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. 1993 Jul;67(7):4070–4077. doi: 10.1128/jvi.67.7.4070-4077.1993

Mutational analysis of the vesicular stomatitis virus glycoprotein G for membrane fusion domains.

Y Li 1, C Drone 1, E Sat 1, H P Ghosh 1
PMCID: PMC237775  PMID: 8389917

Abstract

The spike glycoprotein G of vesicular stomatitis virus (VSV) induces membrane fusion at low pH. We used linker insertion mutagenesis to characterize the domain(s) of G glycoprotein involved in low-pH-induced membrane fusion. Two or three amino acids were inserted in frame into various positions in the extracellular domain of G, and 14 mutants were isolated. All of the mutants expressed fully glycosylated proteins in COS cells. However, only seven mutant G glycoproteins were transported to the cell surface. Two of these mutants, D1 and A6, showed wild-type fusogenic properties. The mutant A2 had a temperature-sensitive defect in the transport of the mutant G glycoprotein to the cell surface. The other four mutants, H2, H5, H10, and A4, although present in cell surface, failed to induce cell fusion when cells expressing these mutant glycoproteins were exposed to acidic pH. These four mutant G proteins could form trimers, indicating that the defect in fusion was not due to defective oligomerization. One of these mutations, H2, is within a region of conserved, uncharged amino acids that has been proposed as a possible fusogenic sequence. The mutation in H5 was about 70 amino acids downstream of the mutation in H2, while mutations in H10 and A4 were about 300 amino acids downstream of the mutation in H2. Conserved sequences were also noted in the H10 and A4 segment. The results suggest that in the case of VSV G glycoprotein, the fusogenic activity may involve several spatially separated regions in the extracellular domain of the protein.

