1887

Abstract

A number of viral membrane fusion proteins can be expressed alone on the surface of host cells, and then triggered to induce cell-to-cell fusion or syncytium formation. Although rapid and easily observed, syncytium formation is not easily quantified and differences in fusion activity are not easily distinguished or measured. To address this problem, we developed a rapid and quantitative cell-to-cell fusion system that is useful for comparative analysis and may be suitable for high throughput screening. In this system, expression of a reporter protein, enhanced green fluorescent protein (EGFP), is dependent on cell-to-cell fusion. (9) insect cells expressing a chimeric Lac repressor-IE1 protein were fused to 9 cells containing an reporter construct under the control of a responsive operator-containing promoter. Membrane fusion efficiency was measured from the resulting EGFP fluorescence activity. 9 cells expressing the multicapsid nucleopolyhedrovirus (MNPV) GP64 envelope fusion protein were used as a model to test this fusion assay. Subtle changes in fusion activities of GP64 proteins containing single amino acid substitutions in a putative membrane fusion domain were distinguished, and decreases in EGFP fluorescence corresponded to decreases in the hydrophobicity in the small putative membrane fusion domain.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-82-10-2519
2001-10-01
2019-12-05
Loading full text...

Full text loading...

/deliver/fulltext/jgv/82/10/0822519a.html?itemId=/content/journal/jgv/10.1099/0022-1317-82-10-2519&mimeType=html&fmt=ahah

