A fusion-defective mutant of the vesicular stomatitis virus glycoprotein (original) (raw)

Abstract

We have recently described an assay in which a temperature-sensitive mutant of vesicular stomatitis virus (VSV; mutant tsO45), encoding a glycoprotein that is not transported to the cell surface, can be rescued by expression of wild-type VSV glycoproteins from cDNA (M. Whitt, L. Chong, and J. Rose, J. Virol. 63:3569-3578, 1989). Here we examined the ability of mutant G proteins to rescue tsO45. We found that one mutant protein (QN-1) having an additional N-linked oligosaccharide at amino acid 117 in the extracellular domain was incorporated into VSV virions but that the virions containing this glycoprotein were not infectious. Further analysis showed that virus particles containing the mutant protein would bind to cells and were endocytosed with kinetics identical to those of virions rescued with wild-type G protein. We also found that QN-1 lacked the normal membrane fusion activity characteristic of wild-type G protein. The absence of fusion activity appears to explain lack of particle infectivity. The proximity of the new glycosylation site to a sequence of 19 uncharged amino acids (residues 118 to 136) that is conserved in the glycoproteins of the two VSV serotypes suggests that this region may be involved in membrane fusion. The mutant glycoprotein also interferes strongly with rescue of virus by wild-type G protein. The strong interference may result from formation of heterotrimers that lack fusion activity.

