Identification and characterization of bovine herpesvirus type 5 glycoprotein H gene and gene products

G. Meyer1, O. Bare1 and E. Thiry1

Department of Virology, Faculty of Veterinary Medicine, University of Liège, Bd de Colonster 20, Bat B43b, B-4000 Liège, Belgium 1

Author for correspondence: Etienne Thiry.Fax +32 4 366 42 61. e-mail Etienne.Thiry{at}ulg.ac.be


   Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Bovine herpesvirus type 5 (BHV-5) is the causative agent of a fatal meningo-encephalitis in calves and is closely related to BHV-1 which causes infectious bovine rhinotracheitis. The gene encoding BHV-5 glycoprotein gH was sequenced. A high degree of conservation was found between BHV-1 and BHV-5 deduced gH amino acid sequences (86·4%), which is also observed for all alphaherpesvirus gH sequences. Transcriptional analysis revealed a 3·1 kb mRNA as the specific gH transcript which was detected 2 h post-infection (p.i.). Twelve out of twenty-one MAbs directed against BHV-1 gH immunoprecipitated a 108–110 kDa glycoprotein, which was then designated BHV-5 gH. Synthesis and intracellular processing of BHV- 5 gH was analysed in infected MDBK cells using gH cross-reacting MAbs. Glycoprotein gH was expressed as a beta-gamma protein, detected by radioimmunoprecipitation as early as 3 h p.i. Glycosylation studies indicated that BHV-5 gH contains N-linked carbohydrates which are essential for the recognition of the protein by the MAbs. This suggests that N-linked glycans are involved in protein folding or are targets for the gH cross-reacting MAbs. Plaque- reduction neutralization assays showed that at least one BHV-1 gH antigenic domain is lacking in BHV-5 which may possibly relate to in vivo differences in virus tropism.


   Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Bovine herpesvirus type 5 (BHV-5), a member of the subfamily Alphaherpesvirinae, is the causative agent of a fatal meningo- encephalitis in calves. This virus is closely related to bovine herpesvirus type 1 (BHV-1) which primarily causes infectious bovine rhinotracheitis and a diverse range of clinical manifestations in cattle (Wyler et al., 1989 ). Although BHV-5 and BHV-1 are both neurotropic viruses which establish latency in sensory ganglia (Rock et al., 1986 ; G. Meyer, unpublished data), they differ markedly in their ability to cause neurological disease in cattle (Bagust & Clark, 1972 ; Belknap et al., 1994 ). Molecular mechanisms which could explain the differences in neuropathogenicity of these two viruses have not been elucidated so far.

The genomes of BHV-5 and BHV-1 have the typical arrangement of herpesvirus group D genomes, consisting of a linear double-stranded DNA molecule of about 140 kb subdivided into a unique long segment (U L, approx. 105 kb) and a short segment (US, approx. 10 kb) flanked by internal and terminal repeats (IRs and TRs, approx. 11 kb each). At the genomic level, BHV-5 and BHV-1 exhibit 85% homology (Engels et al., 1986 ). However, both species can be differentiated by restriction enzyme analysis of genomic DNA, by type-specific antibodies or by type- specific hybridization probes (Metzler et al., 1986 ; Engels et al., 1986 ; Friedli & Metzler, 1987 ). The BHV-1 genome sequence revealed ten genes that could encode glycoproteins (Schwyzer & Ackermann, 1996 ); these were characterized and designated by homology with herpes simplex virus type 1 (HSV-1) gB (Whitbeck et al., 1988 ) as follows: gC (Fitzpatrick et al., 1988 ); gD (Tikoo et al., 1990 ); gH (Baranowski et al., 1995 ); gG (Keil et al., 1996 ); gE (Rijsewijk et al., 1992 ); gI (Rijsewijk et al. , 1995 ); gL (Khattar et al., 1996 ); gK (Khadr et al., 1996 ); and gM (Wu et al. , 1998 ). Up to now, only three BHV-5 genes, encoding glycoproteins gC (Chowdhury, 1995 ), gD (Abdelmagid et al., 1995 ) and gG (Engelhardt & Keil, 1996 ), have been identified and their products characterized, gC being the most divergent between BHV-5 and BHV-1 (Engels et al., 1986 ; Collins et al., 1993 ; Chowdhury, 1995 ). However, it might be assumed that BHV-5 encodes a similar number of glycoproteins as BHV-1.

The BHV-1 gH gene has been sequenced (Meyer et al., 1991 ) and BHV-1 gH has been identified and described as a 108 kDa structural component of the virus, expressed as a beta- gamma protein (Baranowski et al., 1995 ). BHV-1 gH forms a complex with glycoprotein gL, which is necessary for the proper processing and transport of gH but not gL (Khattar et al., 1996 ). In addition, BHV-1 gH–gL complex is also required for induction of neutralizing antibody response and anchoring of gL to the plasma membrane (Khattar et al., 1996 ). Characterization of BHV-1, pseudorabies virus (PrV) and HSV-1 gH null mutants has indicated that gH is an essential viral protein involved in both fusion between virion and cellular membranes during virus entry and in cell-to-cell spread of the virus (Forrester et al., 1992 ; Peeters et al., 1992 ; van Drunen Littel-van den Hurk et al., 1996 ; Meyer et al. , 1998 ). In addition, the absence of gH prevented penetration and propagation of PrV in the nervous system of adult mice after intranasal inoculation, suggesting that transneuronal spread in vivo and direct cell-to-cell spread in cell culture are governed by similar mechanisms (Babic et al., 1996 ). In a similar way, gH is a target for studying the comparative neuropathogenicity of BHV-5 and BHV-1.

The present study reports the identification and characterization of the BHV-5 gH gene and gene products. The BHV-5 gH gene was sequenced and its mRNA characterized. The antigenic structure and the biochemical properties of BHV-5 gH were analysed and compared to those of BHV-1, in order to further elucidate their respective roles in the neuropathogenicity of these viruses.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Cells and viruses.
The BHV-5 N569 and BHV-1 Cooper strains were obtained from M. Studdert (Melbourne University, Australia) and J. T. van Oirschot (ID-DLO, Lelystad, The Netherlands), respectively, and propagated on Madin–Darby bovine kidney (MDBK) cells (ATCC CCL22) as previously described (Baranowski et al., 1993 ). The purification of viral DNA was performed as described by Dubuisson et al. (1988) .

{blacksquare} MAbs.
MAbs directed against BHV-1 gH (6, 8, 10, 14, 18, 20, 23, 25, 60, 61, 64, 67, 71, 83, 92, 95, 123, 126, 129, 136 and 153) were derived previously (Baranowski et al., 1993 ). Among them, MAbs 60, 61, 83, 92, 95, 126 and 153 were shown to possess neutralizing activity (Baranowski et al., 1993 ). MAb 2915 directed against BHV-5 glycoprotein gC was kindly provided by M. Engels (Faculty of Veterinary Medicine, University of Zürich, Switzerland).