References

  1. Bagai, S. & Lamb, R. A. ( 1995; ). Quantitative measurement of paramyxovirus fusion: differences in requirements of glycoproteins between simian virus 5 and human parainfluenza virus 3 or Newcastle disease virus. Journal of Virology 69, 6712-6719.
    [Google Scholar]
  2. Blissard, G. W. & Rohrmann, G. F. ( 1991; ). Baculovirus gp64 gene expression: analysis of sequences modulating early transcription and transactivation by IE1. Journal of Virology 65, 5820-5827.
    [Google Scholar]
  3. Blissard, G. W. & Wenz, J. R. ( 1992; ). Baculovirus GP64 envelope glycoprotein is sufficient to mediate pH dependent membrane fusion. Journal of Virology 66, 6829-6835.
    [Google Scholar]
  4. Camerini, D., Planelles, V. & Chen, I. S. ( 1994; ). CD26 antigen and HIV fusion? Science 264, 1160–1161; discussion 1162–1165.[CrossRef]
    [Google Scholar]
  5. Chernomordik, L., Leikina, E., Cho, M. & Zimmerberg, J. ( 1995; ). Control of baculovirus GP64-induced syncytium formation by membrane lipid composition. Journal of Virology 69, 3049-3058.
    [Google Scholar]
  6. Choe, H., Farzan, M., Sun, Y., Sullivan, N., Rollins, B., Ponath, P. D., Wu, L., Mackay, C. R., LaRosa, G., Newman, W., Gerard, N., Gerard, C. & Sodroski, J. ( 1996; ). The beta-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell 85, 1135-1148.[CrossRef]
    [Google Scholar]
  7. Deng, H., Liu, R., Ellmeier, W., Choe, S., Unutmaz, D., Burkhart, M., Di Marzio, P., Marmon, S., Sutton, R. E., Hill, C. M., Davis, C. B., Peiper, S. C., Schall, T. J., Littman, D. R. & Landau, N. R. ( 1996; ). Identification of a major co-receptor for primary isolates of HIV-1. Nature 381, 661-666.[CrossRef]
    [Google Scholar]
  8. Doms, R. W., Helenius, A. & White, J. ( 1985; ). Membrane fusion activity of the influenza virus hemagglutinin. The low pH-induced conformational change. Journal of Biological Chemistry 260, 2973-2981.
    [Google Scholar]
  9. Doms, R. W., Blumenthal, R. & Moss, B. ( 1990; ). Fusion of intracellular and extracellular forms of vaccinia virus with the cell membrane. Journal of Virology 64, 4884-4892.
    [Google Scholar]
  10. Feng, Y., Broder, C. C., Kennedy, P. E. & Berger, E. A. ( 1996; ). HIV-1 entry cofactor: functional cDNA cloning of a seven- transmembrane, G protein-coupled receptor. Science 272, 872-877.[CrossRef]
    [Google Scholar]
  11. Florkiewicz, R. Z. & Rose, J. K. ( 1984; ). A cell line expressing vesicular stomatitis virus glycoprotein fuses at low pH. Science 225, 721-723.[CrossRef]
    [Google Scholar]
  12. Fuerst, T. R., Niles, E. G., Studier, F. W. & Moss, B. ( 1986; ). Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proceedings of the National Academy of Sciences, USA 83, 8122-8126.[CrossRef]
    [Google Scholar]
  13. Gotte, M. & von Mollard, G. F. ( 1998; ). A new beat for the SNARE drum. Trends in Cell Biology 8, 215-218.[CrossRef]
    [Google Scholar]
  14. Hohmann, A. W. & Faulkner, P. ( 1983; ). Monoclonal antibodies to baculovirus structural proteins: determination of specificities by Western blot analysis. Virology 125, 432-444.[CrossRef]
    [Google Scholar]
  15. Hsu, S. C., Hazuka, C. D., Foletti, D. L. & Scheller, R. H. ( 1999; ). Targeting vesicles to specific sites on the plasma membrane: the role of the sec6/8 complex. Trends in Cell Biology 9, 150-153.[CrossRef]
    [Google Scholar]
  16. Hughson, F. M. ( 1995; ). Structural characterization of viral fusion proteins. Current Biology 5, 265-274.[CrossRef]
    [Google Scholar]
  17. Kingsley, D. H., Behbahani, A., Rashtian, A., Blissard, G. W. & Zimmerberg, J. ( 1999; ). A discrete stage of baculovirus GP64-mediated membrane fusion. Molecular Biology of the Cell 10, 4191-4200.[CrossRef]
    [Google Scholar]
  18. Laemmli, U. K. ( 1970; ). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[CrossRef]
    [Google Scholar]
  19. Leikina, E., Onaran, H. O. & Zimmerberg, J. ( 1992; ). Acidic pH induces fusion of cells infected with baculovirus to form syncytia. FEBS Letters 304, 221-224.[CrossRef]
    [Google Scholar]
  20. Lindau, M. & Almers, W. ( 1995; ). Structure and function of fusion pores in exocytosis and ectoplasmic membrane fusion. Current Opinion in Cell Biology 7, 509-517.[CrossRef]
    [Google Scholar]
  21. Markovic, I., Pulyaeva, H., Sokoloff, A. & Chernomordik, L. V. ( 1998; ). Membrane fusion mediated by baculovirus gp64 involves assembly of stable gp64 trimers into multiprotein aggregates. Journal of Cell Biology 143, 1155-1166.[CrossRef]
    [Google Scholar]
  22. Monsma, S. A. & Blissard, G. W. ( 1995; ). Identification of a membrane fusion domain and an oligomerization domain in the baculovirus GP64 envelope fusion protein. Journal of Virology 69, 2583-2595.
    [Google Scholar]
  23. Monsma, S. A., Oomens, A. G. P. & Blissard, G. W. ( 1996; ). The GP64 envelope fusion protein is an essential baculovirus protein required for cell to cell transmission of infection. Journal of Virology 70, 4607-4616.
    [Google Scholar]
  24. Nussbaum, O., Broder, C. C. & Berger, E. A. ( 1994; ). Fusogenic mechanisms of enveloped-virus glycoproteins analyzed by a novel recombinant vaccinia virus-based assay quantitating cell fusion-dependent reporter gene activation. Journal of Virology 68, 5411-5422.
    [Google Scholar]
  25. Oomens, A. G. P., Monsma, S. A. & Blissard, G. W. ( 1995; ). The baculovirus GP64 envelope fusion protein: synthesis, oligomerization, and processing. Virology 209, 592-603.[CrossRef]
    [Google Scholar]
  26. Plonsky, I. & Zimmerberg, J. ( 1996; ). The initial fusion pore induced by baculovirus GP64 is large and forms quickly. Journal of Cell Biology 135, 1831-1839.[CrossRef]
    [Google Scholar]
  27. Rodriguez, J. F. & Esteban, M. ( 1987; ). Mapping and nucleotide sequence of the vaccinia virus gene that encodes a 14-kilodalton fusion protein. Journal of Virology 61, 3550-3554.
    [Google Scholar]
  28. Rothman, J. E. & Sollner, T. H. ( 1997; ). Throttles and dampers: controlling the engine of membrane fusion [see comments]. Science 276, 1212-1213.[CrossRef]
    [Google Scholar]
  29. Slack, J. M. & Blissard, G. W. ( 1997; ). Identification of two independent transcriptional activation domains in the Autographa californica multicapsid nuclear polyhedrosis virus IE1 protein. Journal of Virology 71, 9579-9587.
    [Google Scholar]
  30. Stegmann, T., Doms, R. W. & Helenius, A. ( 1989; ). Protein-mediated membrane fusion. Annual Review of Biophysics and Biophysical Chemistry 18, 187-212.[CrossRef]
    [Google Scholar]
  31. Steinman, R. M., Mellman, I. S., Muller, W. A. & Cohn, Z. A. ( 1983; ). Endocytosis and the recycling of plasma membrane. Journal of Cell Biology 96, 1-27.[CrossRef]
    [Google Scholar]
  32. Volkman, L. E. & Goldsmith, P. A. ( 1985; ). Mechanism of neutralization of budded Autographa californica nuclear polyhedrosis virus by a monoclonal antibody: inhibition of entry by adsorptive endocytosis. Virology 143, 185-195.[CrossRef]
    [Google Scholar]
  33. Weissenhorn, W., Dessen, A., Calder, L. J., Harrison, S. C., Skehel, J. J. & Wiley, D. C. ( 1999; ). Structural basis for membrane fusion by enveloped viruses. Molecular Membrane Biology 16, 3-9.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-82-10-2519
Loading
/content/journal/jgv/10.1099/0022-1317-82-10-2519
Loading

Data & Media loading...

Most Cited This Month

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