Centre for Equine Virology, School of Veterinary Science, The University of Melbourne, Parkville, Victoria 3010, Australia1
Gluck Equine Research Centre, University of Kentucky, Lexington, Kentucky 40546, USA2
Author for correspondence: Jin-an Huang. Fax +61 383 447 374. e-mail j.huang{at}vet.unimelb.edu.au
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() |
---|
![]() |
Main text |
---|
![]() ![]() ![]() ![]() |
---|
One of the glycoproteins of EHV-1, described originally as gp2 or gp300 (Allen & Yeargan, 1987 ; Whittaker et al., 1990
), was confirmed, after considerable debate and uncertainty, to be encoded by ORF 71 (Sun et al., 1994
; Wellington et al., 1996a
; Whittaker et al., 1992
). ORF 71 is only found in EHV-1, EHV-4 and asinine herpesvirus-3 (Crabb & Studdert, 1990
). The gp2 proteins of both EHV-1 and EHV-4 are rich in serine and threonine residues (Telford et al., 1992
, 1998
) and several studies have shown that EHV-1 gp2 is heavily glycosylated, mostly with O-linked carbohydrates (Sun et al., 1994
; Whittaker et al., 1990
). EHV-1 gp2 was also shown to be proteolytically cleaved into two peptides and the region of O-linked carbohydrates was located in the serine- and threonine-rich N-terminal peptide (Wellington et al., 1996a
, b
). ORF 71 is not essential for virus growth in cell culture and EHV-1 ORF 71 deletion mutants produced smaller plaques (Sun & Brown, 1994
; Sun et al., 1996
). Attenuation of EHV-1 through passages in cell cultures of non-equine origin was correlated with significant changes in the sizes of ORFs 1, 24 and 71 but no detailed analyses of the molecular basis for these changes were reported (Kirisawa et al., 1994
). Mouse model studies of an EHV-1 mutant with an ORF 71 deletion demonstrated that the gene was required for the full expression of virulence (Marshall et al., 1997
), although a subsequent study showed that the same EHV-1 mutant failed to protect the pregnant mice (Fitzmaurice et al., 1997
). EHV-1 and EHV-4 gp2 have been observed to vary remarkably in molecular mass from 200 to 450 kDa (Whittaker et al., 1990
; Zheng et al., 1995
) but no explanation has been offered for the observed differences and the function of the gene remains unknown.
In this study, we report that the ORF 71 sequences of both EHV-1 and EHV-4 are highly heterogeneous due to changes in two regions, designated regions A and B. Variable amounts of glycosylation within region A are proposed as the basis for the significant variation in the molecular mass of gp2 between strains.
The EHV-1 and EHV-4 isolates used in this study and their passage numbers are listed in Table 1. Plaque purification and virus purification were as described before (Crabb & Studdert, 1990
; Studdert & Blackney, 1979
).
|
The PCR products of ORF 71 of EHV-4.405/76.pa2 and EHV-4.405/76.pa80 were used directly for sequencing to obtain consensus sequences and the PCR products of viruses plaque-purified from EHV-4.405/76 were cloned into pUC18 and then sequenced following the ABI Big Dye Terminator sequencing protocol. The nucleotide sequences of ORF 71 from the four viruses plaque-purified from EHV-4.405/76.pa2 showed no variation and completely agreed with the consensus sequence of EHV-4.405/76.pa2. Among the ORF 71 sequences from the six plaque isolates from EHV-4.405/76.pa80, three were identical to each other and to the consensus sequence of EHV-4.405/76.pa80, whereas the others varied in length. Alignment of the deduced amino acid sequences revealed that the ORF 71 sequence is highly conserved except for two regions that contain reiterated sequences (Fig. 1A). Region A, located within the HindIIIEcoRI restriction sites near the 5' end of ORF 71, involved about 500 nucleotides encoding two types of amino acid repeats, TAATT and TADT, with the latter being more variable in copy number. Region B is located near the 3' end of the gene and involved copies of a 114 bp direct nucleotide repeat encoding 38 amino acids. Five out of the six plaque isolates derived from EHV-4.405/76.pa80 had three copies of the 38 amino acid repeat (Fig. 1A
). When searched against sequences in GenBank, the region B repeat had no significant matches other than with EHV-4. The differences in gp2 between EHV-4.405/76 viruses and EHV-4.NS80567 (Telford et al., 1998
) were also confined to the two regions (Fig. 1A
).
|
|
The study was extended to investigate polymorphism of ORF 71 of EHV-1 isolates. Like that of EHV-4, region A of EHV-1 strains at low passages was also polymorphic (data not shown). When the sequences of region A of selected EHV-1 strains were determined and aligned, it was found that region A of EHV-1 strains contained different copies of a TAATT and SSATTATT sequence (Fig. 1B). Sequences from four randomly picked ORF 71 clones of EHV-1.438/77 at passage 12 and three clones of an EHV-1.438/77 glycoprotein G (gG) deletion mutant were identical (Fig. 1B
). The sequence of gp2 of EHV-1.438/77 and EHV-1.Ab4p (Telford et al., 1992
) differed only in the number of repeat sequences in region A (Fig. 1B
). In contrast, EHV-4.405/76 appeared to be far more variable between passages, probably because EHV-4.405/76 was passaged more times, both in a variety of cell lines and at different temperatures. However, the fact that region A differed between strains isolated directly from horses suggested that variation in region A can occur naturally.
Western blotting analysis under reducing conditions revealed that monoclonal antibody (mAb) 1G12 (Allen & Yeargan, 1987 ) recognized a band above 175 kDa for each of the EHV isolates used in the assay (Fig. 2C
). This is much larger than their calculated molecular mass of amino acids alone (<90 kDa). The bands appeared to vary significantly in molecular mass between EHV-1 and EHV-4 and between viruses derived from EHV-4.405/76 in the order of E H V - 1>E H V - 4.405/76.pl1.>E H V - 4.405/76.pa3>E H V-4.405/76.pl5
EHV-4.405/76.pl4 (Fig. 2C
).
O-Glycosidase treatment of purified EHV-4 gp2 resulted in notable reduction of the molecular mass of EHV-4 (and EHV-1) gp2, supporting the findings by other researchers that the protein was modified by O-glycosylation (Sun et al., 1994 ; Wellington et al., 1996a; Whittaker et al., 1990
); however, the completely deglycosylated gp2 was not detected by mAb 1G12 (Fig. 2D
) or by polyclonal anti-EHV-4 sera (data not shown). O-Glycosidase treatment was effective only after gp2 was treated with N-glycosidase F (data not shown), which removes N-linked carbohydrates, supporting the earlier report that N-linked carbohydrates were present (Sun et al., 1994
). However, N-glycosidase F treatment did not seem to have an obvious impact on the molecular mass of EHV-4 gp2 (Fig. 2D
), suggesting that N-linked carbohydrates were a minor component in EHV-4 gp2 and, therefore, unlikely to be a major contributing factor to the molecular mass differences observed for EHV-4 gp2. Attempts to radiolabel EHV-4 gp2 metabolically with [35S]methionine/cysteine for the identification of the completely deglycosylated gp2 were unsuccessful, probably because the C terminus, where almost all of the cysteines and methionines are located, was cleaved off, as is the case for EHV-1 gp2 (Wellington et al., 1996b
).
The region for O-linked glycosylation for EHV-1 gp2 was determined to be in N-terminal peptide where region A is located (Wellington et al., 1996a ). Examination of gp2 sequences of EHV-1 and EHV-4 revealed 5 and 12 seemingly conserved N-linked glycosylation motifs, respectively, that are all located outside the two regions of reiterated sequences. The gp2 sequence differences between plaque 1 and plaque 4 isolates of EHV-4.405/76 are confined to region A only (Fig. 1A
) and the difference in the calculated molecular mass is about 4·5 kDa, without the addition of modifying components such as carbohydrate. The actual difference in molecular mass between plaque 1 and plaque 4 isolates is significantly more than 4·5 kDa (Fig. 2C
), suggesting that the additional copies of the repeat elements in plaque 1 isolates are modified most likely by O-linked glycosylation. The observed differences in gp2 between other EHV-4.405/76 viruses can be explained similarly. The EHV-4.405/76 plaque 3 and plaque 5 isolates showed no obvious difference in the actual molecular mass of the protein (Fig. 2C
), suggesting that the repeat in region B does not impact significantly on the actual molecular mass of gp2.
The threonine and serine residues in region A of both EHV-1 and EHV-4 are clustered together; thus, if these residues are used for O-linked glycosylation, it is unlikely that each one of these residues is utilized. To avoid steric hindrance, it is more likely that other intervening amino acids, such as alanine and aspartic acid, serve as separators between O-linked glycosylation sites.