{blacksquare} Plasmid construction.
All cloning procedures were performed using standard methods (Maniatis et al., 1982 ) and all nucleotide positions of the BHV-1 genome refer to the sequence available under EMBL accession number AJ004801. The location of the gH gene on the BHV- 5 genome and the pertinent restriction sites for cloning the BHV-5 gH gene in plasmids gH-KpnI, pBEK and pLeft are illustrated in Fig. 1 . By collinearity with the BHV-1 sequence, the BHV-5 gH gene should be contained in the 6·9 kb G fragment of the BHV-5 KpnI restriction map (Engels et al., 1986 ). The presence of the BHV- 5 gH gene in this fragment was firstly confirmed by Southern blot hybridization with a BHV-1 gH probe using the colorimetric DIG High Prime DNA Labelling and Detection Starter kit I (Boehringer Mannheim) (data not shown). This 6·9 kb KpnI fragment was consecutively purified after BHV-5 genomic DNA digestion with Kpn I and cloned in the KpnI-linearized pBluescript SK(+) vector to give plasmid gH-KpnI. Comparison of the sequenced extremities of gH-KpnI with the BHV-1 genomic DNA sequence showed that the plasmid contained the entire BHV-5 gH gene. The sub-cloning strategy was performed as shown in Fig. 1.



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Fig. 1. Cloning of the BHV-5 gH genomic region. (a ) The BHV-5 genome (approx. 136 kb) consisting of a unique long segment (UL, 104 kb) and a short segment containing a unique region (US, 10 kb) flanked by internal and terminal inverted repeats (IRs and TRs, 11 kb each). (b) The BHV-5 KpnI restriction map containing the 6·9 kb G segment. (c) Enlarged map of the 6·9 kb segment containing the BHV-5 TK and gH ORFs running from left to right, with BamHI, SalI and Sph I restriction maps. (d) gH-KpnI, pBEK and pLeft clones used in the gH sequencing strategy. Sub-cloning strategy was performed by digestion of gH-KpnI with SalI/BamHI or SphI/ BamHI to generate plasmids pBEK and pLeft, respectively. The presence of the BHV-5 gH gene was confirmed using either terminal sequencing of plasmids or Southern blot hybridization using a BHV-1 gH probe.

 
{blacksquare} Sequence analysis.
The DNA sequences of both strands were determined by the dideoxy chain-termination method (Sanger et al., 1977 ) adjusted to high G+C content genomes. Cycle sequencing was performed at an annealing temperature of 50 °C using Perkin Elmer dye terminator chemistry. The primer walking strategy was used. Ambiguous results due to strong stops in the cycle sequencing reactions were clarified by manual sequencing using {alpha}-33P- radiolabelled dideoxynucleotide terminator (ThermoSequenase sequencing kit; Amersham). The final sequence of the SphI/SalI fragment was compiled and analysed using the PHYLIP software package version 8.0 (UNIX) of the University of Wisconsin Genetics Computer Group (GCG; Devereux et al., 1984 ).

{blacksquare} mRNA isolation.
mRNAs were isolated by cell lysis with guanidine thiocyanate, followed by oligo(dT)–cellulose chromatography (QuickPrep mRNA purification kit; Pharmacia Biotech). RNA concentrations were determined by spectrophotometry.

To study the course of BHV-5 gH mRNA synthesis during BHV-5 replication, MDBK cell monolayers were infected with BHV-5 at an m.o.i. of 10 p.f.u. per cell. After virus adsorption for 1 h at 37 °C, MEM with 4% horse serum was added and cells were incubated at 37 °C. mRNAs were collected at hourly intervals from 0 to 8 h post-infection (p.i.). Metabolic inhibitors were also used as previously described (Seal et al., 1992 ). Briefly, cells were treated for 2 h prior to infection and 8 h p.i. in the presence of cycloheximide (100 µg/ml) to detect immediate-early transcripts or with phosphonoacetic acid (PAA; 400 µg/ml) to detect early transcripts. Late transcripts were classified by their appearance in the absence of metabolic inhibitors.

{blacksquare} Northern blot analysis.
Gel electrophoresis (1% agarose) of mRNA was done under 6% formaldehyde as described previously (Maniatis et al., 1982 ). Northern blot hybridization was performed on mRNAs transferred to pre-wetted nylon membranes (Boehringer Mannheim) by vacuum pressure (70 mbar, 5x SSC, 2 h). Filters with immobilized mRNAs (30 min at 120 °C) were hybridized overnight in 50% formamide buffer at 42 °C, either with specific BHV-5 gH or thymidine kinase (TK) DNA probes. All subsequent steps were performed according to the instructions of the chemiluminescent DIG High Prime DNA Labelling and Detection Starter kit II (Boehringer Mannheim). The BHV-5 gH probe was obtained by digestion of pBEK plasmid with Bam HI and SphI to generate an 852 nt fragment which was digoxigenin-labelled with a random-primed DNA labelling kit (High Prime; Boehringer Mannheim). The BHV-5 TK probe (0·5 kb) was generated using the same method by digestion of gH-KpnI plasmid with BamHI and PpuMI restriction endonucleases selected from the BHV-5 TK sequence (Smith et al., 1991 ). Sizes of mRNAs were verified by using digoxigenin- labelled RNA molecular mass markers with a ladder between 1·6 and 7·4 kb (Boehringer Mannheim).

{blacksquare} Neutralization tests.
Two-fold dilutions of ascitic fluids starting at 1 mg/ml immunoglobulins were mixed (1:1) with 100 p.f.u. BHV-1 or BHV-5 and incubated for 2 h at 37 °C. Each dilution was added in duplicate to confluent MDBK cells grown in 24-well plates and incubated for 2 h at 37 °C. Then plates were overlaid with MEM containing carboxymethylcellulose to a final concentration of 0·6%. Neutralizing activity was measured as the reciprocal of the 50% plaque-reducing end-point.

{blacksquare} Immunoprecipitation and SDS–PAGE.
Preparation of BHV-5 radiolabelled cell lysates was performed as described previously (Baranowski et al., 1993 ). Briefly, an MDBK monolayer was overlaid with methionine-free medium 2 h before infection with BHV-5 (m.o.i. of 10). At 2 h p.i., 20 µCi [35S]methionine per ml medium was added to the cultures.

The expression of glycoprotein gH was studied during each step of BHV-5 protein synthesis using cycloheximide and actinomycin D for alpha protein labelling and PAA for beta protein labelling (Baranowski et al., 1995 ). In this method, infected cells were harvested at 8 h p.i.

Pulse–chase experiments were performed to study the course of glycoprotein gH synthesis during BHV-5 replication in MDBK cells. Proteins were radiolabelled at hourly intervals from 0 to 7 h p.i. Cells were first incubated in methionine-free MEM for 30 min before 1 h labelling with 100 µCi/ml Trans35S- label (ICN Flow) and then harvested after 30 min incubation with unlabelled methionine. Protein radiolabelling in the presence of glycosylation inhibitors was described by van Drunen Littel-van den Hurk & Babiuk (1985) . Infected cells treated with tunicamycin and monensin were harvested at 8 h p.i. Precipitates digested by endoglycosidase F/N-glycosidase F (endo-F; Boehringer Mannheim) and endoglycosidase H (endo-H; Boehringer Mannheim) were obtained from BHV-5-infected cells at 20 h p.i. The immunoprecipitation method (RIP assay) was previously described by Baranowski et al. (1995) .


