Recent studies with isolated M protein from influenza virus have shown that the protein has a high affinity for lipid. The ability of M to partition into lipid vesicles merely by shaking vesicles and M together is suggestive evidence that the protein could be interacting with the lipid in the virus particle. A more direct analysis was carried out here to determine whether M is in contact with the viral lipid in situ, by using the photoactivatable hydrophobic probe, pyrenesulfonyl azide. Covalent linkage of this probe to M indicated that a segment of M resides within the virus membrane in contact with the lipid bilayer. M inserted into lipid vesicles at two locations on the molecule. A major insertion into lipid occurred in the middle of the molecule where a large cluster of 20 hydrophobic and neutral amino acids occurs. A second insertion occurred approximately one fourth in from the amino terminus, where a smaller segment of 13 uncharged amino acids is found. Confirmation that M inserted into lipid at these locations came also from results with cyanogen bromide fragments of M. Of the 12 to 13 fragments produced, 3 specifically bound to lipid vesicles. These were the first, second, and third contiguous segments beginning at the amino terminus and containing the two hydrophobic areas noted above.
The M protein of influenza is the predominant structural component of the virus. The interactions of this protein with the viral lipid or with other proteins are not known. The ability of M to interact with viral or other lipids was investigated. Purified M was mixed with viral lipid or egg phosphatidylcholine and was incorporated into vesicles (i) by addition of sodium deoxycholate followed by dialysis or (ii) by sonication. Between 90 and 100% of the M became firmly associated with the lipid by either of these two methods, whereas nucleoprotein failed to associate with the vesicles. Firm association also occurred if M was mixed with performed vesicles. Most of the M attached to the vesicles could be hydrolyzed with proteolytic enzymes such as trypsin or thermolysin, except for a small fragment of about 5,000 daltons which remained associated with the lipid vesicles. The ability of fragments of M to interact with lipids was also investigated. Of 13 fragments produced by cleavage with cyanogen bromide, 3 specifically associated with lipid vesicles. The data indicate that a specific portion of the M molecule has a high affinity for lipid bilayers of various origins.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.