3-3--dioctadecyloxacarbocyanine and iodonaphthylazide

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.

Topics: 2,2'-Dipyridyl; Animals; Azides; Carbocyanines; Cell Line; Disulfides; Fluorescent Dyes; Glycoproteins; HIV-1; HLA-DR Antigens; Humans; Iodine Radioisotopes; Kinetics; Membrane Fusion; Mice; Photochemistry; Photosensitizing Agents; Simian Immunodeficiency Virus; Sulfhydryl Reagents; Viral Envelope Proteins

Fluorescent lipid analogue 3,3'-dioctadecyloxacarbocyanine incorporated into biological membranes was used to induce photoactivation of a hydrophobic probe 5-[125I]iodonaphthyl-1-azide (125INA) by energy transfer and to thereby confine subsequent radiolabelling of proteins to the lipid bilayer. This approach was applied in bovine chromaffin cells to discover cytosolic proteins that reversibly penetrate into membrane domains. alpha-Glutathione S-transferase (alpha-GST) was identified as the only labelled protein in bovine chromaffin-cell cytosol, indicating that it inserts reversibly into the membrane lipid bilayer. The selectivity of the labelling towards the lipid bilayer is demonstrated by showing that influenza virus haemagglutinin becomes labelled by 125INA only after the insertion of this protein into the target membrane. The molar 125INA:protein ratio was used as a quantitative criterion for evaluation of the penetration of proteins into the membrane lipid bilayer. This ratio was calculated for four integral membrane proteins and four soluble proteins that interact with biological membranes. The values for four integral membrane proteins (erythrocyte anion transporter, multidrug transporter gp-170, dopamine transporter and fusion-competent influenza virus haemagglutinin) were 1, 8, 2 and 2, respectively, whereas for soluble proteins (annexin VII, protein kinase C, BSA and influenza virus haemagglutinin) the values were 0.002, 0, 0.002 and 0.02, respectively. The molar ratio for alpha-GST was found to be 1, compatible with the values obtained for integral membrane proteins.

Topics: Adrenal Medulla; Animals; Azides; Carbocyanines; Cattle; Cell Membrane; Cells, Cultured; Chromaffin Cells; Chromaffin Granules; Cytosol; Energy Transfer; Fluorescent Dyes; Glutathione Transferase; Iodine Radioisotopes; Lipid Bilayers

The interaction of VSV glycoprotein (VSV G) with biological membranes was studied by photosensitized labeling. The method is based on photosensitized activation by the fluorescent lipid analog 3,3'-dioctadecyloxacarbocyanine (DiO) of a hydrophobic probe, [125I]iodonaphthyl azide (125INA), that rapidly partitions into the membrane bilayer of virus and cells. 125INA labeling of proteins and lipids can be confined to the site of chromophore localization by photosensitized labeling. Photoactivation using visible light of target membrane labeled with DiO and 125INA, to which unlabeled virions are bound, results in exclusive labeling of envelope glycoproteins inserted into the target membrane [Pak et al. (1994) J. Biol. Chem. 269, 14614]. In this study, we labeled lipid symmetric erythrocyte ghosts with 125INA and DiO. Photosensitized activation of VSV prebound to labeled ghosts with visible light resulted in VSV G labeling under fusogenic conditions. Photoactivation of 125INA by UV light, which is nonspecific, produced labeled VSV G at both acidic and neutral pH. Photosensitized labeling of VSV G by DiO-125INA-ghosts was also observed at pH 5.5, 4 degrees C, in the absence of mixing between viral and cellular lipids, suggesting insertion of the ectodomain of VSV G. Soluble VSV G lacking the transmembrane domain inserted into DiO-125INA-ghosts under the same conditions as intact VSV G. DiO inserted into intact VSV appeared to be a suitable fluorophore for continuous kinetic measurements of membrane fusion by fluorescence dequenching. Our photosensitized labeling results establish biochemical correlates for the three states of VSV G, which we had proposed based on kinetic data [Clague et al., Biochemistry 29, 1303]. In addition, we found that VSV G insertion into the target membrane is reversible, suggesting a "velcro"-like attachment of the fusogenic domain with the target membrane.

Topics: Affinity Labels; Azides; Carbocyanines; Electrophoresis, Polyacrylamide Gel; Erythrocyte Membrane; Fluorescent Dyes; Glycoproteins; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Iodine Radioisotopes; Membrane Glycoproteins; Photosensitizing Agents; Protein Conformation; Ultraviolet Rays; Viral Envelope Proteins; Viral Fusion Proteins