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Sequence analysis of the BHV-5 gH gene
Since the location of the gH gene is conserved throughout the family Herpesviridae, we decided to clone and sequence the BHV-5 N569 strain SphI/SalI fragment located just after the BHV-5 TK gene (Smith et al., 1991 ). Its sequence (3029 nt) was obtained and compared with the homologous one in BHV-1. Because of space limitations, the sequence is not presented in this paper but has been submitted to GenBank and assigned accession number AF113752. The G+C composition of the SphI/SalI fragment was 74·8%, which was slightly higher than the G+C content of the corresponding BHV-1 Cooper sequence (71·4%). Homology search and codon usage analysis revealed only one 2547 nt complete ORF starting at nt 240 and showing homology with the published gH gene of herpesviruses. The gene is thus hereafter identified as the BHV-5 gH gene. As expected from sequence analysis of the BHV-1 gH gene, the BHV-5 gH ORF was found at a BHV-1 collinear position and starts immediately after the BHV-5 TK gene (Smith et al., 1991 ). The BHV-5 TK and gH ORFs are only separated by a 117 nt segment which contains, in part, the 5' consensus sequence promoter of the gH gene overlapping with the 3'-end consensus sequences of the TK gene. A TATA box (TATAA) is located at position 118–123 before the start codon of the predicted gH ORF and is preceded by GC boxes which are located in the TK ORF. The sequence 5' CGGGAGATGA 3', which resides 40 nt upstream from the TATA box, exhibits similarities to the CAT box consensus sequence. This control element usually lies 70–80 nt upstream from the transcriptional start site (Breathnach & Chambon, 1981 ). Since the transcription of eukaryotic mRNAs often starts at an adenosine residue which is surrounded by pyrimidines (Breathnach & Chambon, 1981 ), the start site of the gH mRNA is predicted to lie 87 nt upstream of the initiation codon. Regarding 3' elements, no polyadenylation signal was detected in this region for the TK gene. In contrast, a poly(A) signal (AATAAA) was found at the 3'-end of the gH gene, 58 nt downstream of the stop codon (nt 2847).

Nucleotide comparisons between the overall BHV-5 and corresponding BHV-1 SphI/SalI fragments revealed 84·6% homology. When comparisons were performed between BHV-5 and BHV-1 gH ORFs, the gaps comprise essentially single or multiple triplets except for two BHV-5-specific segments located at nt 732–756 and 1923–1953 on the BHV-5 sequence. The highest divergence between BHV-5 and BHV-1 (49% homology) was found in the last 170 nt of the SphI/SalI fragment located downstream from the BHV-5 gH poly(A) signal. In BHV-5, this short sequence contains an A+T-rich region characterized by 14 repetitions of the TAT sequence which was not found in BHV-1. In addition, high G+C repeat fragments followed this A+T-rich region.

BHV-5 gH amino acid sequence analysis and comparison
The BHV-5 gH ORF (nt 240–2787) encodes a predicted protein of 849 aa, with a deduced molecular mass of 89·01 kDa. The only start codon of the gH ORF (nt 240) is contained in a consensus sequence of translation initiation (A/GNNATGG; Kozak, 1986 ) with one substitution at +4 reading 5' AGGATGC 3'. The BHV- 5 gH protein has several features that are characteristic of a membrane protein. Based on empirical rules for predicting signal sequences (von Heinje, 1986 ), aa 1–23 have the position, length, hydrophobicity and consensus cleavage site characteristic of a signal sequence. Chou–Fasman analyses (Chou & Fasman, 1978 ) predicted a ß-turn structure preceding the respective glycine residue (position 23), which is characteristic before or after a signal peptidase recognition site (Kreil, 1981 ). Thus, after cleavage of the proposed signal sequence, the non-glycosylated gH should comprise 826 aa and exhibit a molecular mass of 86·8 kDa. Similarly, one hydrophobic region in an {alpha}-helical conformation near the C terminus of the protein from position 810–830 may serve as a transmembrane anchor sequence leading to a small cytoplasmic domain of only 19 aa. The proposed BHV-5 gH ORF surface domain contains six potential N-glycosylation sites and 11 cysteine residues (10 of them located in the extracellular domain) which are completely conserved in the BHV-1 gH sequence. However, the secondary structure predictions calculated by the Chou–Fasman method indicate that the BHV-5 and BHV-1 amino acid backbones may be folded differently, specifically between aa 150–350.

Comparisons with the published sequence of the BHV-1 Cooper strain (Meyer et al., 1991 ) indicated that the BHV-5 gH ORF is 7 aa longer with a molecular mass of 89·01 kDa compared to 88·37 kDa for BHV-1 gH. The BHV-5 and BHV-1 gH amino acid sequences showed 86·4% identity and 87·7% similarity. Alignment of the two sequences revealed that the third C- terminal part of the protein is well conserved (Fig. 2 ) except for one segment of 9 aa (563–573) which is completely different between the two viruses. Sequence alignment showed that this fragment is not conserved among herpesviruses. The most divergence was found in the N-terminal region, consisting essentially of single or double amino acid substitutions except for one specific region located at aa 166–188 (Fig. 2 ). This divergent domain is characterized by an addition of 8 aa for BHV-5 which are not present in the BHV-1 sequence. The first amino acids (166 and 167) of this domain are responsible for two hydrophilic peaks separated by one hydrophobic fragment of 3 aa in BHV- 5 compared to one broad hydrophilic peak for BHV-1.



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Fig. 2. Comparison between BHV-5 and BHV-1 gH (Meyer et al., 1991 ) translation product sequences (Gap program: Gap weight, 3·0; Gap length weight, 0·1). Identical residues are connected by vertical bars and conservative exchanges are marked by dots. Cysteine residues are marked in bold and possible N -glycosylation sites are indicated in boxes. The cleavage site of the signal peptide is indicated by the arrow. The transmembrane domain near the C terminus is marked by a continuous horizontal line.

 
Comparisons of the BHV-5 gH amino acid sequence with the corresponding sequences of alphaherpesviruses revealed 40·7, 39·2, 38·3, 38·3, 33·4, 32·3 and 30·6% similarities with gH of equine herpesvirus type 1 (EHV-1), PrV, equine herpesvirus type 4 (EHV-4), varicella-zoster virus (VZV), HSV-1, Marek's disease virus (MDV) and turkey herpesvirus (HVT), respectively (GCG; Gap program). All BHV-5 cysteine residues were relatively well conserved but the best homology resides in the third C- terminal part of the protein where 5/6 cysteine residues and 2/4 glycosylation sites can be aligned to the homologues of BHV-1, EHV-1, EHV-4, VZV and PrV. By alignment of gH amino acid sequences (Lineup and Pileup programs), a high degree of homology was found in one region corresponding to residues Asn-766 to Val-794, suggesting that this domain might be important for the function of this glycoprotein. The last two potential N-glycosylation sites (positions 768–770 and 792–794) of BHV-5 gH are located in this domain. In particular, the last domain is the only N- glycosylation site of gH which was found in a collinear position for all included alphaherpesviruses.

