next, we synthesized panels of partially overlapping BHV 1 and PrV gIII peptides and examined their reactivity to heparin. The results from these experiments demonstrated five heparin-binding sites between amino acid 129 and 310 of BHV 1 gIII and four heparin-binding sites between amino acid 90 and 275 of PrV gIII.

Analysis of bovine herpesvirus 1 glycoprotein gIV truncations and deletions expressed by recombinant vaccinia viruses.

Glycoprotein gIV is an envelope component of bovine herpesvirus type 1 and appears to be involved in attachment, penetration, and cell fusion. Four antigenic domains which include both continuous and discontinuous epitopes have been previously defined by competition binding assays using gIV-specific monoclonal antibodies (MAbs). Here we describe the construction of C-terminal truncations and internal deletions in the gIV-encoding gene and analyses of the effects of these mutations on the synthesis, processing, transport, and antigenicity of glycoprotein gIV as expressed by recombinant vaccinia viruses. Wild-type gIV expressed by recombinant vaccinia virus STgIV was indistinguishable from authentic gIV produced in bovine herpesvirus 1-infected cells with respect to molecular weight, processing, transport, and antigenicity. Analysis of the mutant proteins showed that the binding sites for MAbs 9D6 and 3D9S, which recognize linear epitopes, lie between amino acids 164 and 216 and amino acids 320 and 355, respectively. Discontinuous epitopes recognized by MAbs 3E7, 4C1, 2C8, and 3C1 were located between amino acids 19 and 320, whereas amino acids 320 to 355 were critical for binding of MAb 136. All mutant proteins containing amino acids 245 to 320 were processed, possess endo-beta-N-acetylglucosaminidase H-resistant oligosaccharides, and were transported to the cell surface or secreted into the medium. In contrast, mutant proteins missing amino acids 245 to 320 were retained in the rough endoplasmic reticulum. These findings suggest that residues 245 to 320 are important for proper processing and transport of gIV to the cell surface.

Role of N-linked glycans in antigenicity, processing, and cell surface expression of bovine herpesvirus 1 glycoprotein gIV.

Glycoprotein gIV, a structural component of bovine herpesvirus type 1, stimulates high titers of virus-neutralizing antibody. The protein contains three potential sites for the addition of N-linked carbohydrates. Three mutants were constructed by oligonucleotide-directed mutagenesis, in each case changing one N-linked glycosylation site from Asn-X-Thr/Ser to Ser-X-Thr/Ser. A fourth mutant was altered at two sites. The altered forms of the gIV gene were cloned into a vaccinia virus transfer vector to generate recombinant vaccinia viruses expressing mutant proteins. Analysis of these mutants revealed that only two (residues 41 and 102) of the three (residues 41, 102, and 411) potential sites for the addition of N-linked glycans are actually utilized. Absence of glycans at residue 41 (gN1) showed no significant effect on the conformation of the protein or induction of a serum neutralizing antibody response. However, mutant proteins lacking glycans at residue 102 (gN2) or residues 41 and 102 (gN1N2) showed altered reactivity with conformation-dependent gIV-specific monoclonal antibodies. These mutants also induced significantly lower serum neutralizing antibody responses than wild-type gIV. Nonetheless, each of the mutant proteins were modified by the addition of O-glycans and transported to the cell surface. Our results demonstrate that absence of N-linked glycans at one (residue 102) or both (residues 41 and 102) utilized N-linked glycosylation sites alters the conformation but does not prevent processing and transport of gIV to the cell surface.

Heat-shock promoter-driven synthesis of secreted bovine herpesvirus glycoproteins in transfected cells.

The bovine hsp70A heat-shock gene promoter was isolated and used to direct the heat-regulated synthesis of bovine herpesvirus glycoproteins gIII and gIV in transfected cultured bovine cells. Sequences encoding the viral glycoproteins incorporated mutations that deleted the transmembrane anchors. Both proteins were efficiently secreted from transfected cells in a temperature-dependent manner and the gIV so produced was found to be antigenically similar to the authentic molecule. Stable cell lines with regulated expression of these proteins were obtained and repeated thermal cycling of the cultures enabled high-yield production of these subunit vaccine antigens. The continuous production demonstrated by this system is highly relevant to the efficient and economic manufacture of vaccines and other protein biopharmaceuticals.

Protection of cattle from BHV-1 infection by immunization with recombinant glycoprotein gIV.

High levels of recombinant bovine herpesvirus-1 (BHV-1) glycoprotein IV were produced in baculovirus, adenovirus, vaccinia virus and Escherichia coli expression systems. The different recombinant forms as well as authentic gIV were injected intramuscularly into seronegative calves. With the exception of E. coli-produced gIV, all forms of gIV induced high levels of neutralizing antibodies both in the serum and in the nasal superficial mucosa. Animals immunized with gIV produced in insect or mammalian cells were completely protected from infection with BHV-1, as demonstrated by the absence of temperature responses, clinical signs or detectable virus in the nasal secretions after challenge exposure. The E. coli-derived gIV induced partial protection from clinical disease, even though it was not glycosylated and did not induce appreciable levels of neutralizing antibodies. This study demonstrated that all forms of glycosylated gIV, whether authentic or recombinant, confer protection from BHV-1 infection and thus may be useful as an effective subunit vaccine.

The effect of subunit or modified live bovine herpesvirus-1 vaccines on the efficacy of a recombinant Pasteurella haemolytica vaccine for the prevention of respiratory disease in feedlot calves.

The efficacy of a Pasteurella haemolytica vaccine (PhV) administered once to calves within 24 hours of arrival at a feedlot was tested for the ability to prevent morbidity and mortality from all bovine respiratory disease (BRD) and specifically from fibrinous pneumonia mortality. The PhV consisted of two immunizing ingredients: outer membrane proteins extracted from P. haemolytica, plus genetically attenuated leukotoxin produced by recombinant DNA technology. This double blind study was conducted at a large Saskatchewan feedlot using 2,324 high-risk calves purchased at auction markets and kept under typical commercial feedlot conditions. The trial design included four vaccine test groups: 1) PhV and a bovine herpesvirus type-1 (BHV-1) subunit vaccine comprised only of the virus glycoprotein IV (gIV); 2) PhV and a commercial modified live vaccine (MLV) containing BHV-1 and parainfluenza-3 viruses; 3) gIV alone; and 4) MLV alone. Calves were assigned to vaccine groups in a random systematic manner, individually identified, and monitored for 90 days after vaccination. The vaccines were given once, on arrival, to reflect common feedlot practice, although vaccination prior to expected risk would be more appropriate.The PhV in combination with gIV reduced BRD morbidity by 20% (p < 0.05) compared to gIV alone and 24% (p < 0.05) compared to MLV alone, and reduced BRD mortality by 88% (p < 0.05) and fibrinous pneumonia mortality by 100% (p < 0.05) when compared to either gIV or MLV alone. Vaccination with PhV in combination with MLV significantly reduced the efficacy of the PhV in preventing BRD morbidity, BRD mortality, and fibrinous pneumonia mortality and also reduced the antibody response to P. haemolytica leukotoxin. These results suggest that the MLV interfered with the protective capacity of the PhV.

An in vivo study of a glycoprotein gIII-negative bovine herpesvirus 1 (BHV-1) mutant expressing beta-galactosidase: evaluation of the role of gIII in virus infectivity and its use as a vector for mucosal immunization.

We constructed a recombinant BHV-1 in which the glycoprotein gIII gene was replaced by the Escherichia coli lacZ gene. The resultant virus mimics the simple gIII deletion mutant in its growth characteristics in cell culture; however, it expresses beta-galactosidase in virus-infected cells. Further characterization of its virulence and the immune responses elicited by it was conducted in cattle. The mutant virus retained the ability to establish an infection when administered intranasally. Infected animals were also capable of transmitting virus to sentinel penmates. However, the mutant virus showed a reduced replication efficiency in the respiratory tract of cattle, as manifested by significantly lower virus shedding and a shorter duration of shedding when compared to wild-type (wt) BHV-1 infections. The mutant virus induced an efficient anti-BHV-1 antibody response and convalescent cattle were fully protected from subsequent wt virus challenge. In addition, cattle infected with the lacZ-expressing virus developed antibodies to beta-galactosidase. Our results demonstrate that the presence of gIII is not a prerequisite for BHV-1 infection; however, gIII does play an important role in maintaining virus replication efficacy in its natural host. With respect to developing BHV-1 as a vaccine vector, our results indicate that deletion of the gIII gene, which partially attenuates the virus and serves as a vaccine virus marker, does not compromise immunogenicity to BHV-1. Most importantly, this vector is effective in delivering foreign antigens to mucosal surfaces of the respiratory tract.

Relationship of bovine herpesvirus 1 immediate-early, early, and late gene expression to host cellular gene transcription.

