Synthesis, cellular location, and immunogenicity of bovine herpesvirus 1 glycoproteins gI and gIII expressed by recombinant vaccinia virus.

Two of the major glycoproteins of bovine herpesvirus 1 (BHV-1) are gI, a polypeptide complex with apparent molecular weights of 130,000, 74,000, and 55,000, and gIII (a 91,000-molecular-weight [91K] glycoprotein), which also exists as a 180K dimer. Vaccinia virus (VAC) recombinants were constructed which carry full-length gI (VAC-I) or gIII (VAC-III) genes. The genes for gI and gIII were each placed under the control of the early VAC 7.5K gene promoter and inserted within the VAC gene for thymidine kinase. The recombinant viruses VAC-I and VAC-III retained infectivity and expressed both precursor and mature forms of glycoproteins gI and gIII. The polypeptide backbones, partially glycosylated precursors, and mature gI and gIII glycoproteins were indistinguishable from those produced in BHV-1-infected cells. Consequently, they were apparently cleaved, glycosylated, and transported in a manner similar to that seen during authentic BHV-1 infection, although the processing efficiencies of both gI and gIII were generally higher in recombinant-infected cells than in BHV-1-infected cells. Immunofluorescence studies further demonstrated that the mature gI and gIII glycoproteins were transported to and expressed on the surface of cells infected with the respective recombinants. Immunization of cattle with recombinant viruses VAC-I and VAC-III resulted in the induction of neutralizing antibodies to BHV-1, which were reactive with authentic gI and gIII. These data demonstrate the immunogenicity of VAC-expressed gI and gIII and indicate the potential of these recombinant glycoproteins as a vaccine against BHV-1.     

Synthesis, processing, and oligomerization of bovine herpesvirus 1 gE and gI membrane proteins.

This study reports the identification and initial characterization of the precursors, modified forms, and oligomers of bovine herpesvirus 1 (BHV-1) gI and gE proteins with polyvalent rabbit serum specific for gI or gE. Our experiments used the Colorado strain of BHV-1 and mutant viruses with insertions of the Escherichia coli lacZ gene into the predicted gE and gI reading frames. We also translated the gE and gI open reading frames in vitro and expressed them in uninfected cells using eukaryotic expression vectors. Precursor-product relationships were established by pulse-chase analysis and endoglycosidase H and glycopeptidase F digestions. Like the homologous glycoproteins of herpes simplex virus type 1, pseudorabies virus, and varicella-zoster virus, BHV-1 gI and gE are modified by N-linked glycosylation and associate with each other soon after synthesis, forming a noncovalent complex in infected and transfected cells. An analysis of mutant and wild-type-virus-infected cells and transfected COS cells expressing gE or gI alone suggested that gE-gI complex formation is necessary for efficient processing of the gE precursor to its mature form. One new finding was that unlike the other alphaherpesvirus gI homologs, a fraction of pulse-labeled gI synthesized in BHV-1-infected cells apparently is cleaved into two relatively stable fragments 2 to 4 h after the pulse. Finally, we incubated BHV-1-infected cell extracts with nonimmune mouse, rabbit, horse, pig, and calf sera and found no evidence that gE or gI functioned as Fc receptors as reported for the herpes simplex virus type 1 and varicella-zoster virus homologs.     

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.     

Bovine natural killer-like cell responses against cell lines expressing recombinant bovine herpesvirus type 1 glycoproteins

The murine retrovirus shuttle vector pZipNeo SV(X)1 was used to construct plasmids encoding the three major surface glycoproteins of bovine herpesvirus-1 (BHV-1). Each plasmid was transfected into D17, a canine osteosarcoma cell line sensitive to lysis by bovine NK-like cells when infected with BHV-1. After selection in G418 sulfate, cell lines expressing the recombinant gene products were sorted by flow microfluorimetry, radioimmunoprecipitated, and analyzed by SDS-PAGE for fidelity as compared to the native viral glycoproteins. Two of the three genetically engineered cell lines (gI and gIV) could successfully serve as targets to detect bovine NK-like cytolysis. These findings support and extend a previous study from our laboratory indicating a role for BHV-1 glycoproteins in the cytolytic response by bovine NK-like cells. Additionally, this study demonstrated that individual proteins are recognized by these effector cells, and that recombinant glycoproteins can direct cytolytic activity in the absence of host cell infection-associated proteins. This is the first known report of Ag directed cytotoxicity by bovine null (non-B, non-T) cells.     

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.     

Temporal control of bovine herpesvirus 1 glycoprotein synthesis.

The gI, gIII, and gIV glycoproteins are major bovine herpesvirus 1 antigens involved in virus neutralization. Results indicate that the gI and gIV glycoproteins were expressed as beta proteins, whereas the gIII glycoprotein was expressed strictly as a gamma protein. These findings suggest that gI and gIV may be superior to gIII as vaccine candidates.     