Northern blot analysis
Northern blot analysis with a BHV-5 gH probe revealed two transcripts of 3·0–3·1 and 4·3–4·4 kb in mRNAs isolated 2 h p.i. which were always present 8 h p.i. (Fig. 3 ). At 2 and 3 h p.i., the 3·0–3·1 and 4·3–4·4 kb transcripts exhibited roughly the same intensity; at later times, the 3·0–3·1 kb transcript was much more prominent (Fig. 3). The two transcripts were detected after synthesis in the presence of PAA (Fig. 4 ). In contrast, no transcripts were isolated from infected cells in the presence of cycloheximide. Upon PAA treatment, a smear due to partial mRNA degradation was observed for the two transcripts which migrate faster than without treatment (Fig. 4 ). In addition, hybridization of BHV-5 mRNA blots with the BHV-5 TK-specific probe only recognized the 4·3–4·4 kb mRNA (Fig. 4).



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Fig. 3. Expression kinetics of BHV-5 gH gene transcripts. Polyadenylated RNAs from BHV-5-infected cells were isolated at hourly intervals from 0 to 8 h p.i. and hybridized with a BHV-5 gH probe labelled by random priming with digoxigenin. Sizes of mRNAs (MW) were verified by using digoxigenin-labelled RNA molecular mass markers with a ladder between 1·6 and 7·4 kb (Boehringer Mannheim).

 


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Fig. 4. Detection of BHV-5 gH mRNA isolated 8 h p.i., from BHV-5-infected cells without treatment (lane nt), in the presence of 100 µg/ml cycloheximide (lane cyclo) or 400 µg/ml PAA (lane PAA). Controls represent mRNAs isolated from mock-infected cells in the presence of cycloheximide (lane mock cyclo) or PAA (lane mock PAA). Hybridization was performed with a BHV-5 gH probe obtained by digestion of plasmid pBEK with BamHI and SphI. Detection of BHV-5 TK mRNA isolated 8 h p.i. from BHV-5 infected cells (lane TK) was also performed with a BHV-5 TK probe generated by digestion of gH-KpnI plasmid with BamHI and PpuMI (Smith et al., 1991 ). Sizes of mRNAs were verified by using digoxigenin-labelled RNA molecular mass markers with a ladder between 1·6 and 7·4 kb (Boehringer Mannheim).

 
Identification of the BHV-5 gH encoded gene product: MAb reactivity
A panel of 21 MAbs directed against BHV-1 gH (Baranowski et al. , 1993 ) were firstly screened for reactivity to BHV-5 by RIP assay. All of the MAbs immunoprecipitated the BHV-1gH, but only twelve were able to recognize a protein of 108 kDa from cells infected with BHV-5 and harvested 20 h p.i. or from viral particles (data not shown). Among them, all but two of the seven MAbs that were previously shown to possess virus neutralizing activity against BHV-1 (Baranowski et al., 1993 ), were reactive with BHV-5. Cross-neutralization assays were performed for BHV- 5 with the seven BHV-1-neutralizing MAbs (Table 1 ). The two neutralizing MAbs (61 and 92) which did not recognize BHV-5 gH by RIP assay also failed to neutralize BHV-5 (Table 1). Four out of the five neutralizing MAbs which reacted with both viruses by RIP assay also neutralized both viruses but with less efficiency for BHV-5. The fifth MAb 95, which possesses a neutralizing titre of 132 against BHV-1, neutralized more than 50 % of BHV-5 plaques only when undiluted. The two non-reacting MAbs 61 and 92 and MAb 95 were directed against epitopes which were previously shown by competitive ELISA to be located in the same antigenic domain II of BHV-1 gH (Baranowski, 1996 ).


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Table 1. Neutralization titres of BHV-1 gH neutralizing MAbs (Baranowski et al., 1993 ) against BHV-1 or BHV- 5

 
Characterization of BHV-5 gH
BHV-5 gH was characterized using the five most reactive BHV-1 MAbs (6, 18, 67, 95 and 153) by RIP assay. Similar results were obtained; MAb 6 was therefore used as a representative of these five MAbs. By RIP assay, MAb 6 recognized a protein with an apparent molecular mass of 108 kDa from cells infected by BHV-5 and harvested at 8 h p.i. (Fig. 5a , lane -) or 20 h p.i (Fig. 5b , lane -). Interestingly, when cells were analysed after 8 h p.i. (Fig. 5a), two bands of 108 kDa and 102 kDa were detected, the lower band with less intensity.



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Fig. 5. Processing of BHV-5 glycoprotein gH. (a) Immunoprecipitation of proteins with MAb 6 from BHV-5- or BHV-1- infected cells collected 8 h p.i., in the presence of 10 µg/ml tunicamycin (tun), 10-6 M monensin (mon) or without treatment (-). (b) Precipitates digested by endo-F, endo-H or without enzyme (-) were obtained from BHV-5-infected cells by MAb 6 at 20 h p.i. (c) Immunoprecipitation of proteins with MAb 6 from BHV-5-infected cells collected 8 h p.i. in the presence of monensin (mon) and subsequently digested by endo-H (mon+endo-H). Precipitates were analysed by SDS–PAGE on a 7·5% acrylamide denaturing gels. Molecular masses (lanes MW) were calculated from at least two different autoradiographs.

 
To analyse the oligosaccharide structure of BHV-5 gH, the sensitivity of BHV-5 gH to digestion with endo-F and endo-H was tested with the five MAbs (Fig. 5b). Results with MAb 6 showed that BHV-5 gH was resistant to endo-H treatment but a molecular mass shift to approximately 92 kDa (sharp lower band) was observed after endo-F digestion. A smear due to proteolysis was observed above the 92 kDa band.

In addition, radiolabelled cells were infected by BHV-5 in the presence of tunicamycin or monensin (Fig. 5a). No bands were detected from cells treated with tunicamycin using either the five most reactive BHV-1 MAbs or the seven additional MAbs which were able to detect BHV-5 gH by RIP assay. According to Baranowski et al. (1995) , only MAbs 10 and 67 recognize gH from tunicamycin-treated BHV-1 infected cells. In this study, gH was not recognized by MAb 10. MAb 67 only immunoprecipitated BHV-5 gH without tunicamycin treatment. The efficacy of tunicamycin was checked by using MAb 2915 raised against BHV-5 glycoprotein gC, a gamma-2 protein. BHV-5 gC was detected as an 87 kDa protein which shifted to a 64 kDa band after tunicamycin treatment. In the presence of monensin, all selected MAbs were able to precipitate only the 102 kDa band when infected cells were harvested at 8 h p.i. (Fig. 5a). Moreover, after endo-H digestion of BHV-5 infected cells treated with monensin, a molecular mass shift from 102 kDa to approximately 92 kDa was observed (Fig. 5c).