Bovine herpesvirus 1 (BHV-1) gene expression was examined by RNA blot hybridization using clones representing immediate-early, early, and late genes. An immediate-early protein gene probe hybridized with two transcripts, 3.4 and 5.8 kb, expressed by infected cells in the presence of cycloheximide (CH). During infection of cells without metabolic inhibitors these transcripts were detected as early as 2 hr postinfection (p.i.) and accumulated to 8 hr p.i. The early gene probe, thymidine kinase, hybridized with a 4.3-kb RNA that was detected in the presence of phosphonoacetic acid (PAA), but not in the presence of CH. The late gene probe, glycoprotein III, (gIII) hybridized with a 1.6-kb transcript that was not expressed by infected cells treated with CH and only in very reduced amounts by infected cells treated with PAA. The gIII RNA was not detected until 4 hr p.i. in total cell RNA. Transcripts for the bovine actin and beta-galactosyltransferase genes did not decrease in BHV-1-infected cells until 6 hr p.i., coincident with the increase of BHV-1 DNA and RNA synthesis. Consequently, shutoff of host cell transcription by BHV-1 may be different than what has been described for herpes simplex virus.

Pseudorabies virus gIII and bovine herpesvirus 1 gIII share complementary functions.

The gIII glycoproteins of bovine herpesvirus 1 (BHV-1) and of pseudorabies virus (PRV) are structurally homologous. Both proteins also play preeminent roles in mediating virus attachment to permissive cells. To directly compare the functional relation between these glycoproteins, we constructed a recombinant BHV-1 in which the BHV-1 gIII coding sequence was replaced by the PRV gene homolog. The resultant recombinant virus efficiently expressed PRV gIII and then incorporated it into its envelope. The levels of PRV gIII expression and incorporation were equivalent to those achieved by the wild-type virus for BHV-1 gIII. The recombinant virus was fully susceptible to neutralization by anti-PRV gIII neutralizing antibody. In addition, the virus attachment and penetration functions, as well as the virus replication efficiency, which were lost by deleting the BHV-1 gIII gene, were restored by expressing the PRV gIII homolog in its place. These results demonstrated that PRV gIII and BHV-1 gIII share complementary functions.

Bovine monoclonal anti-idiotypes induce antibodies specific for a synthetic peptide bearing a neutralizing epitope of bovine herpesvirus 1 glycoprotein gI (gB).

Bovine monoclonal anti-Id mimicking a neutralizing epitope of bovine herpesvirus-1 (BHV-1) glycoprotein gI were developed. An epitope present on the 74K subunit of gI identified by a murine mAb 1E11 was selected for this study. Bovine lymphocytes from the prefemoral lymph node of a heifer immunized with mAb 1E11 were fused with SP-2/0, a nonsecreting murine cell-line. Two bovine x murine hybridomas secreting bovine monoclonal anti-Id specific for the Id of 1E11 were stabilized. These anti-Id inhibited the binding of 1E11 to purified glycoprotein gI in a dose-dependent fashion. Naive mice immunized with the anti-Id produced anti-anti-Id (Ab3) that reacted with BHV-1 glycoprotein gI in a RIA, and neutralized BHV-1 infection in vitro. The Ab3 also showed reactivity to the 74K subunit of authentic gI glycoprotein in a Western blot analysis, and to the synthetic peptide bearing the 1E11 epitope in a RIA. These results substantiate the presence of the population of anti-Ab2 that functionally resemble antibodies specific for the immunizing Ag BHV-1 in Ab3, and demonstrate the ability of these anti-Id to elicit BHV-1-specific antibody response.

Bovine herpesvirus 1 attachment to permissive cells is mediated by its major glycoproteins gI, gIII, and gIV.

A bovine herpesvirus 1 (BHV-1) gIII deletion mutant (gIII-) was produced by means of recombinant DNA that retained the ability to replicate in cell culture. However, the gIII- mutant was functionally defective, showing impaired attachment to permissive cells, a delay in virus replication, and reduced extracellular virus production. The attachment defect exhibited by the gIII- mutant is an indication of the role played by gIII in the normal infection process. This was shown by dramatically decreased binding of radiolabelled gIII- virus to permissive cells and a slower adsorption rate, as measured by plaque formation, than the wild-type (wt) virus. Furthermore, treatment of the gIII- virus with neomycin increased virus adsorption and plaque formation by severalfold, whereas neomycin treatment had no effect on the wt virus. This observation showed that the gIII- mutant was strictly defective in adsorption but fully competent to produce productive infections once induced to attach. The gIII- mutant showed greater sensitivities than did the wt virus to anti-gI and anti-gIV antibody-mediated neutralization. Analyses with panels of monoclonal antibodies to gI and gIV revealed that the epitopes gI-IV and gIV-III were the main targets for enhanced neutralization. This provided evidence that gI and gIV may also participate in virus attachment. Finally, when affinity-purified gI, gIII, and gIV were tested for their ability to inhibit virus adsorption, gIII had the most pronounced inhibitory effect, followed by gI and then gIV. gIII was able to completely inhibit wt virus adsorption, and at a high concentration, it also partially inhibited the gIII- mutant. gI and gIV inhibited wt and gIII- mutant adsorption to a comparable extent. Our results collectively indicate that gIII plays a predominant role in virus attachment, but gI and gIV also contribute to this process. In addition, a potential cooperative mechanism for virus attachment with these three proteins is presented.

