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Bovine herpesvirus 1 (BoHV-1) infects bovine species, causing respiratory infections, genital disorders and abortions. VP8 is the most abundant tegument protein of BoHV-1 and is critical for virus replication in cattle. In this study, the cellular transport of VP8 in BoHV-1-infected cells and its ability to alter the cellular lipid metabolism were investigated. A viral kinase, US3, was found to be involved in regulating these processes. In the early stages of infection VP8 was localized in the nucleus. Subsequently, presumably after completion of its role in the nucleus, VP8 was translocated to the cytoplasm. When US3 was deleted or the essential US3 phosphorylation site of VP8 was mutated in BoHV-1, the majority of VP8 was localized in the nuclei of infected cells. This suggests that phosphorylation by US3 may be critical for cytoplasmic localization of VP8. Eventually, the cytoplasmic VP8 was accumulated in the cis-Golgi apparatus but not in the trans-Golgi network, implying that VP8 was not involved in virion transport toward and budding from the cell membrane. VP8 caused lipid droplet (LD) formation in the nuclei of transfected cells and increased cellular cholesterol levels. Lipid droplets were not found in the nuclei of BoHV-1-infected cells when VP8 was cytoplasmic in the presence of US3. However, when US3 was deleted or phosphorylation residues in VP8 were mutated, nuclear VP8 and LDs appeared in BoHV-1-infected cells. The total cholesterol level was increased in BoHV-1-infected cells but not in ΔU(L)47-BoHV-1-infected cells, further supporting a role for VP8 in altering the cellular lipid metabolism during infection. IMPORTANCE Nuclear localization signals (NLSs) and nuclear export signals (NESs) are important elements directing VP8 to the desired locations in the BoHV-1-infected cell. In this study, a critical regulator that switches the nuclear and cytoplasmic localization of VP8 in BoHV-1-infected cells was identified. BoHV-1 used viral kinase US3 to regulate the cellular localization of VP8. Early during BoHV-1 infection VP8 was localized in the nucleus, where it performs various functions; once US3 was expressed, phosphorylated VP8 was cytoplasmic and ultimately accumulated in the cis-Golgi apparatus, presumably to be incorporated into virions. The Golgi localization of VP8 was only observed in virus-infected cells and not in US3-cotransfected cells, suggesting that this is mediated by other viral factors. Interestingly, VP8 was shown to cause increased cholesterol levels, which is a novel function for VP8 and a potential strategy to supply lipid for viral replication.

The U L 47 gene product, VP8, is the most abundant tegument protein of bovine herpesvirus 1 (BoHV-1). Previously, we demonstrated that a U L 47 -deleted BoHV-1 mutant (BoHV1-ΔU L 47) exhibits 100-fold-reduced virulence in vitro and is avirulent in vivo . In this study, we demonstrated that VP8 expression or BoHV-1 infection inhibits interferon beta (IFN-β) signaling by using an IFN-α/β-responsive plasmid in a luciferase assay. As transducer and activator of transcription (STAT) is an essential component in the IFN-signaling pathways, the effect of VP8 on STAT was investigated. An interaction between VP8 and STAT1 was established by coimmunoprecipitation assays in both VP8-transfected and BoHV-1-infected cells. Two domains of VP8, amino acids 259 to 482 and 632 to 686, were found to be responsible for its interaction with STAT1. The expression of VP8 did not induce STAT1 ubiquitination or degradation. Moreover, VP8 did not reduce STAT1 tyrosine phosphorylation to downregulate IFN-β signaling. However, the expression of VP8 or a version of VP8 (amino acids 219 to 741) that contains the STAT1-interacting domains but not the nuclear localization signal prevented nuclear accumulation of STAT1. Inhibition of nuclear accumulation of STAT1 also occurred during BoHV-1 infection, while nuclear translocation of STAT1 was observed in BoHV1-ΔU L 47-infected cells. During BoHV-1 infection, VP8 was detected in the cytoplasm at 2 h postinfection without any de novo protein synthesis, at which time STAT1 was already retained in the cytoplasm. These results suggest that viral VP8 downregulates IFN-β signaling early during infection, thus playing a role in overcoming the antiviral response of BoHV-1-infected cells. IMPORTANCE Since VP8 is the most abundant protein in BoHV-1 virions and thus may be released in large amounts into the host cell immediately upon infection, we proposed that it might have a function in the establishment of conditions suitable for viral replication. Indeed, while nonessential in vitro , it is critical for BoHV-1 replication in vivo . In this study, we determined that VP8 plays a role in downregulation of the antiviral host response by inhibiting IFN-β signaling. VP8 interacted with and prevented nuclear accumulation of STAT1 at 2 h postinfection in the absence of de novo viral protein synthesis. Two domains of VP8, amino acids 259 to 482 and 632 to 686, were found to be responsible for this interaction. These results provide a new functional role for VP8 in BoHV-1 infection and a potential explanation for the lack of viral replication of the U L 47 deletion mutant in cattle.

