Quantitative measurement of fusion of HIV-1 and SIV with cultured cells using photosensitized labeling.
The fusion of HIV and SIV with biological membranes was studied by photosensitized activation of a hydrophobic probe, [(125)I]iodonaphthylazide ([(125)I]INA), by a fluorescent lipid which is situated in the target membrane. Photosensitized labeling of viral envelope-resident proteins occurs only upon their insertion into target membranes. Photosensitized labeling as a result of HIV-1 Env-mediated cell fusion showed the same kinetics as aqueous dye transfer. We have for the first time measured kinetics of HIV and SIV virus-cell fusion. HIV-1(MN) virions were about 10x less fusion active than SIVmne virions. SIV inactivated by aldrithiol-2 retained fusion activity similar to that seen with untreated virus. The relatively slow time course of SIV-cell fusion (t(1/2) = 19 min) indicates that the fusion events are stochastic. This feature provides a basis for understanding the mode of action of HIV/SIV entry inhibitors that target transition states.
Raviv
Yossef
Y
Laboratory of Experimental and Computational Biology, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702, USA.
Viard
Mathias
M
Bess
Julian
J
Blumenthal
Robert
R
D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives*; D000818:Animals; D001386:Azides; D002232:Carbocyanines; D002460:Cell Line; D004220:Disulfides; D005456:Fluorescent Dyes; D006023:Glycoproteins / Q000378:metabolism; D015497:HIV-1 / Q000502:physiology*; D006684:HLA-DR Antigens / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D008561:Membrane Fusion; D051379:Mice; D010777:Photochemistry; D017319:Photosensitizing Agents; D015302:Simian Immunodeficiency Virus / Q000502:physiology*; D013439:Sulfhydryl Reagents; D014759:Viral Envelope Proteins / Q000378:metabolism
D001386:Azides; D002232:Carbocyanines; D004220:Disulfides; D005456:Fluorescent Dyes; D006023:Glycoproteins; D006684:HLA-DR Antigens; D007457:Iodine Radioisotopes; D017319:Photosensitizing Agents; D013439:Sulfhydryl Reagents; D014759:Viral Envelope Proteins; C001105:2,2'-dipyridyl disulfide; D015082:2,2'-Dipyridyl; C015298:iodonaphthylazide; C098044:3,3'-dioctadecyloxacarbocyanine