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Selected References

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

  1. Ali M. A., Butcher M., Ghosh H. P. Expression and nuclear envelope localization of biologically active fusion glycoprotein gB of herpes simplex virus in mammalian cells using cloned DNA. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5675–5679. doi: 10.1073/pnas.84.16.5675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson R. G., Orci L. A view of acidic intracellular compartments. J Cell Biol. 1988 Mar;106(3):539–543. doi: 10.1083/jcb.106.3.539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Butcher M., Raviprakash K., Ghosh H. P. Acid pH-induced fusion of cells by herpes simplex virus glycoproteins gB an gD. J Biol Chem. 1990 Apr 5;265(10):5862–5868. [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. 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]
  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. Freed E. O., Myers D. J., Risser R. Characterization of the fusion domain of the human immunodeficiency virus type 1 envelope glycoprotein gp41. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4650–4654. doi: 10.1073/pnas.87.12.4650. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  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. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  12. Guan J. L., Ruusala A., Cao H., Rose J. K. Effects of altered cytoplasmic domains on transport of the vesicular stomatitis virus glycoprotein are transferable to other proteins. Mol Cell Biol. 1988 Jul;8(7):2869–2874. doi: 10.1128/mcb.8.7.2869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Horvath C. M., Lamb R. A. Studies on the fusion peptide of a paramyxovirus fusion glycoprotein: roles of conserved residues in cell fusion. J Virol. 1992 Apr;66(4):2443–2455. doi: 10.1128/jvi.66.4.2443-2455.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hurtley S. M., Helenius A. Protein oligomerization in the endoplasmic reticulum. Annu Rev Cell Biol. 1989;5:277–307. doi: 10.1146/annurev.cb.05.110189.001425. [DOI] [PubMed] [Google Scholar]
  15. Kielian M., Helenius A. pH-induced alterations in the fusogenic spike protein of Semliki Forest virus. J Cell Biol. 1985 Dec;101(6):2284–2291. doi: 10.1083/jcb.101.6.2284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kondor-Koch C., Burke B., Garoff H. Expression of Semliki Forest virus proteins from cloned complementary DNA. I. The fusion activity of the spike glycoprotein. J Cell Biol. 1983 Sep;97(3):644–651. doi: 10.1083/jcb.97.3.644. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kornfeld R., Kornfeld S. Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem. 1985;54:631–664. doi: 10.1146/annurev.bi.54.070185.003215. [DOI] [PubMed] [Google Scholar]
  18. Kotwal G. J., Capone J., Irving R. A., Rhee S. H., Bilan P., Toneguzzo F., Hofmann T., Ghosh H. P. Viral membrane glycoproteins: comparison of the amino terminal amino acid sequences of the precursor and mature glycoproteins of three serotypes of vesicular stomatitis virus. Virology. 1983 Aug;129(1):1–11. doi: 10.1016/0042-6822(83)90390-2. [DOI] [PubMed] [Google Scholar]
  19. Kreis T. E., Lodish H. F. Oligomerization is essential for transport of vesicular stomatitis viral glycoprotein to the cell surface. Cell. 1986 Sep 12;46(6):929–937. doi: 10.1016/0092-8674(86)90075-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lefrancios L., Lyles D. S. The interactionof antiody with the major surface glycoprotein of vesicular stomatitis virus. I. Analysis of neutralizing epitopes with monoclonal antibodies. Virology. 1982 Aug;121(1):157–167. [PubMed] [Google Scholar]
  21. Levy-Mintz P., Kielian M. Mutagenesis of the putative fusion domain of the Semliki Forest virus spike protein. J Virol. 1991 Aug;65(8):4292–4300. doi: 10.1128/jvi.65.8.4292-4300.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Masters P. S., Bhella R. S., Butcher M., Patel B., Ghosh H. P., Banerjee A. K. Structure and expression of the glycoprotein gene of Chandipura virus. Virology. 1989 Jul;171(1):285–290. doi: 10.1016/0042-6822(89)90540-0. [DOI] [PubMed] [Google Scholar]
  24. Paterson R. G., Hiebert S. W., Lamb R. A. Expression at the cell surface of biologically active fusion and hemagglutinin/neuraminidase proteins of the paramyxovirus simian virus 5 from cloned cDNA. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7520–7524. doi: 10.1073/pnas.82.22.7520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Puri A., Winick J., Lowy R. J., Covell D., Eidelman O., Walter A., Blumenthal R. Activation of vesicular stomatitis virus fusion with cells by pretreatment at low pH. J Biol Chem. 1988 Apr 5;263(10):4749–4753. [PubMed] [Google Scholar]
  26. Raviprakash K., Rasile L., Ghosh K., Ghosh H. P. Shortened cytoplasmic domain affects intracellular transport but not nuclear localization of a viral glycoprotein. J Biol Chem. 1990 Jan 25;265(3):1777–1782. [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. 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]
  30. Schneider M., Graham F. L., Prevec L. Expression of the glycoprotein of vesicular stomatitis virus by infectious adenovirus vectors. J Gen Virol. 1989 Feb;70(Pt 2):417–427. doi: 10.1099/0022-1317-70-2-417. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Tanese N., Goff S. P. Domain structure of the Moloney murine leukemia virus reverse transcriptase: mutational analysis and separate expression of the DNA polymerase and RNase H activities. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1777–1781. doi: 10.1073/pnas.85.6.1777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tanese N., Roth M., Epstein H., Goff S. P. An insertion mutation in the pol gene of Moloney murine leukemia virus results in temperature-sensitive pol maturation and viral replication. Virology. 1989 Jun;170(2):378–384. doi: 10.1016/0042-6822(89)90428-5. [DOI] [PubMed] [Google Scholar]
  34. Vandepol S. B., Lefrancois L., Holland J. J. Sequences of the major antibody binding epitopes of the Indiana serotype of vesicular stomatitis virus. Virology. 1986 Jan 30;148(2):312–325. doi: 10.1016/0042-6822(86)90328-4. [DOI] [PubMed] [Google Scholar]
  35. Wharton S. A., Ruigrok R. W., Martin S. R., Skehel J. J., Bayley P. M., Weis W., Wiley D. C. Conformational aspects of the acid-induced fusion mechanism of influenza virus hemagglutinin. Circular dichroism and fluorescence studies. J Biol Chem. 1988 Mar 25;263(9):4474–4480. [PubMed] [Google Scholar]
  36. White J. M. Membrane fusion. Science. 1992 Nov 6;258(5084):917–924. doi: 10.1126/science.1439803. [DOI] [PubMed] [Google Scholar]
  37. White J. M. Viral and cellular membrane fusion proteins. Annu Rev Physiol. 1990;52:675–697. doi: 10.1146/annurev.ph.52.030190.003331. [DOI] [PubMed] [Google Scholar]
  38. White J., Helenius A., Gething M. J. Haemagglutinin of influenza virus expressed from a cloned gene promotes membrane fusion. Nature. 1982 Dec 16;300(5893):658–659. doi: 10.1038/300658a0. [DOI] [PubMed] [Google Scholar]
  39. White J., Matlin K., Helenius A. Cell fusion by Semliki Forest, influenza, and vesicular stomatitis viruses. J Cell Biol. 1981 Jun;89(3):674–679. doi: 10.1083/jcb.89.3.674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Whitt M. A., Zagouras P., Crise B., Rose J. K. A fusion-defective mutant of the vesicular stomatitis virus glycoprotein. J Virol. 1990 Oct;64(10):4907–4913. doi: 10.1128/jvi.64.10.4907-4913.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wiley D. C., Skehel J. J. The structure and function of the hemagglutinin membrane glycoprotein of influenza virus. Annu Rev Biochem. 1987;56:365–394. doi: 10.1146/annurev.bi.56.070187.002053. [DOI] [PubMed] [Google Scholar]
  42. Yang Y. C., Ciarletta A. B., Temple P. A., Chung M. P., Kovacic S., Witek-Giannotti J. S., Leary A. C., Kriz R., Donahue R. E., Wong G. G. Human IL-3 (multi-CSF): identification by expression cloning of a novel hematopoietic growth factor related to murine IL-3. Cell. 1986 Oct 10;47(1):3–10. doi: 10.1016/0092-8674(86)90360-0. [DOI] [PubMed] [Google Scholar]
  43. de Silva A. M., Balch W. E., Helenius A. Quality control in the endoplasmic reticulum: folding and misfolding of vesicular stomatitis virus G protein in cells and in vitro. J Cell Biol. 1990 Sep;111(3):857–866. doi: 10.1083/jcb.111.3.857. [DOI] [PMC free article] [PubMed] [Google Scholar]

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