ORF 71 has been shown to be non-essential for EHV-1 growth in cell culture but deletion of the gene was found to impact significantly on virus adsorption, penetration and virus egress and resulted in smaller plaques (Sun & Brown, 1994 ; Sun et al., 1996
). Further studies are required to elucidate the importance of expansion and contraction of repeat sequences in region A and the modification of the repeats in immune evasion and pathogenicity of EHV-1 and EHV-4. It will also be interesting to compare the ORF 71 sequence of the cell culture-attenuated EHV-1 isolates (Flowers & OCallaghan, 1992
; Kirisawa et al., 1994
) to investigate if the changes are confined to the two defined regions.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() |
---|
Allen, G. P. & Yeargan, M. R. (1987). Use of lambda gt11 and monoclonal antibodies to map the genes for the six major glycoproteins of equine herpesvirus 1. Journal of Virology 61, 2454-2461.[Medline]
Allen, G. P., Yeargan, M. R. & Bryans, J. T. (1983). Alterations in the equine herpesvirus 1 genome after in vitro and in vivo virus passage. Infection and Immunity 40, 436-439.[Medline]
Crabb, B. S. & Studdert, M. J. (1990). Comparative studies of the proteins of equine herpesviruses 4 and 1 and asinine herpesvirus 3: antibody response of the natural hosts. Journal of General Virology 71, 2033-2041.[Abstract]
Crabb, B. S. & Studdert, M. J. (1995). Equine herpesviruses 4 (equine rhinopneumonitis virus) and 1 (equine abortion virus). Advances in Virus Research 45, 153-190.[Medline]
Fitzmaurice, T., Walker, C., Kukreja, A., Sun, Y., Brown, S. M. & Field, H. J. (1997). The pathogenesis of ED71, a defined deletion mutant of equine herpesvirus-1, in a murine intranasal infection model for equine abortion. Journal of General Virology 78, 2167-2169.[Abstract]
Flowers, C. C. & OCallaghan, D. J. (1992). The equine herpesvirus type 1 (EHV-1) homolog of herpes simplex virus type 1 US9 and the nature of a major deletion within the unique short segment of the EHV-1 KyA strain genome. Virology 190, 307-315.[Medline]
Hubert, P. H., Birkenmaier, S., Rziha, H. J. & Osterrieder, N. (1996). Alterations in the equine herpesvirus type-1 (EHV-1) strain RacH during attenuation. Zentralblatt fuer Veterinaermedizin Reihe B 43, 1-14.
Kirisawa, R., Ui, S., Takahashi, A., Kawakami, Y. & Iwai, H. (1994). Comparison of the genomes of attenuated equine herpesvirus-1 strains with their parent virulent strain. Virology 200, 651-660.[Medline]
Marshall, K. R., Sun, Y., Brown, S. M. & Field, H. J. (1997). An equine herpesvirus-1 gene 71 deletant is attenuated and elicits a protective immune response in mice. Virology 231, 20-27.[Medline]
Studdert, M. J. & Blackney, M. H. (1979). Equine herpesviruses: on the differentiation of respiratory from foetal strains of type 1. Australian Veterinary Journal 55, 488-492.[Medline]
Studdert, M. J., Fitzpatrick, D. R., Horner, G. W., Westbury, H. A. & Gleeson, L. J. (1984). Molecular epidemiology and pathogenesis of some equine herpesvirus type 1 (equine abortion virus) and type 4 (equine rhinopneumonitis virus) isolates. Australian Veterinary Journal 61, 345-348.[Medline]
Studdert, M. J., Crabb, B. S. & Ficorilli, N. (1992). The molecular epidemiology of equine herpesvirus 1 (equine abortion virus) in Australasia 1975 to 1989. Australian Veterinary Journal 69, 104-111.[Medline]
Sun, Y. & Brown, S. M. (1994). The open reading frames 1, 2, 71, and 75 are nonessential for the replication of equine herpesvirus type 1 in vitro. Virology 199, 448-452.[Medline]
Sun, Y., MacLean, A. R., Dargan, D. & Brown, S. M. (1994). Identification and characterization of the protein product of gene 71 in equine herpesvirus 1. Journal of General Virology 75, 3117-3126.[Abstract]
Sun, Y., MacLean, A. R., Aitken, J. D. & Brown, S. M. (1996). The role of the gene 71 product in the life cycle of equine herpesvirus 1. Journal of General Virology 77, 493-500.[Abstract]
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]
Telford, E. A. R., Watson, M. S., Perry, J., Cullinane, A. A. & Davison, A. J. (1998). The DNA sequence of equine herpesvirus-4. Journal of General Virology 79, 1197-1203.[Abstract]
Wellington, J. E., Allen, G. P., Gooley, A. A., Love, D. N., Packer, N. H., Yan, J. X. & Whalley, J. M. (1996a). The highly O-glycosylated glycoprotein gp2 of equine herpesvirus 1 is encoded by gene 71. Journal of Virology 70, 8195-8198.[Abstract]
Wellington, J. E., Gooley, A. A., Love, D. N. & Whalley, J. M. (1996b). N-terminal sequence analysis of equine herpesvirus 1 glycoproteins D and B and evidence for internal cleavage of the gene 71 product. Journal of General Virology 77, 75-82.[Abstract]
Whittaker, G. R., Wheldon, L. A., Giles, L. E., Stocks, J.-M., Halliburton, I. W., Killington, R. A. & Meredith, D. M. (1990). Characterization of the high Mr glycoprotein (gP300) of equine herpesvirus type 1 as a novel glycoprotein with extensive O-linked carbohydrate. Journal of General Virology 71, 2407-2416.[Abstract]
Whittaker, G. R., Bonass, W. A., Elton, D. M., Halliburton, I. W., Killington, R. A. & Meredith, D. M. (1992). Glycoprotein 300 is encoded by gene 28 of equine herpesvirus type 1: a new family of herpesvirus membrane proteins? Journal of General Virology 73, 2933-2940.[Abstract]
Zheng, M., Love, D. N. & Sabine, M. (1995). High molecular weight polypeptide bands specific for equine herpesvirus 4. Veterinary Microbiology 46, 203-211.[Medline]
Received 12 July 2001;
accepted 23 November 2001.