Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1985 May;54(2):374–382. doi: 10.1128/jvi.54.2.374-382.1985

A single amino acid substitution in a hydrophobic domain causes temperature-sensitive cell-surface transport of a mutant viral glycoprotein.

C J Gallione, J K Rose
PMCID: PMC254807  PMID: 2985803

Abstract

DNA sequences were determined for three cDNA clones encoding vesicular stomatitis virus glycoproteins from the tsO45 mutant (which encodes a glycoprotein that exhibits temperature-sensitive cell-surface transport), the wild-type parent strain, and a spontaneous revertant of tsO45. The DNA sequence analysis showed that as many as three amino acid changes could be responsible for the transport defect. By recombining the cDNA clones in vitro and expressing the recombinants in COS cells, we were able to trace the critical lesion in tsO45 to a single substitution of a polar amino acid (serine) for a hydrophobic amino acid (phenylalanine) in a hydrophobic domain. We suggest that this nonconservative substitution may block protein transport by causing protein denaturation at the nonpermissive temperature. Comparison of the predicted glycoprotein sequences from two vesicular stomatitis virus strains suggests a possible basis for the differential carbohydrate requirement in transport of the two glycoproteins.

Full text

PDF
374

Images in this article

378Image
on p.378
380Image
on p.380
381Image
on p.381

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arias C., Bell J. R., Lenches E. M., Strauss E. G., Strauss J. H. Sequence analysis of two mutants of Sindbis virus defective in the intracellular transport of their glycoproteins. J Mol Biol. 1983 Jul 25;168(1):87–102. doi: 10.1016/s0022-2836(83)80324-6. [DOI] [PubMed] [Google Scholar]
  2. Bergmann J. E., Tokuyasu K. T., Singer S. J. Passage of an integral membrane protein, the vesicular stomatitis virus glycoprotein, through the Golgi apparatus en route to the plasma membrane. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1746–1750. doi: 10.1073/pnas.78.3.1746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Briley P. A., Sibilli L., Chalvignac M. A., Cossart P., Le Bras G., De Wolf A., Cohen G. N. The primary structure of Escherichia coli K12 aspartokinase I-homoserine dehydrogenase I. Site of limited proteolytic cleavage by subtilisin. J Biol Chem. 1978 Dec 25;253(24):8867–8871. [PubMed] [Google Scholar]
  4. Cartwright B., Smale C. J., Brown F. Surface structure of vesicular stomatitis virus. J Gen Virol. 1969 Jul;5(1):1–10. doi: 10.1099/0022-1317-5-1-1. [DOI] [PubMed] [Google Scholar]
  5. Crimmins D. L., Mehard W. B., Schlesinger S. Physical properties of a soluble form of the glycoprotein of vesicular stomatitis virus at neutral and acidic pH. Biochemistry. 1983 Dec 6;22(25):5790–5796. doi: 10.1021/bi00294a017. [DOI] [PubMed] [Google Scholar]
  6. Flamand A. Etude génétique du virus de la stomatite vésiculaire: classement de mutants thermosensibles spontanés en groupes de complémentation. J Gen Virol. 1970 Sep;8(3):187–195. doi: 10.1099/0022-1317-8-3-187. [DOI] [PubMed] [Google Scholar]
  7. Florkiewicz R. Z., Rose J. K. A cell line expressing vesicular stomatitis virus glycoprotein fuses at low pH. Science. 1984 Aug 17;225(4663):721–723. doi: 10.1126/science.6087454. [DOI] [PubMed] [Google Scholar]
  8. Gallione C. J., Rose J. K. Nucleotide sequence of a cDNA clone encoding the entire glycoprotein from the New Jersey serotype of vesicular stomatitis virus. J Virol. 1983 Apr;46(1):162–169. doi: 10.1128/jvi.46.1.162-169.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gibson R., Leavitt R., Kornfeld S., Schlesinger S. Synthesis and infectivity of vesicular stomatitis virus containing nonglycosylated G protein. Cell. 1978 Apr;13(4):671–679. doi: 10.1016/0092-8674(78)90217-9. [DOI] [PubMed] [Google Scholar]
  10. Gibson R., Schlesinger S., Kornfeld S. The nonglycosylated glycoprotein of vesicular stomatitis virus is temperature-sensitive and undergoes intracellular aggregation at elevated temperatures. J Biol Chem. 1979 May 10;254(9):3600–3607. [PubMed] [Google Scholar]
  11. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  12. Holland J., Spindler K., Horodyski F., Grabau E., Nichol S., VandePol S. Rapid evolution of RNA genomes. Science. 1982 Mar 26;215(4540):1577–1585. doi: 10.1126/science.7041255. [DOI] [PubMed] [Google Scholar]