4907

Images in this article

Selected References

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

  1. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Crise B., Ruusala A., Zagouras P., Shaw A., Rose J. K. Oligomerization of glycolipid-anchored and soluble forms of the vesicular stomatitis virus glycoprotein. J Virol. 1989 Dec;63(12):5328–5333. doi: 10.1128/jvi.63.12.5328-5333.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Doms R. W., Keller D. S., Helenius A., Balch W. E. Role for adenosine triphosphate in regulating the assembly and transport of vesicular stomatitis virus G protein trimers. J Cell Biol. 1987 Nov;105(5):1957–1969. doi: 10.1083/jcb.105.5.1957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Doms R. W., Ruusala A., Machamer C., Helenius J., Helenius A., Rose J. K. Differential effects of mutations in three domains on folding, quaternary structure, and intracellular transport of vesicular stomatitis virus G protein. J Cell Biol. 1988 Jul;107(1):89–99. doi: 10.1083/jcb.107.1.89. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Felgner P. L., Gadek T. R., Holm M., Roman R., Chan H. W., Wenz M., Northrop J. P., Ringold G. M., Danielsen M. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7413–7417. doi: 10.1073/pnas.84.21.7413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Florkiewicz R. Z., Smith A., Bergmann J. E., Rose J. K. Isolation of stable mouse cell lines that express cell surface and secreted forms of the vesicular stomatitis virus glycoprotein. J Cell Biol. 1983 Nov;97(5 Pt 1):1381–1388. doi: 10.1083/jcb.97.5.1381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fuerst T. R., Niles E. G., Studier F. W., Moss B. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8122–8126. doi: 10.1073/pnas.83.21.8122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Garoff H., Frischauf A. M., Simons K., Lehrach H., Delius H. Nucleotide sequence of cdna coding for Semliki Forest virus membrane glycoproteins. Nature. 1980 Nov 20;288(5788):236–241. doi: 10.1038/288236a0. [DOI] [PubMed] [Google Scholar]
  10. Gething M. J., Doms R. W., York D., White J. Studies on the mechanism of membrane fusion: site-specific mutagenesis of the hemagglutinin of influenza virus. J Cell Biol. 1986 Jan;102(1):11–23. doi: 10.1083/jcb.102.1.11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gething M. J., White J. M., Waterfield M. D. Purification of the fusion protein of Sendai virus: analysis of the NH2-terminal sequence generated during precursor activation. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2737–2740. doi: 10.1073/pnas.75.6.2737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Guan J. L., Rose J. K. Conversion of a secretory protein into a transmembrane protein results in its transport to the Golgi complex but not to the cell surface. Cell. 1984 Jul;37(3):779–787. doi: 10.1016/0092-8674(84)90413-6. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Machamer C. E., Florkiewicz R. Z., Rose J. K. A single N-linked oligosaccharide at either of the two normal sites is sufficient for transport of vesicular stomatitis virus G protein to the cell surface. Mol Cell Biol. 1985 Nov;5(11):3074–3083. doi: 10.1128/mcb.5.11.3074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Machamer C. E., Rose J. K. Influence of new glycosylation sites on expression of the vesicular stomatitis virus G protein at the plasma membrane. J Biol Chem. 1988 Apr 25;263(12):5948–5954. [PubMed] [Google Scholar]
  16. Machamer C. E., Rose J. K. Vesicular stomatitis virus G proteins with altered glycosylation sites display temperature-sensitive intracellular transport and are subject to aberrant intermolecular disulfide bonding. J Biol Chem. 1988 Apr 25;263(12):5955–5960. [PubMed] [Google Scholar]
  17. Marsh M., Helenius A. Virus entry into animal cells. Adv Virus Res. 1989;36:107–151. doi: 10.1016/S0065-3527(08)60583-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Matlin K. S., Reggio H., Helenius A., Simons K. Pathway of vesicular stomatitis virus entry leading to infection. J Mol Biol. 1982 Apr 15;156(3):609–631. doi: 10.1016/0022-2836(82)90269-8. [DOI] [PubMed] [Google Scholar]
  19. Puddington L., Machamer C. E., Rose J. K. Cytoplasmic domains of cellular and viral integral membrane proteins substitute for the cytoplasmic domain of the vesicular stomatitis virus glycoprotein in transport to the plasma membrane. J Cell Biol. 1986 Jun;102(6):2147–2157. doi: 10.1083/jcb.102.6.2147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Riedel H., Kondor-Koch C., Garoff H. Cell surface expression of fusogenic vesicular stomatitis virus G protein from cloned cDNA. EMBO J. 1984 Jul;3(7):1477–1483. doi: 10.1002/j.1460-2075.1984.tb01999.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56(1):125–135. doi: 10.1016/0378-1119(87)90165-x. [DOI] [PubMed] [Google Scholar]
  24. Schlegel R., Wade M. Biologically active peptides of the vesicular stomatitis virus glycoprotein. J Virol. 1985 Jan;53(1):319–323. doi: 10.1128/jvi.53.1.319-323.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Skehel J. J., Bayley P. M., Brown E. B., Martin S. R., Waterfield M. D., White J. M., Wilson I. A., Wiley D. C. Changes in the conformation of influenza virus hemagglutinin at the pH optimum of virus-mediated membrane fusion. Proc Natl Acad Sci U S A. 1982 Feb;79(4):968–972. doi: 10.1073/pnas.79.4.968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Stegmann T., Doms R. W., Helenius A. Protein-mediated membrane fusion. Annu Rev Biophys Biophys Chem. 1989;18:187–211. doi: 10.1146/annurev.bb.18.060189.001155. [DOI] [PubMed] [Google Scholar]
  27. Trono D., Feinberg M. B., Baltimore D. HIV-1 Gag mutants can dominantly interfere with the replication of the wild-type virus. Cell. 1989 Oct 6;59(1):113–120. doi: 10.1016/0092-8674(89)90874-x. [DOI] [PubMed] [Google Scholar]
  28. White J., Kielian M., Helenius A. Membrane fusion proteins of enveloped animal viruses. Q Rev Biophys. 1983 May;16(2):151–195. doi: 10.1017/s0033583500005072. [DOI] [PubMed] [Google Scholar]
  29. Whitt M. A., Chong L., Rose J. K. Glycoprotein cytoplasmic domain sequences required for rescue of a vesicular stomatitis virus glycoprotein mutant. J Virol. 1989 Sep;63(9):3569–3578. doi: 10.1128/jvi.63.9.3569-3578.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wilson I. A., Skehel J. J., Wiley D. C. Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature. 1981 Jan 29;289(5796):366–373. doi: 10.1038/289366a0. [DOI] [PubMed] [Google Scholar]
  31. Woodgett C., Rose J. K. Amino-terminal mutation of the vesicular stomatitis virus glycoprotein does not affect its fusion activity. J Virol. 1986 Aug;59(2):486–489. doi: 10.1128/jvi.59.2.486-489.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]