Expression of bovine herpesvirus 1 glycoprotein gIV by recombinant baculovirus and analysis of its immunogenic properties.

The gene encoding the gIV glycoprotein of bovine herpesvirus 1 has been inserted into the genome of Autographa californica baculovirus in lieu of the coding region of the A. californica baculovirus polyhedrin gene. Recombinant protein was identified by its reactivity with gIV-specific monoclonal antibodies and expressed at high levels (about 85 micrograms per 2.5 x 10(6) cells) in Spodoptera frugiperda (SF9) cells. The recombinant glycoprotein had an apparent molecular mass of 63 kDa, indicating that it was incompletely glycosylated. However, it was transported to and expressed on the cell surface of infected SF9 cells. Furthermore, reactivity with polyclonal and monoclonal antibodies specific for gIV suggested that most epitopes were functionally unaltered on the recombinant gIV. Immunization of cattle with recombinant gIV in crude, partially purified, or pure form resulted in the induction of neutralizing antibodies to BHV-1, which were reactive with authentic gIV. However, the neutralizing antibody titers were lower than those elicited by an equivalent amount of affinity-purified authentic gIV, which appeared to be mainly due to reduced recognition of one of the neutralizing antigenic domains of gIV, designated domain I. The potential use of this recombinant gIV glycoprotein as a vaccine to bovine herpesvirus 1 infection in cattle is discussed.

Molecular cloning, sequencing, and expression of functional bovine herpesvirus 1 glycoprotein gIV in transfected bovine cells.

The gene encoding bovine herpesvirus 1 (BHV-1) glycoprotein gIV was mapped, cloned, and sequenced. The gene is situated between map units 0.892 and 0.902 and encodes a predicted protein of 417 amino acids with a signal sequence cleavage site between amino acids 18 and 19. Comparison of the BHV-1 amino acid sequence with the homologous glycoproteins of other alphaherpesviruses, including herpes simplex virus type 1 glycoprotein gD, revealed significant homology in the amino-terminal half of the molecules, including six invariant cysteine residues. The identity of the open reading frame was verified by expression of the authentic recombinant BHV-1 gIV in bovine cells by using eucaryotic expression vectors pRSDneo (strong, constitutive promoter) and pMSG (weak, dexamethasone-inducible promoter). Constitutive expression of gIV proved toxic to cells, since stable cell lines could only be established when the gIV gene was placed under the control of an inducible promoter. Expression of gIV was cell associated and localized predominantly in the perinuclear region, although nuclear and plasma membrane staining was also observed. Radioimmunoprecipitation revealed that the recombinant glycoprotein was efficiently processed and had a molecular weight similar to that of the native form of gIV expressed in BHV-1-infected bovine cells. Recombinant gIV produced in the transfected bovine cells induced cell fusion, polykaryon formation, and nuclear fusion. In addition, expression of gIV interfered with BHV-1 replication in the transfected bovine cells.

Molecular mimicry: a herpes virus glycoprotein antigenically related to a cell-surface glycoprotein expressed by macrophages, polymorphonuclear leucocytes, and platelets.

Bovine herpes virus type 1 (BHV-1) gIII is a major virion glycoprotein with homology to the immunoglobulin superfamily. We have investigated the possibility that gIII is related to host molecules and have identified a gIII-specific monoclonal antibody (mAb) that cross-reacts with normal bovine cells. The cross-reactive entity was expressed mainly on monocyte/macrophages (M phi), polymorphonuclear leucocytes (PMN) and platelets, and was identified as a 43,000-63,000 molecular weight (MW) cell-surface glycoprotein. For M phi, the glycoprotein appears to be a general lineage marker, rather than a maturation or activation marker, and may be a functional receptor, as evidenced by its endocytosis via coated pits and its involvement in proliferation of mononuclear cells in vitro. This novel leucocyte marker was also detected on subsets of human, ovine and canine M phi. Competitive binding assays with sera from cattle immunized with BHV-1 or gIII revealed apparent low responsiveness to the cross-reactive epitope. The results suggest that BHV-1 gIII is antigenically related to a novel host leucocyte receptor and that evasion and/or interference with leucocyte function may be a consequence of this molecular mimicry relationship.