VP8, the UL47 gene product in bovine herpesvirus-1 (BoHV-1), is a major tegument protein that is essential for virus replication in vivo The major DNA damage response protein, ataxia telangiectasia mutated (ATM), phosphorylates Nijmegen breakage syndrome (NBS1) and structural maintenance of chromosome-1 (SMC1) proteins during the DNA damage response. VP8 was found to interact with ATM and NBS1 during transfection and BoHV-1 infection. However, VP8 did not interfere with phosphorylation of ATM in transfected or BoHV-1-infected cells. In contrast, VP8 inhibited phosphorylation of both NBS1 and SMC1 in transfected cells, as well as in BoHV-1-infected cells, but not in cells infected with a VP8 deletion mutant (BoHV-1ΔUL47). Inhibition of NBS1 and SMC1 phosphorylation was observed at 4 h postinfection by nuclear VP8. Furthermore, UV light-induced cyclobutane pyrimidine dimer (CPD) repair was reduced in the presence of VP8, and VP8 in fact enhanced etoposide or UV-induced apoptosis. This suggests that VP8 blocks the ATM/NBS1/SMC1 pathway and inhibits DNA repair. VP8 induced apoptosis in VP8-transfected cells through caspase-3 activation. The fact that BoHV-1 is known to induce apoptosis through caspase-3 activation is in agreement with this observation. The role of VP8 was confirmed by the observation that BoHV-1 induced significantly more apoptosis than BoHV-1ΔUL47. These data reveal a potential role of VP8 in the modulation of the DNA damage response pathway and induction of apoptosis during BoHV-1 infection.IMPORTANCE To our knowledge, the effect of BoHV-1 infection on the DNA damage response has not been characterized. Since BoHV-1ΔUL47 was previously shown to be avirulent in vivo, VP8 is critical for the progression of viral infection. We demonstrated that VP8 interacts with DNA damage response proteins and disrupts the ATM-NBS1-SMC1 pathway by inhibiting phosphorylation of DNA repair proteins NBS1 and SMC1. Furthermore, interference of VP8 with DNA repair was correlated with decreased cell viability and increased DNA damage-induced apoptosis. These data show that BoHV-1 VP8 developed a novel strategy to interrupt the ATM signaling pathway and to promote apoptosis. These results further enhance our understanding of the functions of VP8 during BoHV-1 infection and provide an additional explanation for the reduced virulence of BoHV-1ΔUL47.