Pseudorabies virus recombinants expressing functional virulence determinants gE and gI from bovine herpesvirus 1.1.

In the Alphaherpesvirinae subfamily, the gE and gI genes are conserved and encode membrane glycoproteins required for efficient pathogenesis (virulence). The molecular mechanism(s) responsible is not well understood, but the existence of similar phenotypes of gE and gI mutations in diverse Alphaherpesvirinae implies conservation of function(s). In this report, we describe construction of pseudorabies virus (PRV) recombinants that efficiently express the bovine herpesvirus 1 (BHV-1) membrane proteins gI and gE at the PRV gG locus. Each BHV-1 gene was cloned in a PRV mutant lacking both the PRV gI and gE coding sequences. All recombinant viruses expressed the BHV-1 proteins at levels similar to or greater than that observed after infection with parental BHV-1, and there were no observable differences in processing or ability to form gE-gI oligomers. The important observation resulting from this report is that the BHV-1 gE and gI proteins functioned together to complement the virulence defect of PRV lacking its own gE and gI genes in a rodent model, despite being derived from a highly restricted host range virus with a different pathogenic profile.     

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.     

Stable rescue of a glycoprotein gII deletion mutant of pseudorabies virus by glycoprotein gI of bovine herpesvirus 1.

Glycoproteins homologous to glycoprotein B (gB) of herpes simplex virus constitute the most highly conserved group of herpesvirus glycoproteins. This strong conservation of amino acid sequences might be indicative of a common functional role. Indeed, gB homologs have been implicated in the processes of viral entry and virus-mediated cell-cell fusion. Recently, we showed that pseudorabies virus (PrV) lacking the essential gB-homologous glycoprotein gII could be propagated on a cell line expressing the gB homolog of bovine herpesvirus 1, gI(BHV-1), leading to a phenotypic complementation of the gII defect (I. Rauh, F. Weiland, F. Fehler, G. Keil, and T.C. Mettenleiter, J. Virol. 65:621-631, 1991). However, this pseudotypic virus could still replicate only on complementing cell lines, thereby limiting experimental approaches to analyze the effects of the gB exchange in detail. We describe here the construction and isolation of a PrV recombinant, 9112C2, that lacks gII(PrV) but instead stably carries and expresses the gene encoding gI(BHV-1). The recombinant is able to replicate on noncomplementing cells with growth kinetics and final titers similar to those of its gII-positive wild-type PrV parent. Neutralization tests and immunoprecipitation analyses demonstrated incorporation of gI(BHV-1) into 9112C2 virions with concomitant absence of gII(PrV). Analysis of in vitro host ranges of wild-type PrV, BHV-1, and recombinant 9112C2 showed that in cells of pig, rabbit, canine, monkey, or human origin, the plating efficiency of 9112C2 was similar to that of its PrV parent. Exchange of gII(PrV) for gI(BHV-1) in recombinant 9112C2 or by phenotypic complementation of gII- PrV propagated on gI(BHV-1)-expressing cell lines resulted in penetration kinetics intermediate between those of wild-type PrV and BHV-1. In conclusion, we report the first isolation of a viral recombinant in which a lethal glycoprotein mutation has been rescued by a homologous glycoprotein of a different herpesvirus. Our data show that in gII- PrV, gI(BHV-1) in vitro fully complements the lethal defect associated with lack of gII(PrV). These results conclusively demonstrate that gI(BHV-1) in a PrV background can execute all essential functions normally provided by gII(PrV). They also indicate that the origin of gB-homologous glycoproteins influences the penetration kinetics of herpesviruses.     

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.     

Pseudorabies virus mutants lacking the essential glycoprotein gII can be complemented by glycoprotein gI of bovine herpesvirus 1.

