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© The Rockefeller University Press, 0021-9525/1998//315 $5.00
The Journal of Cell Biology, Volume 140, Number 2, , 1998 315-323

Article

Dilation of the Human Immunodeficiency Virus–1 Envelope Glycoprotein Fusion Pore Revealed by the Inhibitory Action of a Synthetic Peptide from gp41



Isabel Muñoz-Barroso*, Stewart Durell*, Kazuyasu Sakaguchi{ddagger}, Ettore Appella{ddagger}, and Robert Blumenthal*

* Laboratory of Experimental and Computational Biology, National Institutes of Health, Frederick, MD; and {ddagger} Laboratory of Cell Biology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 21702-1201

We have monitored fusion between cell pairs consisting of a single human immunodeficiency virus–1 (HIV-1) envelope glycoprotein–expressing cell and a CD4+ target cell, which had been labeled with both a fluorescent lipid in the membrane and a fluorescent solute in the cytosol. We developed a new three-color assay to keep track of the cell into which fluorescent lipids and/or solutes are redistributed. Lipid and solute redistribution occur as a result of opening a lipid-permissive fusion pore and a solute-permissive fusion pore (FPS), respectively. A synthetic peptide (DP178) corresponding to residues 643–678 of the HIV-1LAI gp120-gp41 sequence (Wild, C.T., D.C. Shugars, T.K. Greenwell, C.B. McDanal, and T.J. Matthews. 1994. Proc. Natl. Acad. Sci. USA. 91:12676–12680) completely inhibited FPS at 50 ng/ml, whereas at that concentration there was 20–30% fusion activity measured by the lipid redistribution. The differences detected in lipid mixing versus contents mixing are maintained up to 6 h of coculture of gp120-41–expressing cells with target cells, indicating that DP178 can "clamp" the fusion complex in the lipid mixing intermediate for very long time periods. A peptide from the NH2-terminal of gp41, DP107, inhibited HIV-1LAI gp120-gp41–mediated cell fusion at higher concentrations, but with no differences between lipid and aqueous dye redistribution at the different inhibitor concentrations. The inhibition of solute redistribution by DP178 was complete when the peptide was added to the fusion reaction mixture during the first 15 min of coculture. We have analyzed the inhibition data in terms of a fusion pore dilation model that incorporates the recently determined high resolution structure of the gp41 core.

Abbreviations used in this paper: CMAC, 7-amino-4-chloromethylcoumarin; DiI, 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate; DiO, 3,3'-dioctadecyloxaocarbocyanine perchlorate; FPL, lipid-permissive fusion pore; FPS, solute permissive fusion pore; HA, hemagglutinin; HIV-1, human immunodeficiency virus type–1; IC50, 50% effective inhibitory concentration; RBCs, red blood cells.

The work was supported by the AIDS Intramural Targeted Antiviral Program. I. Muñoz-Barroso is a postdoctoral fellow of the Direccion General de Investigacion Cientifica y Enseñanza Superior, Spain.

Address all correspondence to Robert Blumenthal, Miller Drive, P.O. Box B, Building 469, Room 213, Frederick, MD 21702-1201. Tel.: (301) 846-1446. Fax: (301) 846-6192. E-mail: blumen{at}helix.nih.gov


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