  13. Hubbard S. C., Ivatt R. J. Synthesis and processing of asparagine-linked oligosaccharides. Annu Rev Biochem. 1981;50:555–583. doi: 10.1146/annurev.bi.50.070181.003011. [DOI] [PubMed] [Google Scholar]
  14. Knipe D. M., Baltimore D., Lodish H. F. Maturation of viral proteins in cells infected with temperature-sensitive mutants of vesicular stomatitis virus. J Virol. 1977 Mar;21(3):1149–1158. doi: 10.1128/jvi.21.3.1149-1158.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Lafay F. Envelope proteins of vesicular stomatitis virus: effect of temperature-sensitive mutations in complementation groups III and V. J Virol. 1974 Nov;14(5):1220–1228. doi: 10.1128/jvi.14.5.1220-1228.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Leavitt R., Schlesinger S., Kornfeld S. Impaired intracellular migration and altered solubility of nonglycosylated glycoproteins of vesicular stomatitis virus and Sindbis virus. J Biol Chem. 1977 Dec 25;252(24):9018–9023. [PubMed] [Google Scholar]
  19. Magee A. I., Koyama A. H., Malfer C., Wen D., Schlesinger M. J. Release of fatty acids from virus glycoproteins by hydroxylamine. Biochim Biophys Acta. 1984 Apr 10;798(2):156–166. doi: 10.1016/0304-4165(84)90298-8. [DOI] [PubMed] [Google Scholar]
  20. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. McGeoch D. J. Structure of the gene N:gene NS intercistronic junction in the genome of vesicular stomatitis virus. Cell. 1979 Jul;17(3):673–681. doi: 10.1016/0092-8674(79)90274-5. [DOI] [PubMed] [Google Scholar]
  22. Reading C. L., Penhoet E. E., Ballou C. E. Carbohydrate structure of vesicular stomatitis virus glycoprotein. J Biol Chem. 1978 Aug 25;253(16):5600–5612. [PubMed] [Google Scholar]
  23. Rose J. K., Adams G. A., Gallione C. J. The presence of cysteine in the cytoplasmic domain of the vesicular stomatitis virus glycoprotein is required for palmitate addition. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2050–2054. doi: 10.1073/pnas.81.7.2050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rose J. K., Bergmann J. E. Altered cytoplasmic domains affect intracellular transport of the vesicular stomatitis virus glycoprotein. Cell. 1983 Sep;34(2):513–524. doi: 10.1016/0092-8674(83)90384-7. [DOI] [PubMed] [Google Scholar]
  25. Rose J. K., Bergmann J. E. Expression from cloned cDNA of cell-surface secreted forms of the glycoprotein of vesicular stomatitis virus in eucaryotic cells. Cell. 1982 Oct;30(3):753–762. doi: 10.1016/0092-8674(82)90280-x. [DOI] [PubMed] [Google Scholar]
  26. Rose J. K. Complete intergenic and flanking gene sequences from the genome of vesicular stomatitis virus. Cell. 1980 Feb;19(2):415–421. doi: 10.1016/0092-8674(80)90515-2. [DOI] [PubMed] [Google Scholar]
  27. Rose J. K., Gallione C. J. Nucleotide sequences of the mRNA's encoding the vesicular stomatitis virus G and M proteins determined from cDNA clones containing the complete coding regions. J Virol. 1981 Aug;39(2):519–528. doi: 10.1128/jvi.39.2.519-528.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rothman J. E., Lodish H. F. Synchronised transmembrane insertion and glycosylation of a nascent membrane protein. Nature. 1977 Oct 27;269(5631):775–780. doi: 10.1038/269775a0. [DOI] [PubMed] [Google Scholar]
  29. Sprague J., Condra J. H., Arnheiter H., Lazzarini R. A. Expression of a recombinant DNA gene coding for the vesicular stomatitis virus nucleocapsid protein. J Virol. 1983 Feb;45(2):773–781. doi: 10.1128/jvi.45.2.773-781.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tarentino A. L., Maley F. Purification and properties of an endo-beta-N-acetylglucosaminidase from Streptomyces griseus. J Biol Chem. 1974 Feb 10;249(3):811–817. [PubMed] [Google Scholar]
  31. Wu G. E., Hozumi N., Murialdo H. Secretion of a lambda 2 immunoglobulin chain is prevented by a single amino acid substitution in its variable region. Cell. 1983 May;33(1):77–83. doi: 10.1016/0092-8674(83)90336-7. [DOI] [PubMed] [Google Scholar]
  32. Zilberstein A., Snider M. D., Porter M., Lodish H. F. Mutants of vesicular stomatitis virus blocked at different stages in maturation of the viral glycoprotein. Cell. 1980 Sep;21(2):417–427. doi: 10.1016/0092-8674(80)90478-x. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES