1887

Journal of General Virology header logo

Volume 75, Issue 11

Identification and Characterization of the Protein Product of Gene 71 in Equine Herpesvirus 1 Free

Abstract

Equine herpesvirus 1 (EHV-1) strain Ab4 gene 71 is predicted to encode a primary product with a of 80·1K. We have previously constructed a deletion/Z insertion mutant, ED71, and demonstrated that gene 71 is dispensable for growth of virus in cell culture. We have now constructed a gene 71 revertant, Re71. To identify and characterize the product of gene 71, we produced a specific antiserum, anti-71, against a -galactosidase fusion protein containing the carboxy terminus of the gene 71 polypeptide. Using the anti-71 serum, mutant ED71 and the revertant Re71, we have demonstrated that gene 71 encodes a 192K polypeptide. Experiments with glycosylation inhibitors revealed that the protein product of gene 71 is -glycosylated and heavily glycosylated. When the 192K polypeptide is synthesized in the presence of monensin, the of the polypeptide is reduced to 80K, the predicted unmodified of the gene 71 polypeptide. The gene 71 product is found in virions and L particles in a fully processed form that runs as a diffuse band in electrophoresis, with a in excess of 200K. Immunofluorescence and virion surface labelling experiments showed that the polypeptide product of gene 71 is located on cellular membranes and the virion envelope. A time course of infection confirmed that gene 71 is regulated as a leaky late gene in infected cells. Finally, using wild-type EHV-1 Ab4, mutant ED71, revertant Re71 and two antibodies (P19 against EHV-1 glycoprotein gp300, and anti-71) we conclusively demonstrated that gene 71 encodes gp300. This contradicts published results with P19 alone, which indicated gp300 was the product of EHV-1 gene 28.

  • Received:
  • Accepted:
  • Published Online:
© The Journal of General Virology 1994
Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-75-11-3117
1994-11-01
2024-04-19
Loading full text...

Full text loading...

/deliver/fulltext/jgv/75/11/JV0750113117.html?itemId=/content/journal/jgv/10.1099/0022-1317-75-11-3117&mimeType=html&fmt=ahah

