The gene encoding the capsid protein P82 of the Choristoneura fumiferana multicapsid nucleopolyhedrovirus: sequencing, transcription and characterization by immunoblot analysis Free

Abstract

A gene encoding a capsid-associated viral structural protein has been identified and sequenced in the genome of the multicapsid nucleopolyhedrovirus (CfMNPV). The gene has a 1872 nucleotide open reading frame (ORF) encoding 624 amino acids with a predicted molecular mass of 71·4 kDa. Transcription, which appeared to be initiated from a conserved GTAAG motif of baculovirus late genes, was detected at 12 h, reached a maximum at 48 h and declined at 72 h post-infection (p.i.). Part of the ORF was cloned in frame into a prokaryotic expression vector, pMAL- c2, and the fusion protein was used to generate antibodies in rabbits. It was shown, with the aid of the polyclonal antiserum, that this viral protein was detectable at 24 h p.i. in infected cells. The protein appeared as an 82 kDa band in occlusion-derived virus and as an 82 kDa band and a 72 kDa band in budded virus. Amino acid sequence comparisons revealed that this ORF had high homology with the ORF p87 (77% similarity) of (Op) MNPV and the ORF p80 (60% similarity) of (Ac) MNPV. Immunoblots confirmed that the CfMNPV protein had antigenic similarities to the P87 protein of OpMNPV, but not to the P80 of AcMNPV.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-78-10-2665
1997-10-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/jgv/78/10/9349489.html?itemId=/content/journal/jgv/10.1099/0022-1317-78-10-2665&mimeType=html&fmt=ahah

References

  1. Arif B. M., Kuzio J., Faulkner P., Doerfler W. 1984; The genome of Choristoneura fumiferana nuclear polyhedrosis virus: molecular cloning and mapping of the Eco RI, Bam HI, Sma I, Xba I and Bgl II restriction sites. Virus Research 1:605–614
    [Google Scholar]
  2. Barrett J. W., Krell P. J., Arif B. M. 1995; Characterization, sequencing and phylogeny of the ecdysteroid UDP-glucosyltransferase gene from two distinct nuclear polyhedrosis viruses isolated from Choristoneura fumiferana . Journal of General Virology 76:2447–2456
    [Google Scholar]
  3. Barrett J. W., Lauzon H. A. M., Mercuri P. S., Krell P. J., Sohi S. S., Arif B. M. 1996; The putative LEF-1 proteins from two distinct Choristoneura fumiferana multiple nucleopolyhedroviruses share domain homology to eukaryotic primases. Virus Genes 13:229–237
    [Google Scholar]
  4. Braunagel S. C., Summers M. D. 1994; Autographa californica nuclear polyhedrosis virus, PDV, and ECV viral envelopes and nucleocapsids: structural proteins, antigens, lipid and fatty acid profiles. Virology 202:315–328
    [Google Scholar]
  5. Braunagel S. C., Elton D. M., Ma H., Summers M. D. 1996a; Identification and analysis of an Autographa californica nuclear polyhedro- sis virus structural protein of the occlusion-derived virus envelope: ODV-E56. Virology 217:97–110
    [Google Scholar]
  6. Braunagel S. C., He H., Ramamurthy P., Summers M. D. 1996b; Transcription, translation, and cellular localization of three Autographa californica nuclear polyhedrosis virus structural proteins: ODV-E18, ODV-E35, and ODV-EC27. Virology 222:100–114
    [Google Scholar]
  7. Clarke E. E., Tristem M., Cory J. S., O’Reilly D. R. 1996; Characterization of the ecdysteroid UDP-glucosyltransferase gene from Mamestra brassicae nucleopolyhedrovirus. Journal of General Virology 77:2865–2871
    [Google Scholar]
  8. Harlow E., Lane D. 1988 Antibodies. A Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  9. Hawkins J. D. 1991 Gene Structure and Expression, 2nd edn.. Cambridge: Cambridge University Press;
    [Google Scholar]
  10. Higgins D. G., Sharp P. M. 1988; CLUSTAL: a package for performing multiple sequence alignment on a microcomputer. Gene 73:237–244
    [Google Scholar]
  11. Hong T., Braunagel S. C., Summers M. D. 1994; Transcription, translation, and cellular localization of PDV-E66 : a structural protein of the PDV envelope of Autographa californica nuclear polyhedrosis virus. Virology 204:210–222
    [Google Scholar]
  12. Hu Z. H., Broer R., Westerlaken J., Martens J. W. M., Jin F., Jehle J. A., Wang L. M., Vlak J. M. 1997; Characterization of the ecdysteroid UDP-glucosyl-transferase gene of a single nucleocapsid nucleopoly- hedrovirus of Buzura suppressaria . Virus Research 47:91–97
    [Google Scholar]
  13. King L. A., Possee R. D. 1992 The Baculovirus Expression System. A Laboratory Guide New York: Chapman & Hall;
    [Google Scholar]
  14. Kozak M. 1986; Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44:283–292
    [Google Scholar]
  15. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
    [Google Scholar]
  16. Lu A., Carstens E. B. 1992; Nucleotide sequence and transcriptional analysis of the p80 gene of Autographa californica nuclear polyhedrosis virus : a homologue of the Orgyia pseudotsugata nuclear polyhedrosis virus capsid-associated gene. Virology 190:201–209
    [Google Scholar]
  17. Müller R., Pearson M. N., Russell R. L. Q., Rohrmann G. F. 1990; A capsid-associated protein of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata : genetic location, sequence, transcriptional mapping, and immunocytochemical characterization. Virology 176:133–144
    [Google Scholar]
  18. O’Reilly D. R., Miller L. K., Luckow V. A. 1992 Baculovirus Expression Vectors. A Laboratory Manual New York: W. H. Freeman and Company;
    [Google Scholar]
  19. Rohrmann G. F. 1992; Baculovirus structural proteins. Journal of General Virology 73:749–761
    [Google Scholar]
  20. Russell R. L. Q., Pearson M. N., Rohrmann G. F. 1991; Immuno- electron microscopic examination of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus-infected Lymantria dispar cells : time course and localization of major polyhedron-associated proteins. Journal of General Virology 72:275–283
    [Google Scholar]
  21. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: A Laboratory Manual, 2nd edn.. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  22. Summers M. D., Smith G. E. 1978; Baculovirus structural polypeptides. Virology 84:390–402
    [Google Scholar]
  23. Theilmann D. A., Chantler J. K., Stewart S., Flipsen H. T. M., Vlak J. M., Crook N. E. 1996; Characterization of a highly conserved baculovirus structural protein that is specific for occlusion-derived virions. Virology 218:148–158
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-78-10-2665
Loading
/content/journal/jgv/10.1099/0022-1317-78-10-2665
Loading

Data & Media loading...

Most cited Most Cited RSS feed