Localization of Membrane-Associated Proteins in Vesicular Stomatitis Virus by Use of Hydrophobic Membrane Probes and Cross-Linking Reagents

Publisher Website
Google Scholar
Abstract
The location of membrane-associated proteins of vesicular stomatitis virus was investigated using 2 monofunctional and 3 bifunctional probes that differ in the degree to which they partition into membranes and in their specific group reactivity. Two hydrophobic aryl azide probes, [125I]5-iodonaphthyl-1-azide and [3H]pyrenesulfonylazide, readily partitioned into virion membrane and, when activated to nitrenes by UV irradiation, formed stable covalent adducts to membrane constituents. Both monofunctional probes labeled the glycoprotein G and matrix M proteins; [125I]5-iodonaphthyl-1-azide labeled the nucleocapsid N protein and an unidentified low MW component. Protein labeling of intact virions was unaffected by the presence of cytochrome c or glutathione, but disruption of membrane by sodium dodecyl sulfate greatly enhanced the labeling of all viral proteins except G. Labeling of G protein was essentially restricted to the membrane-embedded, thermolysin-resistant tail fragment. Three bifunctional reagents (tartryl diazide, dimethylsuberimidate and 4,4''-dithiobisphenylazide) were tested for their capacity to cross-link proteins to membrane phospholipids of virions grown in the presence of [3H]palmitate. Only G and M proteins of intact virions were labeled with 3H-phospholipid by these cross-linkers; the reactions were not affected by cytochrome c but were abolished by disruption of virus with sodium dodecyl sulfate. Dimethylsuberimidate, which reacts with free amino groups, cross-linked 3H-phospholipid to G and M protein. The hydrophilic tartryl diazide cross-linked phospholipid primarily to the M protein; the hydrophobic 4,4''-dithiobisphenylazide cross-linked phospholipid primarily to the intrinsic G protein. The G protein probably traverses the virion membrane, and the M protein is probably membrane-associated but does not penetrate deeply, if at all.