References

  1. Abodeely R. A., Palmer E., Lawson L. A. C. C. 1971; The proteins of enveloped and de-enveloped equine abortion (herpes) virus and the separated envelope. Virology 44:146–152
     [Google Scholar]
  2. Allen G. P., Yeargan M. R. 1987; Use of lgtll and monoclonal antibodies to map the genes for the six major glycoproteins of equine herpesvirus type 1. Journal of Virology 61:2454–2461
     [Google Scholar]
  3. Atherton E., Sheppard R. C. 1989 Solid Phase Peptide Synthesis - A Practical Approach Oxford: IRL Press;
     [Google Scholar]
  4. Audonnet J. -C., Winslow J., Allen G., Paoletti E. 1990; Equine herpesvirus type 1 unique short fragment encodes glycoproteins with homology to herpes simplex virus type 1 gD, gl and gE. Journal of General Virology 71:2969–2978
     [Google Scholar]
  5. Brown S. M., Ritchie D. A., Subak-Sharpe J. H. 1973; Genetic studies with herpes simplex virus type 1. The isolation of temperature-sensitive mutants, their arrangement into complementation groups and recombination analysis leading to a linkage map. Journal of General Virology 18:329–346
     [Google Scholar]
  6. Bryans J. T., Allen G. P. 1989; Herpesviral diseases of the horse. In Herpesviral Disease of Cattle, Horses and Pigs pp. 176–229 Wittman G. Edited by Dordrecht: Kluwer;
     [Google Scholar]
  7. Bryant M., Ratner L. 1990; Myristylation dependent replication and assembly of human immunodeficienty virus 1. Proceedings of the National Academy of Sciences, U.S.A 87:523–527
     [Google Scholar]
  8. Clements J. B., Watson R. J., Wilkie N. M. 1977; Temporal regulation of herpes simplex 1 transcription: location of transcripts on the viral genome. Cell 12:275–285
     [Google Scholar]
  9. Cohen J. C., Randall C. C., O’Callaghan D. J. 1975; Transcription of equine herpesvirus type 1: evidence for classes of transcripts differing in abundance. Virology 68:561–565
     [Google Scholar]
  10. Colle C. F. III Flowers C. C., O’Callaghan D. J. 1992; Open reading frames encoding a protein kinase, homologue of glycoprotein gX of pseudorabies virus, and a novel glycoprotein map within the unique short segment of equine herpesvirus type 1. Virology 188:545–557
     [Google Scholar]
  11. Frame M. C., Purves F. C., McGeoch D. J., Marsden H. S., Leader D. P. 1987; Identification of the herpes simplex virus protein kinase as the product of viral gene US3. Journal of General Virology 68:2699–2704
     [Google Scholar]
  12. Gibson R., Kornfeld S., Schlesinger S. 1980; The role of oligosaccharides in glycoprotein biosynthesis. Trends in Biochemical Sciences 5:290–293
     [Google Scholar]
  13. Goodrich L. D., Rixon F. J., Parris D. S. 1989; Kinetics of expression of the gene encoding the 65-kilodalton DNA binding protein of herpes simplex virus type 1. Journal of Virology 63:137–147
     [Google Scholar]
  14. Harlow E., Lane D. 1988 Antibodies: A Laboratory Manual New York: Cold Spring Harbor Laboratory;
     [Google Scholar]
  15. Johnson D. C., Spear P. D. 1983; O-linked oligosaccharides are acquired by herpes simplex virus glycoproteins in the golgi apparatus. Cell 32:987–997
     [Google Scholar]
  16. Johnson P. A., MacLean C., Marsden H. S., Dalziel R. G., Everett R. D. 1986; The product of gene US11 of herpes simplex virus type 1 is expressed as a true late gene. Journal of General Virology 67:871–883
     [Google Scholar]
  17. Kemp M. C., Perdue M. L., Roger H. W., O’Callaghan D. J., Randall C. W. 1974; Structural polypeptides of the hamster strain of equine herpes virus type 1: products associated with purification. Virology 61:361–375
     [Google Scholar]
  18. Kyte J., Doolittle R. F. 1982; A simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology 157:105–132
     [Google Scholar]
  19. McGeogh D. J. 1985; On the predictive recognition of signal peptide sequences. Virus Research 3:271–286
     [Google Scholar]
  20. McGeoch D. J., Dolan A., Donald S., Rixon F. J. 1985; Sequence determination and genetic content of the short unique region in the genome of herpes simplex virus type 1. Journal of Molecular Biology 181:1–13
     [Google Scholar]
  21. McGeoch D. J., Moss H. W. M., McNab D., Frame M. C. 1987; DNA sequence and genetic content of HindIII l region in the short unique component of the herpes simplex virus type 2 genome: identification of the gene encoding glycoprotein G, and evolutionary comparisons. Journal of General Virology 68:19–38
     [Google Scholar]
  22. McGeoch D. J., Barnett B. C., MacLean C. A. 1993; Emerging functions of alphaherpesvirus genes. Seminars in Virology 4:125–134
     [Google Scholar]