The expression of glycoprotein gH was studied during each step of BHV-5 protein synthesis. Glycoprotein gH was not immunoprecipitated from infected cells treated with actinomycin D and cycloheximide suggesting that this protein is not expressed during expression of alpha protein (Fig. 6). The treatment of BHV-5 infected cells with PAA did not influence immunoprecipitation of gH. Consequently, the gH synthesis is not dependent on DNA replication. The efficacy of PAA was checked by using MAb 2915 raised against BHV-5 glycoprotein gC, a gamma-2 protein. After PAA treatment, the 87 kDa band corresponding to gC was not detected.



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Fig. 6. Expression of glycoprotein gH during each step of BHV-5 protein synthesis. For BHV-5 alpha protein labelling (cyclo lane), cells were treated with cycloheximide (100 µg/ml) from 1 h before infection until 4 h p.i. Then, infected cells were washed and radiolabelled in medium containing actinomycin D (10 µg/ml). For BHV-5 beta protein labelling (PAA lane), infected cells were treated with 400 µg/ml PAA. For alpha and beta labelling, proteins were precipitated after 8 h p.i. using MAb 6. For gamma labelling (Late lane), infected cells were analysed by RIP assay 20 h p.i. Proteins were analysed by SDS–PAGE on 7·5% acrylamide denaturing gels.

 
To study the course of BHV-5 gH synthesis during virus replication, pulse–chase experiments were performed by radiolabelling proteins at hourly intervals from 0 to 7 h p.i. with excess MAb 6. A BHV-1 pulse–chase experiment was carried out for comparison. As shown in Fig. 7, gH of BHV-5 and BHV-1 were immunoprecipitated in two bands of 108 and 102 kDa as early as 3 h p.i. and were still detected 7 h p.i. The rate of gH synthesis was relatively similar from 4 to 7 h p.i. Since BHV-5 synthesis started during beta protein expression and did not decline significantly during viral DNA replication, this glycoprotein could be classified as a beta-gamma-regulated protein.



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Fig. 7. Temporal appearance of BHV-5 and BHV-1 gH at 0, 1, 2, 3, 4, 5, 6 and 7 h (lanes 0–7, respectively) after infection. Proteins radiolabelled at hourly intervals were precipitated by MAb 6.

 

   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
This study describes the localization and characterization of the BHV-5 N569 gH gene and gene products. The BHV-5 gH gene is located within a block of conserved genes and maps immediately downstream of the TK gene like all herpesviruses which encode this enzyme, including BHV-1. Glycoprotein gH, an essential component of the virion, constitutes the second most highly conserved glycoprotein which is present in nearly all members of the Herpesviridae family (Baranowski et al., 1996 ). The only exception is channel catfish virus, which shares few common genomic characteristics with the other herpesviruses (Davison, 1992 ). Comparison of amino acid sequences of herpesvirus gH genes by Gompels et al. (1988) indicated a greater diversity of sequence in the N- terminal region of the protein and highlighted several features of the gH protein conserved throughout the herpesvirus family which are also shared by BHV-5. Indeed, the BHV-5 proposed cytoplasmic domain is 19 aa in length, the four implicated BHV-5 cysteines are conserved at similar position relative to the putative transmembrane domain and the C- terminal glycosylation site is located within the conserved sequence NGTV (aa 790–794). The strong conservation of cysteine residues between all alphaherpesvirus gH sequences investigated implies some degree of conservation of the secondary and tertiary structure of the proteins presumably involving disulfide bonds.

Up to now, only three BHV-5 genes encoding glycoproteins gC (Chowdhury, 1995 ), gD (Abdelmagid et al., 1995 ) and gG (Engelhardt & Keil, 1996 ) have been identified and their products characterized. Amino acid comparisons showed that gH is the most conserved between BHV-5 and BHV-1 (86·4% identity). The primary structure of the putative transmembrane anchor domain is highly conserved, as are the positions of all cysteine residues and glycosylation sites found in BHV-5 gH. However, the secondary structure prediction indicated that BHV-5 and BHV-1 gH may be folded differently between aa 150–350. In addition, two different domains located at aa 166–174 and 563–573 of the BHV-5 sequence clearly differentiate BHV-5 and BHV- 1 gH amino acid sequences. The first divergent domain, located in the divergent secondary structure predictive region, is characterized by a sequence of 8 aa for BHV-5, which is not present in the BHV-1 sequence. Comparisons of amino acid sequences of BHV-5 gH over this region with those of other alphaherpesviruses suggest that the 8 aa lacking in BHV- 1 gH are the result of a deletion during evolution. Considering this point, the BHV-5 gH glycoprotein, in contrast to BHV-1 gH, is clearly similar to that of other mammalian alphaherpesviruses.

Nucleotide analysis revealed one specific BHV-5 domain located just after the polyadenylation signal of the gH transcript. This BHV-5 sequence contains an A+T-rich region which was directly followed by high G+C repeat fragments. It was previously reported that this region, between UL21 and UL22 (gH) of PrV, EHV-1 and EHV-4, encompasses an origin of replication containing repeated sequences around a A+T-rich stretch of DNA (Klupp & Mettenleiter, 1991 ; Nicolson et al., 1990 ; Baumann et al., 1989 ). Alphaherpesvirus origins are characterized by the presence of a 9 nt conserved sequence element CGTTCGCAC, proximal to the A+T- rich region. No consensus BHV-5 origin sequence could be found after the gH gene despite the fact that this region contains A+T-rich sequences. However, since repeat elements in other herpesvirus genomes are known to be unstable when cloned, sequencing needs to be performed on BHV-5 genomic DNA to determine the possible location of an origin of replication between UL21 and UL22.

The BHV-5 gH ORF is contained within two early-late transcripts of 3·0–3·1 and 4·3–4·4 kb detected from 2 h p.i. Sequence analysis revealed no polyadenylation signal after the TK stop codon. Based on these observations, it was suggested that the 3·0–3·1 kb transcript constitutes the gH mRNA and that the 4·3–4·4 kb mRNA most likely represents a structurally bicistronic transcript which encompasses the TK ORF and terminates at the gH polyadenylation signal. Confirmation was obtained by hybridization of BHV-5 mRNA blots with a BHV-5 TK probe which only recognized the 4·3–4·4 kb mRNA. Similar results were obtained for PrV, BHV-1 and MDV/HVT (Klupp & Mettenleiter, 1991 ; Bello et al., 1992 ; Scott et al., 1993 ) but not for HSV-1, VZV, EHV- 1 or EHV-4 (Davison & Scott, 1986 ; McGeoch & Davison, 1986 ; Nicolson et al., 1990 ; Telford et al., 1992 ), suggesting distinct transcriptional regulation in these alphaherpesviruses.