VP8 is a major tegument protein of bovine herpesvirus 1 (BoHV-1) and is essential for viral replication in cattle. The protein undergoes phosphorylation after transcription through cellular casein kinase 2 (CK2) and a viral kinase, US3. In this study, a virus containing a mutated VP8 protein that is not phosphorylated by CK2 and US3 (BoHV-1-YmVP8) was constructed by homologous recombination in mammalian cells. When BoHV-1-YmVP8-infected cells were observed by transmission electron microscopy, blocking phosphorylation of VP8 was found to impair viral DNA encapsidation, resulting in release of incomplete viral particles to the extracellular environment. Consequently, less infectious virus was produced by the mutant virus than by wild-type (WT) virus. A comparison of mutant and WT VP8 by confocal microscopy revealed that mutant VP8 is nuclear throughout infection while WT VP8 is nuclear early during infection and is associated with the Golgi apparatus at later stages. This, together with the observation that mutant VP8 is present in virions, albeit in smaller amounts, suggests that the incorporation of VP8 may occur at two stages. The first takes place without the need for phosphorylation and before or during nuclear egress of capsids, whereas the second occurs in the Golgi apparatus and requires phosphorylation of VP8. The results indicate that phosphorylated VP8 plays a role in viral DNA encapsidation and in the secondary virion incorporation of VP8. To perform these functions, the cellular localization of VP8 is adjusted based on the phosphorylation status. IMPORTANCE In this study, phosphorylation of VP8 was shown to have a function in BoHV-1 replication. A virus containing a mutated VP8 protein that is not phosphorylated by CK2 and US3 (BoHV-1-YmVP8) produced smaller numbers of infectious virions than wild-type (WT) virus. The maturation and egress of WT and mutant BoHV-1 were studied, showing a process similar to that reported for other alphaherpesviruses. Interestingly, lack of phosphorylation of VP8 by CK2 and US3 resulted in reduced incorporation of viral DNA into capsids during mutant BoHV-1 infection, as well as lower numbers of extracellular virions. Furthermore, mutant VP8 remained nuclear throughout infection, in contrast to WT VP8, which is nuclear at early stages and Golgi apparatus associated late during infection. This correlates with smaller amounts of mutant VP8 in virions and suggests for the first time that VP8 may be assembled into the virions at two stages, with the latter dependent on phosphorylation.

Tegument protein VP8 encoded by the U L 47 gene of bovine herpesvirus type 1 (BHV-1) is the most abundant constituent of mature virions. In the present report, we describe the characterization of U L 47 gene-deleted BHV-1 in cultured cells and its natural host. The U L 47 deletion mutant exhibited reduced plaque size and more than 100-fold decrease in intracellular and extracellular viral titers in cultured cells. Ultrastructural observations of infected cells showed normal maturation of BHV-1 virions in the absence of VP8. There was no evidence for a change in immediate-early gene activator function of VP16 in the U L 47 deletion mutant virus-infected cells, since bovine ICP4 mRNA and protein levels were similar to those in the wild-type and revertant virus-infected cells throughout the course of infection. Whereas VP16, glycoprotein C (gC), gB, and VP5 were expressed to wild-type levels in the U L 47 deletion mutant-infected cells, the gD and VP22 protein levels were significantly reduced. The reduction in gD protein was associated with increased turnover of the protein. Furthermore, some of the analyzed early and late proteins were expressed with earlier kinetics in the absence of VP8. Extracellular virions of the U L 47 deletion mutant contained reduced amounts of gD, gB, gC, and VP22 but similar amounts of VP16 compared to those of wild-type or revertant virus particles. In addition, the U L 47 gene product was indispensable for BHV-1 replication in vivo, since no clinical manifestations or viral shedding were detected in the U L 47 deletion mutant-infected calves, and the virus failed to induce significant levels of humoral and cellular immunity.

The major tegument protein of bovine herpesvirus 1 (BoHV-1), VP8, is essential for virus replication in cattle. VP8 is phosphorylated in vitro by casein kinase 2 (CK2) and BoHV-1 unique short protein 3 (U S 3). In this study, VP8 was found to be phosphorylated in both transfected and infected cells but was detected as a nonphosphorylated form in mature virions. This suggests that phosphorylation of VP8 is strictly controlled during different stages of the viral life cycle. The regulation and function of VP8 phosphorylation by U S 3 and CK2 were further analyzed. An in vitro kinase assay, site-directed mutagenesis, and liquid chromatography-mass spectrometry were used to identify the active sites for U S 3 and CK2. The two kinases phosphorylate VP8 at different sites, resulting in distinct phosphopeptide patterns. S 16 is a primary phosphoreceptor for U S 3, and it subsequently triggers phosphorylation at S 32 . CK2 has multiple active sites, among which T 107 appears to be the preferred residue. Additionally, CK2 consensus motifs in the N terminus of VP8 are essential for phosphorylation. Based on these results, a nonphosphorylated VP8 mutant was constructed and used for further studies. In transfected cells phosphorylation was not required for nuclear localization of VP8. Phosphorylated VP8 appeared to recruit promyelocytic leukemia (PML) protein and to remodel the distribution of PML in the nucleus; however, PML protein did not show an association with nonphosphorylated VP8. This suggests that VP8 plays a role in resisting PML-related host antiviral defenses by redistributing PML protein and that this function depends on the phosphorylation of VP8. IMPORTANCE The progression of VP8 phosphorylation over time and its function in BoHV-1 replication have not been characterized. This study demonstrates that activation of S 16 initiates further phosphorylation at S 32 by U S 3. Additionally, VP8 is phosphorylated by CK2 at several residues, with T 107 having the highest level of phosphorylation. Evidence for a difference in the phosphorylation status of VP8 in host cells and mature virus is presented for the first time. Phosphorylation was found to be a critical modification, which enables VP8 to attract and to redistribute PML protein in the nucleus. This might promote viral replication through interference with a PML-mediated antiviral defense. This study provides new insights into the regulation of VP8 phosphorylation and suggests a novel, phosphorylation-dependent function for VP8 in the life cycle of BoHV-1, which is important in view of the fact that VP8 is essential for virus replication in vivo .

The bovine herpesvirus 1 (BHV-1) tegument protein VP22 is predominantly localized in the nucleus after viral infection. To analyze subcellular localization in the absence of other viral proteins, a plasmid expressing BHV-1 VP22 fused to enhanced yellow fluorescent protein (EYFP) was constructed. The transient expression of VP22 fused to EYFP in COS-7 cells confirmed the predominant nuclear localization of VP22. Analysis of the amino acid sequence of VP22 revealed that it does not have a classical nuclear localization signal (NLS). However, by constructing a series of deletion derivatives, we mapped the nuclear targeting domain of BHV-1 VP22 to amino acids (aa) 121 to 139. Furthermore, a 4-aa motif, 130 PRPR 133 , was able to direct EYFP and an EYFP dimer (dEYFP) or trimer (tEYFP) predominantly into the nucleus, whereas a deletion or mutation of this arginine-rich motif abrogated the nuclear localization property of VP22. Thus, 130 PRPR 133 is a functional nonclassical NLS. Since we observed that the C-terminal 68 aa of VP22 mediated the cytoplasmic localization of EYFP, an analysis was performed on these C-terminal amino acid sequences, and a leucine-rich motif, 204 LDRMLKSAAIRIL 216 , was detected. Replacement of the leucines in this putative nuclear export signal (NES) with neutral amino acids resulted in an exclusive nuclear localization of VP22. Furthermore, this motif was able to localize EYFP and dEYFP in the cytoplasm, and the nuclear export function of this NES could be blocked by leptomycin B. This demonstrates that this leucine-rich motif is a functional NES. These data represent the first identification of a functional NLS and NES in a herpesvirus VP22 homologue.

Commercial killed bovine respiratory syncytial virus (K-BRSV) and formalin-inactivated BRSV (FI-BRSV) tend to induce Th2-type immune responses, which may not be protective and may even be detrimental during subsequent exposure to the virus. In this study we assessed the ability of CpG oligodeoxynucleotides (ODNs) to aid in the generation of effective and protective BRSV-specific immune responses. Mice were immunized subcutaneously with FI-BRSV formulated with CpG ODN, Emulsigen (Em), CpG ODN and Em, or non-CpG ODN and Em. Two additional groups were immunized with K-BRSV or K-BRSV and CpG ODN. After two vaccinations, the mice were challenged with BRSV. FI-BRSV induced Th2-biased immune responses characterized by production of serum immunoglobulin G1 (IgG1) and IgE, as well as interleukin-4 (IL-4), by in vitro-restimulated splenocytes. Formulation of FI-BRSV with CpG ODN, but not with non-CpG ODN, enhanced serum IgG2a and IFN-γ production by splenocytes, whereas serum IgE was reduced. Although the immune response induced by K-BRSV was not as strongly Th2 biased, the addition of CpG ODN to this commercial vaccine also resulted in a more Th1-type response. Furthermore, the addition of CpG ODN to the BRSV vaccine formulations resulted in enhanced neutralizing antibody responses. Significant production of IL-5, eotaxin, and eosinophilia was observed in the lungs of FI-BRSV- and K-BRSV-immunized mice. However, IL-5 and eotaxin levels, as well as the number of eosinophils, were decreased in the mice vaccinated with the CpG ODN-formulated vaccines. Finally, when formulated with CpG ODN, both FI-BRSV and K-BRSV significantly reduced virus production after challenge with BRSV.