  23. McLauchlan J., Rixon F. J. 1992; Characterization of envelope tegument structures (L particles) produced by alphaherpesviruses: integrity of the tegument does not depend on the presence of capsid or envelope. Journal of General Virology 73:269–276
     [Google Scholar]
  24. MacLean C. A., Rixon F. J., Marsden H. S. 1987; The products of gene Usll of herpes simplex virus type 1 are DNA-binding and localize to the nucleoli of infected cells. Journal of General Virology 68:1921–1937
     [Google Scholar]
  25. MacLean C. A., Dolan A., Jamieson F. E., McGeoch D. J. 1992; The myristylated virion proteins of herpes simplex virus type 1: investigation of their role in the virus life cycle. Journal of General Virology 73:539–547
     [Google Scholar]
  26. MacPherson I., Stoker M. G. 1962; Polyoma transformation of hamster cell clones - an investigation of genetic factors affecting cell competence. Virology 16:147–151
     [Google Scholar]
  27. Masahira H., Yoshiyuki S. 1988; Dideoxy sequencing methods using denatured plasmid templates. Analytical Biochemistry 152:232–238
     [Google Scholar]
  28. Meredith D. M., Stocks J. -M., Whittaker G. R., Halliburton I. W., Snowden B. W., Killington R. A. 1989; Identification of the gB homologues of equine herpesvirus types 1 and 4 as disulphide-linked heterodimers and their characterization using monoclonal antibodies. Journal of General Virology 70:1161–1172
     [Google Scholar]
  29. Parris D. S., Cross A., Haar L., Orr A., Frame M. C., Murphy M., McGeoch D. J., Marsden H. S. 1988; Identification of the gene encoding a 65-kilodalton DNA-binding protein of herpes simplex virus type 1. Journal of Virology 62:818–825
     [Google Scholar]
  30. Perdue M. L., Kemp M. C., Randall C. C., O’Callaghan D. J. 1974; Studies of the molecular anatomy of L-M cell strain of equine herpesvirus type 1: proteins of the nucleocapsid and intact virion. Virology 59:201–216
     [Google Scholar]
  31. Rüther U., Müller-Hill B. 1983; Easy identification of cDNA clones. EMBO Journal 2:1791–1794
     [Google Scholar]
  32. Serafini-Cessi F., Malagolini N., Dall’Olio F., Pereira L., Campadelli-Fiume G. 1985; Oligosaccharide chains of herpes simplex virus type 2 glycoprotein gG2. Archives of Biochemistry and Biophysics 240:866–876
     [Google Scholar]
  33. Sodora D. L., Cohen G. H., Eisenberg R. J. 1989; Influence of asparagine linked oligosaccharides on antigenicity, processing and cell surface expression of herpes simplex virus type-1 glycoprotein D. Journal of Virology 63:5184–5193
     [Google Scholar]
  34. Spear P. G. 1993; Entry of alphaherpesviruses into cells. Seminars in Virology 4:167–180
     [Google Scholar]
  35. Stow N. D., Wilkie N. M. 1976; An improved technique for obtaining enhanced infectivity with herpes simplex virus type 1 DNA. Journal of General Virology 33:447–458
     [Google Scholar]
  36. Sun Y., Brown S. M. 1994; The open reading frames 1, 2, 71 and 75 are non-essential for the replication of equine herpes virus type 1 in vitro . Virology 199:448–452
     [Google Scholar]
  37. Szilágyi J. F., Cunningham C. 1991; Identification and characterization of a novel non-infectious herpes simplex virus-related particle. Journal of General Virology 72:661–668
     [Google Scholar]
  38. Telford E. A. R., Watson M. S., McBride K., Davison A. J. 1992; The DNA sequence of equine herpesvirus-1. Virology 189:304–316
     [Google Scholar]
  39. Turtinen L. W., Allen G. P. 1982; Identification of the envelope surface glycoproteins of equine herpesvirus type 1. Journal of General Virology 63:481–485
     [Google Scholar]
  40. Wallenfels S. 1979; Development and mutational changes of glycoproteins in the mouse neuronal retina: studies with bovine galactosyl transferase. Proceedings of the National Academy of Sciences, U.S.A 76:3223–3227
     [Google Scholar]
  41. Whalley J. M., Robertson G. R., Davison A. J. 1981; Analysis of the genome of equine herpesvirus type 1: arrangement of cleavage sites for restriction endonucleases EcoRI, BglII and BamHI. Journal of General Virology 57:307–323
     [Google Scholar]
  42. 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
     [Google Scholar]
  43. 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
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-75-11-3117
Loading
Identification and Characterization of the Protein Product of Gene 71 in Equine Herpesvirus 1
J. Gen. Virol. 75, 3117 (1994); https://doi.org/10.1099/0022-1317-75-11-3117
/content/journal/jgv/10.1099/0022-1317-75-11-3117
/content/journal/jgv/10.1099/0022-1317-75-11-3117
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error