By RIP assay, MAbs directed against BHV-1 gH detected specifically a 108 kDa glycoprotein in both virus particles and infected cells. This glycoprotein was then designated BHV-5 gH. The expression of BHV-5 gH during each step of protein synthesis showed that BHV-5 gH could be classified as a beta-gamma regulated protein. In the pulse–chase experiments, BHV-1 (Jura strain) and BHV-5 (N569 strain) gH were immunoprecipitated as early as 3 h p.i. and were still detected 7 h p.i. Previous studies (van Drunen Littel-van den Hurk et al., 1986 ; Baranowski et al., 1995 ) detected BHV-1 Cooper strain gH at 2 h p.i. This difference in time could be due to the sensitivity of the RIP assay. Nevertheless, the results are quite similar due to the fact that samples were collected at hourly intervals. Moreover, the expression of gH starting at 3 h p.i. correlated with the expression kinetics of BHV-5 gH transcripts which were detected as early as 2 h p.i.

The acquisition of resistance to endo-H corresponds to the intracellular transport time required for the high mannose glycosylated peptides to move from the rough endoplasmic reticulum to elements of the Golgi, where processing reactions convert them to endo-H-resistant complex chains (Kornfeld & Kornfeld, 1985 ; Tarentino et al., 1989 ). In the present study, radiolabelled BHV-5 gH was resistant to endo-H but an apparent molecular mass shift from 108 to 92 kDa was observed after endo-F digestion. This molecular mass reduction with endo-F is consistent with all six potential N -linked glycosylation sites being used, assuming 3000 Da for each site.

Interestingly, when BHV-5 gH was detected at 8 h p.i. or pulse- labelled between 3–8 h p.i., a second specific band of 102 kDa was recognized by MAbs using RIP assay. This band showed the same electrophoretic mobility as BHV-5 gH expressed under monensin treatment. Moreover, BHV-5 gH expressed under monensin treatment appeared to be sensitive to endo-F and endo-H digestions with a molecular mass shift from 102 kDa to 92 kDa. These results suggest that BHV-5 gH is processed in MDBK cells by N- glycosylation from an endo-H-sensitive precursor of 102 kDa to an endo-H-resistant form of 108 kDa, containing complex and hybrid type oligosaccharides. Similar results were described for BHV-1 gH in Georgia bovine kidney or MDBK cells (van Drunen Littel-van den Hurk & Babiuk, 1986 ; Baranowski et al., 1995 ). The MAbs used in this study were unable to precipitate BHV-1 or BHV-5 gH when radiolabelling was performed in the presence of tunicamycin. This suggests that the presence of N-linked precursors on BHV-1 and BHV-5 gH is crucial for epitope conformation and recognition by all of the MAbs used, although processing in the Golgi apparatus is dispensable. Another possibility would be that gH without carbohydrate is unstable and is consequently degraded when cell extracts are made.

Complex formation between glycoproteins gH and gL has been described in several alphaherpesviruses, including BHV-1 (Khattar et al. , 1996 ). This association is essential for gH transport, glycosylation and epitope formation (Khattar et al., 1996 ). The BHV-1 MAbs used in this study were previously shown to be unable to detect a BHV-1 gH–gL complex (Baranowski et al., 1995 ). Similar results were obtained with a potential BHV-5 gH–gL complex because no band corresponding to hypothetical BHV-5 gL could be immunoprecipitated with the BHV-1 gH MAbs using either [35S]methionine, [35S]cysteine, [3H]leucine and/or [3H]glucosamine (data not shown).

Differences in the antigenic structure of the major glycoproteins gB, gC and gD of BHV-1 and BHV-5 were previously demonstrated with a panel of MAbs prepared against BHV-1 or BHV-5 glycoproteins (Metzler et al., 1986 ; Friedli & Metzler, 1987 ; Collins et al., 1993 ). Of the three glycoproteins, gC showed the main antigenic differences between the two viruses, while gB and gD are closely related antigenically. In this study, we report differences in the antigenic structure of gH between BHV-1 and BHV-5. A panel of 21 MAbs directed against BHV-1 gH (Baranowski et al., 1993 ) were tested for reactivity to lysates of BHV-5- infected cells. Twelve out of the 21 MAbs were able to specifically recognize BHV-5 gH by RIP assay, indicating the same antigenic relationship to BHV-1 as for gB and gD. The differences observed in the viral glycoproteins involve several biological functions. Glycoprotein gC has a predominant role in attachment of BHV-1 to cells (Okazaki et al., 1991 , 1994 ; Liang et al., 1991 ), gB and gD participate in attachment and penetration (Liang et al., 1991 ), while gH only plays a role in the penetration of virus into target cells (Meyer et al., 1998 ). In addition, gH is involved in the neuroinvasiveness of PrV (Babic et al., 1996 ). Some of the functional epitopes on these BHV-1 glycoproteins are absent from those of BHV-5. For BHV-1 gH, at least two antigenic domains were shown to be involved in penetration (Baranowski, 1996 ). From our virus neutralization data, one of these domains, binding with MAbs 61, 92 and 95, differed between BHV- 1 and BHV-5. MAbs 61 and 92 did not immunoprecipitate BHV-5 gH suggesting that amino acid differences within the epitopes could prevent DNA binding. MAb 95 binds to BHV-1 and BHV-5. Binding of MAb 95 to BHV-1 gH interferes with its function, resulting in virus neutralization. In contrast, due to amino acid differences, binding of MAb 95 to BHV-5 gH may not interfere with its function. These antigenic differences between BHV-5 and BHV-1 gH, as well as sequence data comparisons which essentially focused on divergence between BHV-5 and BHV-1 for one amino acid domain located between aa 150–250, may possibly relate to in vivo differences in virus tropism.


   Acknowledgments
 
The authors extend special thanks to Gilles Callens, Laurence Nols and Maria Loncar for their technical assistance. This investigation was financially supported by the European Commission, Agro-Industrial Research grant AIR3-BM92-OO8 and Food and Agro- Industrial Research project FAIR1-PL95-0316.


   Footnotes
 
The GenBank accession number of the sequence reported in this paper is AF113752.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Abdelmagid, O. Y. , Minocha, H. C. , Collins, J. K. & Chowdhury, S. I. (1995). Fine mapping of bovine herpesvirus 1 (BHV-1) glycoprotein D (gD) neutralising epitopes by type-specific monoclonal antibodies and sequence comparison with BHV- 5 gD. Virology 206, 242-253.[Medline]

Babic, N. , Klupp, B. G. , Makoschey, B. , Karger, A. , Flamand, A. & Mettenleiter, T. C. (1996). Glycoprotein gH of pseudorabies virus is essential for penetration and propagation in cell culture and in the nervous system of mice. Journal of General Virology 77, 2277-2285 .[Abstract]

Bagust, T. J. & Clark, L. (1972). Pathogenesis of meningo-encephalitis produced in calves by infectious bovine rhinotracheitis herpesvirus. Journal of Comparative Pathology 82, 375-383.[Medline]

Baranowski, E. (1996). Identification et caract érisation des glycoprotéines gH, gE, gG et gp42 du bovine herpèsvirus 1. PhD thesis, University of Li ège.

