1887

Abstract

Negative-stranded virion RNA and oligonucleotide primers complementary to fusion (F) protein gene sequences were used to generate cDNA clones, revealing that the gene 5′-proximal to the F protein corresponded to the M2 (22K) gene, as in respiratory syncytial (RS) virus. The transcription start signal, GGGACAAGU, was identical to that of the F and matrix (M) proteins of turkey rhinotracheitis virus (TRTV). There were two sequences with the potential to function as transcription termination/poly(A) signals, located at nucleotides 751 to 762 and 777 to 787; 15 clones derived from mRNA indicated that the first of these sequences formed the major signal. Part of the next downstream (5′) gene was sequenced; unlike mammalian pneumoviruses the TRTV M2 gene did not overlap the beginning of the 5′-proximal gene. Northern blotting indicated that infected Vero cells contained less M2 mRNA than F mRNA and that about half of the M2 mRNA was present as a F-M2 dicistronic mRNA. The M2 gene contained two overlapping open reading frames (ORFs 1 and 2), as with RS virus. ORF 1 comprised 558 nucleotides with the coding potential for a 186 amino acid polypeptide, 20959, eight or nine residues shorter than for human RS virus strains. The overall amino acid identity was 40%, the N-terminal one-third of the proteins sharing 62% of residues, the remainder 29%. A hydropathy plot of the TRTV M2 protein had close similarity to that of the M2 of RS virus. The protein was predicted to have a basic character with no N-terminal signal sequence or other major highly hydrophobic sequences. translation of a transcript comprising both ORFs 1 and 2 produced a single product of apparent 23000, corresponding to the M2 product of ORF 1. Site-directed mutagenesis confirmed that this product was derived from ORF 1 and that frameshifting was not involved. The second ORF was expressed only from a transcript which lacked the AUG codons of ORF 1 and, although occupying a similar position to that in the RS virus M2 gene, had virtually no amino acid identity in its 73 residue length and was approximately 25% shorter than the corresponding RS virus ORF 2. The hydropathy plot of the potential products of the second ORFs of TRTV and RS virus showed little resemblance. Taken together these results suggest that ORF 2 is unlikely to be expressed . Our accumulated data show that TRTV has the partial gene order 3′ M-F-M2 5′, whereas the corresponding RS virus genes are arranged 3′ M-SH-G-F-M2 5′.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-73-6-1355
1992-06-01
2021-10-25
Loading full text...

Full text loading...

/deliver/fulltext/jgv/73/6/JV0730061355.html?itemId=/content/journal/jgv/10.1099/0022-1317-73-6-1355&mimeType=html&fmt=ahah

References

  1. Barr J., Chambers P., Pringle C. R., Easton A. J. 1991; Sequence of the major nucleocapsid protein gene of pneumonia virus of mice: sequence comparisons suggest structural homology between nucleocapsid proteins of pneumoviruses, paramyxoviruses, rhabdo-viruses and filoviruses. Journal of General Virology 72:677–685
    [Google Scholar]
  2. Baybutt H. N., Pringle C. R. 1987; Molecular cloning and sequencing of the F and 22K membrane protein genes of the RSS-2 strain of respiratory syncytial virus. Journal of General Virology 68:2789–2796
    [Google Scholar]
  3. Cavanagh D., Barrett T. 1988; Pneumovirus-like characteristics of the mRNA and proteins of turkey rhinotracheitis virus. Virus Research 11:241–256
    [Google Scholar]
  4. Cavanagh D., Davis P. J. 1988; Evolution of avian coronavirus IBV: sequence of the matrix glycoprotein gene and intergenic region of several serotypes. Journal of General Virology 69:621–629
    [Google Scholar]
  5. Chambers P., Barr J., Pringle C. R., Easton A. J. 1990; Molecular cloning of pneumonia virus of mice. Journal of Virology 64:1869–1872
    [Google Scholar]
  6. Collins P. L. 1991; The molecular biology of human respiratory syncytial virus (RSV) of the genus Pneumovirus. In The Paramyxoviruses pp 103–162 Edited by Kingsbury D. W. New York: Plenum Press;
    [Google Scholar]
  7. Collins M. S., Gough R. E. 1988; Characterization of a virus associated with turkey rhinotracheitis. Journal of General Virology 69:909–916
    [Google Scholar]
  8. Collins P. L., Wertz G. W. 1985; The envelope-associated 22K protein of human respiratory syncytial virus: nucleotide sequence of the mRNA and a related polytranscript. Journal of Virology 54:65–71
    [Google Scholar]
  9. Collins P. L., Huang Y. T., Wertz G. W. 1984; Identification of a tenth mRNA of respiratory syncytial virus and assignment of polypeptides to the 10 viral genes. Journal of Virology 49:572–578
    [Google Scholar]
  10. Collins P. L., Hill M. G., Johnson P. R. 1990; The two open reading frames of the 22K mRNA of human respiratory syncytial virus: sequence comparison of antigenic subgroups A and B and expression in vitro. Journal of General Virology 71:3015–3020
    [Google Scholar]
  11. Elango N., Satake M., Venkatesan S. 1985; mRNA sequence of three respiratory syncytial virus genes encoding two non-structural proteins and a 22K structural protein. Journal of Virology 55:101–110
    [Google Scholar]
  12. Huang Y. T., Collins P. L., Wertz G. W. 1985; Characterization of the 10 proteins of human respiratory syncytial virus: identification of a fourth envelope-associated protein. Virus Research 2:157–173
    [Google Scholar]
  13. Jackson R. J., Hunt T. 1983; Preparation and use of nuclease-treated rabbit reticulocyte lysate for the translation of eukaryotic messenger RNA. Methods in Enzymology 96:50–74
    [Google Scholar]
  14. Krieg P. A., Melton D. A. 1984; Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Research 12:7057–7070
    [Google Scholar]
  15. Kunkel T. A. 1985; Rapid and efficient site-specific mutagenesis without phenotypic selection. Proceedings of the National Academy of Sciences, U.S.A 82:488–492
    [Google Scholar]
  16. Kunkel T. A., Roberts J. D., Zakour R. A. 1987; Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods in Enzymology 154:367–382
    [Google Scholar]
  17. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227:680–685
    [Google Scholar]
  18. Lerch R. A., Anderson K., Amann V. L., Wertz G. W. 1991; Nucleotide sequence analysis of the bovine respiratory syncytial virus fusion protein mRNA and expression from a recombinant vaccinia virus. Virology 181:118–131
    [Google Scholar]
  19. Ling R., Pringle C. R. 1988; Turkey rhinotracheitis virus: in vivo and in vitro polypeptide synthesis. Journal of General Virology 69:917–923
    [Google Scholar]
  20. Ling R., Pringle C. R. 1989; Polypeptides of pneumonia virus of mice. I. Immunological cross-reactions and post-translational modifications. Journal of General Virology 70:1427–1440
    [Google Scholar]
  21. Peeples M., Levine S. 1979; Respiratory syncytial virus polypeptides: their location in the virion. Virology 95:137–145
    [Google Scholar]
  22. Pringle C. R. 1991; Paramyxoviridae. Archives of Virology supplement 2242–246.
    [Google Scholar]
  23. Routledge E. G., McQuillin J., Samson A. C. R., Toms G. L. 1985; The development of monoclonal antibodies to respiratory syncytial virus and their use in diagnosis by indirect immunofluorescence. Journal of Medical Virology 15:305–320
    [Google Scholar]
  24. Routledge E. G., Willcocks M. M., Morgan L., Samson A. C. R., Scott R., Toms G. L. 1987; Expression of the respiratory syncytial virus 22K protein on the surface of infected HeLa cells. Journal of General Virology 68:1217–1222
    [Google Scholar]
  25. Samal S. K., Zamora M. 1991; Nucleotide sequence analysis of a matrix and small hydrophobic protein dicistronic mRNA of bovine respiratory syncytial virus demonstrates extensive sequence divergence of the small hydrophobic protein from that of human respiratory syncytial virus. Journal of General Virology 72:1715–1720
    [Google Scholar]
  26. Samal S. K., Zamora M., McPhillips T. H., Mohanty S. B. 1991; Molecular cloning and sequence analysis of bovine respiratory syncytial virus mRNA encoding the major nucleocapsid protein. Virology 180:453–456
    [Google Scholar]
  27. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: A Laboratory Manual 2nd edn New York: Cold Spring Harbor Laboratory;
    [Google Scholar]
  28. Satake M., Venkatesan S. 1984; Nucleotide sequence of the gene encoding the respiratory syncytial virus matrix protein. Journal of Virology 50:92–99
    [Google Scholar]
  29. Thomas S. M., Lamb R. A., Paterson R. G. 1988; Two mRNAs that differ by two non-templated nucleotides encode the amino coterminal proteins P and V of the paramyxovirus SV5. Cell 54:891–902
    [Google Scholar]
  30. Vidal S., Curran J., Kolakofsky D. 1990; A stuttering model for paramyxovirus P mRNA editing. EMBO Journal 9:2017–2022
    [Google Scholar]
  31. Wunner W. H., Pringle C. R. 1976; Respiratory syncytial virus proteins. Virology 73:228–243
    [Google Scholar]
  32. Yu Q., Davis P. J., Barrett T., Binns M. M., Boursnell M. E. G., Cavanagh D. 1991; Deduced amino acid sequence of the fusion glycoprotein of turkey rhinotracheitis virus has greater identity with that of human respiratory syncytial virus, a pneumovirus, than that of paramyxoviruses and morbilliviruses. Journal of General Virology 72:75–81
    [Google Scholar]
  33. Yu Q., Davis P. J., Li J., Cavanagh D. 1992; Cloning and sequencing of the matrix protein gene of turkey rhinotracheitis virus reveal a gene order different from that of respiratory syncytial virus. Virology 186:426–434
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-73-6-1355
Loading
/content/journal/jgv/10.1099/0022-1317-73-6-1355
Loading

Data & Media loading...

Most cited this month 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