For this study, the intercellular trafficking ability of bovine herpesvirus 1 (BHV-1) VP22 was applied to improve the efficacy of a DNA vaccine in calves. A plasmid encoding a truncated version of glycoprotein D (tgD) fused to VP22 was constructed. The plasmid encoding tgD-VP22 elicited significantly enhanced and more balanced immune responses than those induced by a plasmid encoding tgD. Furthermore, protection against a BHV-1 challenge was obtained in calves immunized with the plasmid encoding tgD-VP22, as shown by significant reductions in viral excretion. However, less significant protection was observed for animals vaccinated with the tgD-expressing plasmid, correlating with the lower level of immunity observed prechallenge. This is the first report of the use of VP22 as a transport molecule in the context of a DNA vaccine for a large animal species.

The immunogenicity and protective efficacy of a bovine herpesvirus 1 (BHV-1) subunit vaccine formulated with Emulsigen (Em) and a synthetic oligodeoxynucleotide containing unmethylated CpG dinucleotides (CpG ODN) was determined in cattle. A truncated, secreted version of BHV-1 glycoprotein D (tgD) formulated with Em and CpG ODN at concentrations of 25, 2.5, or 0.25 mg/dose produced a more balanced immune response, higher levels of virus neutralizing antibodies, and greater protection after BHV-1 challenge compared to tgD adjuvanted with either Em or CpG ODN alone. In contrast, tgD formulated with Em and either 25 mg of a non-CpG ODN or another immunostimulatory compound, dimethyl dioctadecyl ammonium bromide, induced similar immunity and protection compared to tgD formulated with Em alone, a finding which confirms the immunostimulatory effect of ODN to be CpG motif mediated. Our results demonstrate the ability of CpG ODN to induce a strong and balanced immune response in a target species.

Vaccination by a mucosal route is an excellent approach to the control of mucosally acquired infections. Several reports on rodents suggest that DNA vaccines can be used to achieve mucosal immunity when applied to mucosal tissues. However, with the exception of one study with pigs and another with horses, there is no information on mucosal DNA immunization of the natural host. In this study, the potential of inducing mucosal immunity in cattle by immunization with a DNA vaccine was demonstrated. Cattle were immunized with a plasmid encoding bovine herpesvirus 1 (BHV-1) glycoprotein B, which was delivered with a gene gun either intradermally or intravulvomucosally. Intravulvomucosal DNA immunization induced strong cellular immune responses and primed humoral immune responses. This was evident after BHV-1 challenge when high levels of both immunoglobulin G (IgG) and IgA were detected. Intradermal delivery resulted in lower levels of immunity than mucosal immunization. To determine whether the differences between the immune responses induced by intravulvomucosal and intradermal immunizations might be due to the efficacy of antigen presentation, the distributions of antigen and Langerhans cells in the skin and mucosa were compared. After intravulvomucosal delivery, antigen was expressed early and throughout the mucosa, but after intradermal administration, antigen expression occurred later and superficially in the skin. Furthermore, Langerhans cells were widely distributed in the mucosal epithelium but found primarily in the basal layers of the epidermis of the skin. Collectively, these observations may account for the stronger immune response induced by mucosal administration.

The potential for DNA vaccines encoding mutated versions of the same antigen to modulate immune responses in C3H/HeN mice was investigated. We created expression plasmids that encoded several versions of glycoprotein D (gD) from bovine herpesvirus 1, including authentic membrane-anchored glycoprotein (pSLRSV.AgD), a secreted glycoprotein (pSLRSV.SgD), and an intracellular protein (pSLRSV.CgD). Immunization of an inbred strain of mice with these plasmids resulted in highly efficacious and long-lasting humoral and cell-mediated immunity. We also demonstrated that the cell compartment in which plasmid-encoded gD was expressed caused a deviation in the serum immunoglobulin (Ig) isotype profile as well as the predominant cytokines secreted from the draining lymph node. Immunization of C3H/HeN mice with DNA vaccines encoding cell-associated forms of gD resulted in a predominance of serum IgG2a and gamma interferon-secreting cells within the spleens and draining lymph nodes. In contrast, mice immunized with a secreted form of this same antigen displayed immune responses characterized by greater levels of interleukin 4 in the draining lymph node and IgG1 as the predominant serum isotype. We also showed evidence of compartmentalization of distinct immune responses within different lymphoid organs.