Baranowski, E. , Dubuisson, J. , Pastoret, P.-P. & Thiry, E. (1993). Identification of a 108K, 93K, and 42K glycoproteins of bovine herpesvirus-1 by monoclonal antibodies. Archives of Virology 113, 97-111.

Baranowski, E. , Dubuisson, J. , van Drunen Littel-van den Hurk, S. , Babiuk, L. A. , Michel, A. , Pastoret, P.-P. & Thiry, E. (1995). Synthesis and processing of bovine herpesvirus-1 glycoprotein H. Virology 206, 651-654.[Medline]

Baranowski, E. , Keil, G. , Lyaku, J. , Rijsewijk, F. A. , van Oirschot, J. T. , Pastoret, P. P. & Thiry, E. (1996). Structural and functional analysis of bovine herpesvirus 1 minor glycoproteins. Veterinary Microbiology 53, 91-101.[Medline]

Baumann, R. P. , Yalamanchili, V. R. R. & O'Callaghan, D. J. (1989). Functional mapping and DNA sequence of an equine herpesvirus 1 origin of replication. Journal of Virology 63, 1275-1283 .[Medline]

Belknap, E. B. , Collins, J. K. , Ayers, V. K. & Schulteiss, P. C. (1994). Experimental infection of neonatal calves with neurovirulent bovine herpesvirus type 1.3. Veterinary Pathology 31, 358-365.[Abstract]

Bello, L. J. , Whitbeck, J. C. & Lawrence, W. C. (1992). Sequence and transcript analysis of the bovine herpesvirus 1 thymidine kinase locus. Virology 189, 407-414.[Medline]

Breathnach, R. & Chambon, R. (1981). Organisation and expression of eucaryotic split genes coding for proteins. Annual Review in Biochemistry 50, 349-383.[Medline]

Chou, P. Y. & Fasman, G. D. (1978). Prediction of the secondary structure of proteins from their amino acid sequence. Advances in Enzymology Relating Areas of Molecular Biology 47, 145-148.

Chowdhury, S. I. (1995). Molecular basis of antigenic variation between the glycoprotein C of respiratory bovine herpesvirus 1 (BHV-1) and neurovirulent (BHV-5). Virology 213, 558-568.[Medline]

Collins, J. K. , Ayers, V. K. , Whetstone, C. A. & van Drunen Littel-van den Hurk, S. (1993). Antigenic differences between the major glycoproteins of bovine herpesvirus type 1.1 and bovine encephalitis herpesvirus type 1.3. Journal of General Virology 74, 1509-1517 .[Abstract]

Davison, A. J. (1992). Channel catfish virus: a new type of herpesvirus. Virology 186, 9-14.[Medline]

Davison, A. J. & Scott, J. E. (1986). The complete DNA sequence of varicella-zoster virus. Journal of General Virology 67, 1759-1816 .[Abstract]

Devereux, J. , Haeberli, P. & Smithies, O. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Research 12, 387-395.[Abstract]

Dubuisson, J. , Thiry, E. , Thalasso, F. , Bublot, M. & Pastoret, P.-P. (1988). Biological and biochemical comparison of bovid herpesvirus-4 strains. Veterinary Microbiology 16, 339-349.[Medline]

Engelhardt, T. & Keil, G. M. (1996). Identification and characterisation of the bovine herpesvirus 5 US4 gene and gene products. Virology 225, 126-135.[Medline]

Engels, M. , Giuliani, C. , Wild, P. , Beck, P. , Loepfe, T. M. & Wyler, R. (1986). The genome of bovine herpesvirus 1 (BHV-1) strains exhibiting a neuropathogenic potential compared to known BHV-1 strains by restriction site mapping and cross- hybridisation. Virus Research 6, 57-73.[Medline]

Fitzpatrick, D. R. , Zamb, T. J. , Parker, M. D. , Van Drunen Littel-van den Hurk, S. , Babiuk, L. A. & Lawman, M. J. P. (1988). Expression of bovine herpesvirus 1 glycoproteins gI and gIII in transfected murine cells. Journal of Virology 62, 4239-4248 .[Medline]

Forrester, A. , Farrell, H. , Wilkinson, G. , Kaye, J. , Davis-Poynter, N. & Minson, T. (1992). Construction and properties of a mutant of herpes simplex virus type 1 with glycoprotein H coding sequences deleted. Journal of Virology 66, 341-348.[Abstract]

Friedli, K. & Metzler, A. E. (1987). Reactivity of monoclonal antibodies to proteins of a neurotropic bovine herpesvirus 1 (BHV-1) strain and to proteins of representative BHV-1 strains. Archives of Virology 94, 109-122.[Medline]

Gompels, U. A. , Craxton, M. A. & Honess, R. W. (1988). Conservation of glycoprotein H (gH) in herpesviruses: nucleotide sequence of the gH gene from herpesvirus saimiri. Journal of General Virology 69, 2819-2829 .[Abstract]

Keil, G. M. , Engelhardt, T. , Karger, A. & Enz, M. (1996). Bovine herpesvirus 1 Us open reading frame 4 encodes a glycoproteoglycan. Journal of Virology 70, 3032-3038 .[Abstract]

Khadr, A. , Tikoo, S. K. , Babiuk, L. A. & van Drunen Littel-van den Hurk, S. (1996). Sequence and expression of a bovine herpesvirus-1 gene homologous to the glycoprotein K-encoding gene of herpes simplex virus-1. Gene 168, 189-193.[Medline]

Khattar, S. K. , van Drunen Littel-van den Hurk, S. , Attah-Poku, S. K. , Babiuk, L. A. & Tikoo, S. K. (1996). Identification and characterisation of a bovine herpesvirus-1 (BHV-1) glycoprotein gL which is required for proper antigenicity, processing, and transport of BHV-1 glycoprotein gH. Virology 219, 66-76.[Medline]

Klupp, B. G. & Mettenleiter, T. C. (1991). Sequence and expression of the glycoprotein gH gene of pseudorabies virus. Virology 182, 732-741.[Medline]

Kornfeld, R. & Kornfeld, S. (1985). Assembly of asparagine-linked oligosaccharides. Annual Review of Biochemistry 54, 631-664.[Medline]

Kozak, M. (1986). Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eucaryotic ribosomes. Cell 44, 283-292.[Medline]

Kreil, G. (1981). Transfer of proteins across membranes. Annual Review of Biochemistry 50, 317-348.[Medline]

Liang, X. , Babiuk, L. A. , van Drunen Littel-van den Hurk, S. , Fitzpatrick, D. R. & Zamb, T. J. (1991). Bovine herpesvirus-1 attachment to permissive cells is mediated by its major glycoproteins gI, gIII and gIV. Journal of Virology 65, 1124-1132 .[Medline]