The genome of pseudorabies virus (PrV) encodes at least seven glycoproteins. The glycoprotein complex gII consists of three related polypeptides, two of them derived by proteolytic cleavage from a common precursor and linked via disulfide bonds. It is homologous to herpes simplex virus (HSV) gB and is therefore thought to be essential for PrV replication, as is gB for HSV replication. To isolate PrV mutants deficient in gII expression, we established cell lines that stably carry the PrV gII gene. Line N7, of Vero cell origin, contains the gII gene under its own promoter and expresses gII after transactivation by herpesviral functions after infection. MDBK-derived line MT3 contains the gII gene under control of the mouse metallothionein promoter. However, it has essentially lost inducibility and constitutively produces high amounts of correctly processed glycoprotein gII. We used a beta-galactosidase expression cassette inserted into a partially deleted cloned copy of the gII gene for cotransfection with PrV DNA. gII- PrV mutants were isolated from viral progeny by taking advantage of their blue-plaque phenotype when incubated under an agarose overlay containing a chromogenic substrate. Analysis of these mutants proved that gII is indeed essential for PrV replication, since the gII- mutants grew normally on gII-complementing cells but were unable to produce plaques on noncomplementing cells. Surprisingly the PrV gII- mutants were also able to grow on a cell line constitutively expressing the gB-homologous glycoprotein gI from bovine herpesvirus 1 (BHV-1) to the same extent as on cells expressing PrV gII. gII- PrV propagated on cells expressing BHV-1 gI became susceptible to neutralization by anti-BHV-1 gI monoclonal antibodies. We also found that BHV-1 gI is present in the envelope of purified gII- pseudorabies virions grown on cells expressing BHV-1 gI, as judged by radioimmunoprecipitation and immunoelectron microscopy. These results prove that BHV-1 gI is integrated into the PrV envelope and can functionally replace glycoprotein gII of PrV.     

The gE and gI homologs from two alphaherpesviruses have conserved and divergent neuroinvasive properties.

The membrane glycoproteins gE and gI are encoded by pseudorabies virus (PRV), a neurotropic, broad-host-range alphaherpesvirus of swine. PRV gE and gI are required for anterograde spread to a restricted set of retinorecipient neurons in the brain after infection of the rat retina. A related alphaherpesvirus, encoding gE and gI homologs, is called bovine herpesvirus 1.1 (BHV-1.1). BHV-1.1 is a respiratory pathogen of highly restricted host range and, in contrast to PRV, is unable to propagate in or cause disease in rodents. We have shown previously that the BHV-1.1 gE and gI proteins are capable of complementing the virulence functions of PRV gE and gI in a rodent model (A. C. Knapp and L. W. Enquist, J. Virol. 71:2731-2739, 1997). We examined the ability of the BHV-1.1 gE and gI homologs to direct circuit-specific invasion of the rat central nervous system by PRV. Both complete open reading frames were cloned into a PRV mutant lacking the PRV gE and gI genes. Recombinant viruses were analyzed for the ability to invade the rat brain after infection of the retina. Surprisingly, in a portion of the animals tested, the BHV-1.1 gE and gI proteins functioned autonomously to promote spread of PRV to a subset of retinorecipient regions of the brain. First, the presence of BHV-1.1 gI alone, but not PRV gI alone, promoted viral invasion of the optic tectum. Second, expression of BHV-1.1 gE alone facilitated PRV infection of a subset of neurons in the hippocampus not normally infected by PRV. When both BHV-1.1 proteins were expressed in a coinfection, all retinorecipient regions of the rat brain were infected. Therefore, depending on the viral source, homologs of gE and gI differentially affect spread between synaptically connected neurons in the rat.     

Release of pseudorabies virus from infected cells is controlled by several viral functions and is modulated by cellular components.

The role of the nonessential glycoproteins gI, gp63, and gIII in the release of pseudorabies virus from different cell lines was investigated. We show that these glycoproteins may have a beneficial or deleterious effect on virus release depending on the type of cell in which the virus is grown. Inactivation of the genes encoding either gI, gp63, or gIII has no detectable effect on virus release from rabbit kidney cells. Inactivation of gI or gp63 strongly promotes virus release from chicken embryo fibroblasts, whereas inactivation of gIII reduces virus release from these cells. A defect in both gI and gIII or in both gp63 and gIII diminishes virus release from rabbit kidney cells but improves release from chicken embryo fibroblasts. We demonstrate that all three nonessential glycoproteins contribute to one specific aspect of viral growth, namely, virus release, and that they affect virus release in conjunction with each other. Furthermore, our results show that the manifestation of the role of each of these viral functions in virus growth may differ in different cell types, i.e., that release is affected by these viral functions in conjunction with some unknown cellular function.     

Expression in recombinant vaccinia virus of the equine herpesvirus 1 gene encoding glycoprotein gp13 and protection of immunized animals.

The equine herpesvirus 1 (EHV-1) gene encoding glycoprotein 13 (gp13) was cloned into the hemagglutinin (HA) locus of vaccinia virus (Copenhagen strain). Expression of the gp13 gene was driven by the early/late vaccinia virus H6 promoter. Metabolically radiolabeled polypeptides of approximately 47 and 44 kilodaltons and 90 kilodaltons (glycosylated form) were precipitated with both polyclonal and gp13-specific monoclonal antibodies. Presentation of gp13 on the cytoplasmic membrane of cells infected with the recombinant gp13 vaccinia virus was demonstrated by immunofluorescence of unfixed cells. Inoculation of the recombinant gp13 vaccinia virus into guinea pigs induced neutralizing antibodies to both EHV-1 and vaccinia virus. Hamsters vaccinated with the recombinant gp13 vaccinia virus survived a lethal challenge with the hamster-adapted Kentucky strain of EHV-1. These results indicate that expression in vaccinia virus vectors of EHV-1 gp13, the glycoprotein homolog of herpes simplex virus gC-1 and gC-2, pseudorabies virus gIII, and the varicella-zoster virus gpV may provide useful vaccine candidates for equine herpesvirus infections.     

