Progressive truncations C terminal to the membrane-spanning domain of simian immunodeficiency virus Env reduce fusogenicity and increase concentration dependence of Env for fusion.
The simian immunodeficiency virus (SIV) transmembrane (TM) protein, gp41, has multiple functions, which include anchoring the glycoprotein complex in the lipid envelope of the virus and mediating fusion of the virus and host cell membranes. Recently, a series of mutants of the SIVmac239 TM protein that have truncations at the carboxyl terminus of the membrane-spanning domain (MSD) have been characterized (J. T. West, P. Johnston, S. R. Dubay, and E. Hunter, J. Virol. 75:9601-9612, 2001). These mutants retained membrane anchorage but demonstrated reduced fusogenicity and infectivity as the MSD length was shortened. We have established a novel three-color fluorescence assay, which allows qualitative confocal and quantitative flow cytometric analyses, to further characterize the nature of the fusion defect in five of the MSD mutants: TM185, TM186, TM187, TM188, and TM189. Our analysis showed that each mutant could mediate complete lipid and aqueous dye transfer at early time points after effector and target cell mixing. No hemifusion with only lipid dye flux was detected. However, another intermediate fusion stage, which appears to involve small-fusion-pore formation that allowed small aqueous dye transfer but prevented the exchange of large cytoplasmic components, was identified infrequently in mutant-Env-expressing cell and target cell mixtures. Quantitative flow cytometric analysis of these mutants demonstrated that the TM187, TM188, and TM189 mutants were significantly more fusogenic than TM185 and TM186 but remained significantly impaired compared to the wild type. Moreover, fusion efficiency showed an increased dependence on the expression level of glycoproteins, suggesting that, for these mutants, formation of an active fusion complex was an increasingly stochastic event.
Lin
Xiaoxu
X
Department of Microbiology, The University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Derdeyn
Cynthia A
CA
Blumenthal
Robert
R
West
John
J
Hunter
Eric
E
D000818:Animals; D019556:COS Cells; D002459:Cell Fusion; D002462:Cell Membrane / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D008561:Membrane Fusion; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal / Q000379:methods; D009154:Mutation; D012191:Retroviridae Proteins / Q000235:genetics / Q000378:metabolism*; D015302:Simian Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*
D005456:Fluorescent Dyes; D008562:Membrane Glycoproteins; D012191:Retroviridae Proteins; C082614:SIV envelope protein gp41