McGeoch, D. J. & Davison, A. J. (1986). DNA sequence of the herpes simplex virus type 1 gene encoding glycoprotein gH, and identification of homologues in the genomes of varicella-zoster virus and Epstein–Barr virus. Nucleic Acids Research 14, 4281-4292 .[Abstract]

Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Metzler, A. E. , Schudel, A. A. & Engels, M. (1986). Bovine herpesvirus 1: molecular and antigenic characteristics of variants isolated from calves with neurological disease. Archives of Virology 87, 205-217.[Medline]

Meyer, A. L. , Petrovskis, E. A. , Duffus, W. P. H. , Thomsen, D. R. & Post, L. E. (1991). Cloning and sequence of an infectious bovine rhinotracheitis virus (BHV-1) gene homologous to glycoprotein H of herpes simplex virus. Biochimica et Biophysica Acta 1090, 267-269 .[Medline]

Meyer, G. , Hanon, E. , Georlette, D. , Pastoret, P.-P. & Thiry, E. (1998). Bovine herpesvirus type 1 glycoprotein H is essential for penetration and propagation in cell culture. Journal of General Virology 79, 1983-1987 .[Abstract]

Nicolson, L. , Cullinane, A. A. & Onions, D. E. (1990). The nucleotide sequence of an equine herpesvirus 4 gene homologue of the herpes simplex virus 1 glycoprotein H gene. Journal of General Virology 71, 1793-1800 .[Abstract]

Okazaki, K. , Matsuzaki, T. , Sugahara, Y. , Okada, J. , Hasebe, M. , Iwamura, Y. , Ohnishi, M. , Kanno, T. , Shimizu, M. & Honda, E. (1991). BHV-1 adsorption is mediated by the interaction of glycoprotein gIII with heparin-like moiety on the cell surface. Virology 181, 666-670.[Medline]

Okazaki, K. , Honda, E. & Kono, Y. (1994). Heparin-binding domain of bovid herpesvirus 1 glycoprotein gIII. Archives of Virology 134, 413-419.[Medline]

Peeters, B. , de Wind, N. , Broer, R. , Gielkens, A. & Moormann, R. (1992). Glycoprotein H of pseudorabies virus is essential for entry and cell-to-cell spread of the virus. Journal of Virology 66, 3888-3892 .[Abstract]

Rijsewijk, F. A. M., Magdalena, J., Kaashoek, M., Maris-Veldhuis, M. A., Gielkens, A. L. J. & van Oirschot, J. T. (1992). Identification and functional analysis of the glycoprotein E (gE) of bovine herpesvirus 1. In 17th International Herpesvirus Workshop, Edinburgh, Scotland, p. 245.

Rijsewijk, F., Kaashoek, M., Keil, G., Paal, H., Ruuls, R., van Engelenburg, F. & van Oirschot, J. T. (1995). In vitro and in vivo role of the non essential glycoproteins gC, gG, gI and gE of bovine herpesvirus 1. In Symposium on IBR and other ruminant herpesvirus infections. European Society for Virology, Liège, Belgium, p.27.

Rock, D. L. , Hagemoser, W. A. , Osorio, F. A. & Reed, D. E. (1986). Detection of bovine herpesvirus type 1 RNA in trigeminal ganglia of latently infected rabbits by in situ hybridization. Journal of General Virology 67, 2515-2520 .[Abstract]

Sanger, F. , Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences, USA 74, 5463-5467 .[Abstract]

Schwyzer, M. & Ackermann, M. (1996). Molecular virology of ruminant herpesviruses. Veterinary Microbiology 53, 17-29.[Medline]

Scott, S. D. , Smith, G. D. , Ross, N. L. J. & Binns, M. M. (1993). Identification and sequence of the homologues of the herpes simplex virus type 1 glycoprotein H in Marek's disease virus and the herpesvirus of turkeys. Journal of General Virology 74, 1185-1190 .[Abstract]

Seal, B. S. , Whetstone, C. A. , Zamb, T. J. , Bello, L. J. & Lawrence, W. C. (1992). Relationship of bovine herpesvirus 1 immediate-early, early and late gene expression to host cellular gene transcription. Virology 188, 152-159.[Medline]

Smith, G. A. , Young, P. L. & Mattick, J. S. (1991). Nucleotide and amino acid sequence analysis of the thymidine kinase gene of a bovine encephalitis herpesvirus. Archives of Virology 119, 199-210.[Medline]

Tarentino, A. L. , Trimble, R. B. , Thomas, H. & Plummer, T. H. (1989). Enzymatic approaches for studying the structure, synthesis, and processing of glycoproteins. Methodology in Cellular Biology 32, 111-139.

Telford, E. A. R. , Watson, M. S. , McBride, K. & Davison, A. J. (1992). The DNA sequence of equine herpesvirus-1. Virology 189, 304-316.[Medline]

Tikoo, S. K. , Fitzpatrick, D. R. , Babiuk, L. A. & Zamb, T. J. (1990). Molecular cloning, sequencing, and expression of functional bovine herpesvirus 1 glycoprotein IV in transfected bovine cells. Journal of Virology 64, 5132-5142 .[Medline]

van Drunen Littel-van den Hurk, S. & Babiuk, L. A. (1985). Effect of tunicamycin and monensin on biosynthesis, transport, and maturation of bovine herpesvirus type-1 glycoproteins. Virology 143, 104-118.[Medline]

van Drunen Littel-van den Hurk, S. & Babiuk, L. A. (1986). Synthesis and processing of bovine herpesvirus-1 glycoproteins. Journal of Virology 59, 401-410.[Medline]

van Drunen Littel-van den Hurk, S. , Khattar, S. , Tikoo, S. K. , Babiuk, L. A. , Baranowski, E. , Plainchamp, D. & Thiry, E. (1996). Glycoprotein H (gII/gp108) and glycoprotein L form a functional complex which plays a role in penetration, but not in attachment, of bovine herpesvirus 1. Journal of General Virology 77, 1515-1520 .[Abstract]

von Heinje, G. (1986). A new method for predicting signal sequence cleavage sites. Nucleic Acids Research 14, 4683-4690 .[Abstract]

Whitbeck, J. C. , Bello, L. J. & Lawrence, W. C. (1988). Comparison of the bovine herpesvirus type 1 gI gene and the herpes simplex virus type 1 B gene. Journal of Virology 62, 3319-3327 .[Medline]

Wu, S. X. , Zhu, X. P. & Letchworth, G. J. (1998). Bovine herpesvirus 1 glycoprotein M forms a disulfide-linked heterodimer with the U(L)49.5 protein. Journal of Virology 72, 3029-3036 .[Abstract/Free Full Text]

Wyler, R. , Engels, M. & Schwyzer, M. (1989). Infectious bovine rhinotracheitis/vulvovaginitis (BHV-1). In Herpesvirus Diseases of Cattle, Horses and Pigs. Developments in Veterinary Virology, pp. 1-72. Edited by G. Wittmann. Boston: Kluwer.

Received 10 May 1999; accepted 26 July 1999.