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.

Previous studies have suggested that the attachment of bovine herpesvirus 1 (BHV-1) to permissive cells is mediated by its major glycoproteins B (gB), C (gC), and D (gD). In order to gain further insight into the mechanism of the BHV-1 attachment process, we purified authentic gB, gC, and gD from BHV-1-infected cells and membrane anchor-truncated, soluble gB, gC, and gD from stably transfected cell lines by affinity chromatography and examined their cell-binding properties on Madin-Darby bovine kidney cells. All of the glycoproteins tested exhibited saturable binding to Madin-Darby bovine kidney cells. All of the glycoproteins tested exhibited saturable binding to Madin-Darby bovine kidney cells. Addition of exogenous heparin or treatment of cells with heparinase to remove cellular heparan sulfate (HS) prevented both gC and gB from binding to cells but had no effect on gD binding. An assessment of competition between gB, gC, and gD for cell binding revealed that gC was able to inhibit gB binding, whereas other combinations showed no effect. Cell-bound gC could be dissociated by heparin or heparinase treatment. The response of bound gB to heparin and heparinase treatments differed for the authentic and soluble forms; while soluble gB was susceptible to the treatment, a significant portion of cell-bound authentic gB was resistant to the treatment. Binding affinity analysis showed that soluble gB and both forms of gC and gD each had single binding kinetics with comparable dissociation constants (Kds), ranging from 1.5 x 10(-7) to 5.1 x 10(-7) M, whereas authentic gB exhibited dual binding kinetics with Kd1 = 5.2 x 10(-7) M and Kd2 = 4.1 x 10(-9) M. These results demonstrate that BHV-1 gC binds only to cellular HS, gD binds to a non-HS component, and gB initially binds to HS and then binds with high affinity to a non-HS receptor. Furthermore, we found that while authentic gB was able to inhibit viral plaque formation, soluble gB, which retains the HS-binding property but lacks the high-affinity binding property, was defective in this respect. These results suggest that the interaction between gB and its high-affinity receptor may play a critical role in the virus entry process.

Four unique glycoproteins or glycoprotein complexes were recognized by a panel of monoclonal antibodies to bovine herpesvirus 1 (BHV-1), i.e., GVP 6/11a/16 (130,000-molecular-weight glycoprotein [130K glycoprotein]/74K/55K), GVP 7 (108K), GVP 3/9 (180K/91K), and GVP 11b (71K). The absence of any antigenic or structural relationship between GVP 11a and GVP 11b, which were previously identified as one glycoprotein, GVP 11, demonstrated that these two GVP 11 species are unique glycoproteins. GVP 3 and GVP 9 showed complete sequence homology, as shown by the identity of their antigenic determinants and by partial peptide mapping. This observation, as well as the ratio of their apparent molecular weights, indicated that GVP 3 (180K) is a dimeric form of GVP 9 (91K). GVP 6 and GVP 11a, as well as GVP 6 and GVP 16, showed at least partial sequence homology, since they shared several antigenic determinants and peptides. In addition, GVP 6, GVP 11a, and GVP 16 were derived from one primary precursor. These results, as well as the ratio of their apparent molecular weights, indicated that the GVP 6/11a/16 complex consists of two forms: one in which GVP 6 (130K) is uncleaved and the other one in which GVP 6 is cleaved and composed of GVP 11a (74K) and GVP 16 (55K), linked by disulfide bridges. An antigenically distinct precursor to each of the four BHV-1 glycoproteins or glycoprotein complexes was identified by monoclonal antibodies. These precursors, pGVP 6 (117K), pGVP 11a (62K), pGVP 7 (100K), pGVP 9 (69K), and pGVP 11b (63K) were sensitive to endo-beta-N-acetylglucosaminidase H treatment, indicating that they represent the partially glycosylated high-mannose-type intermediate forms generated by cotranslational glycosylation of the primary, unglycosylated precursors to GVP 6/11a/16, GVP 7, GVP 3/9, and GVP 11b, which were identified as having apparent molecular weights of 105,000, 90,000, 61,000, and 58,000, respectively. A new nomenclature for the BHV-1 glycoproteins, based on roman numerals, is proposed.