Lymphocyte proliferative responses to separated bovine herpesvirus 1 proteins in immune cattle.

The immune response to bovine herpesvirus 1 (BHV-1) infection can protect cattle from subsequent challenge with the virus. This protection involves a variety of defensive strategies, and the activation of most of these defenses requires the recognition of viral proteins by the cellular immune system. To identify some of the BHV-1 proteins recognized by T lymphocytes, we measured in vitro proliferative responses to individual proteins. Viral proteins were separated by gel electrophoresis followed by Western immunoblotting, and immunoblots were evaluated for serological reactions. Unstained blotted fractions were processed into antigen-bearing particles for analysis in blastogenesis assays. Purified BHV-1 proteins obtained by immunoadsorbent chromatography were processed and included for comparison in both enzyme-linked immunosorbent and proliferation assays. The tegument protein VP8 and the glycoprotein gIV appeared to be the antigens which most consistently stimulated the proliferation of lymphocytes from BHV-1-immunized animals. Positive blastogenic responses were also detected to gI, gIII, and to one or more uncharacterized, low-molecular-weight proteins in some of the cattle tested. These results indicate that T-lymphocyte proliferative responses to BHV-1 proteins are detectable in immune cattle and may be important in protection from BHV-1 infection.     

Role of glycoprotein gIII of pseudorabies virus in virulence.

Deletion mutants of pseudorabies virus unable to express glycoprotein gIII, gI, or gp63 or double and triple mutants defective in these glycoproteins were constructed, and their virulence for day-old chickens inoculated intracerebrally was determined. Mutants of wild-type pseudorabies virus defective in glycoprotein gIII, gI, or gp63 were only slightly less virulent (at most, fivefold) for chickens than was the wild-type virus. However, mutants defective in both gIII and gI or gIII and gp63 were avirulent for chickens, despite their ability to grow in cell culture in vitro to about the same extent as mutants defective in gIII alone (which were virulent). These results show that gIII plays a role in virulence and does so in conjunction with gI or gp63. The effect of gIII on virulence was also shown when the resident gIII gene of variants of the Bartha vaccine strain (which codes for gIIIB) was replaced with a gIII gene derived from a virulent wild-type strain (which codes for gIIIKa); gIIIKa significantly enhanced the virulence of a variant of the Bartha strain to which partial virulence had been previously restored by marker rescue. Our results show that viral functions that play a role in the virulence of the virus (as measured by intracerebral inoculation of chickens) may act synergistically to affect the expression of virulence and that the ability of the virus to grow in cell culture is not necessarily correlated with virulence.     

Characterization of envelope proteins of infectious bovine rhinotracheitis virus (bovine herpesvirus 1) by biochemical and immunological methods.

Ten glycoproteins of molecular weights of 180,000, 150,000, 130,000, 115,000, 97,000, 77,000, 74,000, 64,000, 55,000, and 45,000 (designated as 180K, 150K, etc.) and a single nonglycosylated 107,000-molecular-weight (107K) protein were quantitatively removed from purified bovine herpesvirus 1 (BHV-1) virions by detergent treatment. Immunoprecipitations with monospecific and monoclonal antibodies showed that three sets of coprecipitating glycoproteins, 180K/97K, 150K/77K, and 130K/74K/55K, were the major components of the BHV-1 envelope. These glycoproteins were present in the envelope of the virion and on the surface of BHV-1-infected cells and reacted with neutralizing monoclonal and monospecific antibodies. Antibodies to 150K/77K protein had the largest proportion of virus-neutralizing antibodies, followed by antibodies to 180K/97K protein. Monoclonal antibodies to 130K/74K/55K protein were neutralizing but only in the presence of complement; however, monospecific antisera produced with 55K protein did not have neutralizing activity. Analysis under nonreducing conditions showed that the 74K and 55K proteins interact through disulfide bonds to form the 130K molecule. Partial proteolysis studies showed that the 180K protein was a dimeric form of the 97K protein and that the 150K protein was a dimer of the 77K protein, but these dimers were not linked by disulfide bonds. The 107K protein was not glycosylated and induced antibodies that did not neutralize BHV-1. The 64K protein was not precipitated by anti-BHV-1 convalescent antisera, and monospecific antisera to this protein precipitated several polypeptides from uninfected cell lysates, suggesting that 64K is a protein of cellular origin associated with the BHV-1 virion envelope.