title: Amphipathic alpha-helices and putative cholesterol binding domains of the influenza virus matrix M1 protein are crucial for virion structure organisation.
abstract: The influenza virus matrix M1 protein is an amphitropic membrane-associated protein, forming the matrix layer immediately beneath the virus raft membrane, thereby ensuring the proper structure of the influenza virion. The objective of this study was to elucidate M1 fine structural characteristics, which determine amphitropic properties and raft membrane activities of the protein, via 3D in silico modelling with subsequent mutational analysis. Computer simulations suggest the amphipathic nature of the M1 α-helices and the existence of putative cholesterol binding (CRAC) motifs on six amphipathic α-helices. Our finding explains for the first time many features of this protein, particularly the amphitropic properties and raft/cholesterol binding potential. To verify these results, we generated mutants of the A/WSN/33 strain via reverse genetics. The M1 mutations included F32Y in the CRAC of α-helix 2, W45Y and W45F in the CRAC of α-helix 3, Y100S in the CRAC of α-helix 6, M128A and M128S in the CRAC of α-helix 8 and a double L103I/L130I mutation in both a putative cholesterol consensus motif and the nuclear localisation signal. All mutations resulted in viruses with unusual filamentous morphology. Previous experimental data regarding the morphology of M1-gene mutant influenza viruses can now be explained in structural terms and are consistent with the pivotal role of the CRAC-domains and amphipathic α-helices in M1-lipid interactions.
authors.affiliation: Mechnikov Research Institute for Vaccines and Sera, Russian Academy of Medical Sciences, Mal. Kazenij per. Moscow, Russia; Institut Gustave Roussy, 39, rue Camille-Desmoulins, 94805 Villejuif, France.
authors.forename: Tatyana
authors.initials: T
authors.lastname: Tsfasman
authors.affiliation: Mechnikov Research Institute for Vaccines and Sera, Russian Academy of Medical Sciences, Mal. Kazenij per. Moscow, Russia.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Kost
authors.affiliation: Mechnikov Research Institute for Vaccines and Sera, Russian Academy of Medical Sciences, Mal. Kazenij per. Moscow, Russia.
authors.forename: Stanislav
authors.initials: S
authors.lastname: Markushin
authors.affiliation: Mechnikov Research Institute for Vaccines and Sera, Russian Academy of Medical Sciences, Mal. Kazenij per. Moscow, Russia.
authors.forename: Vera
authors.initials: V
authors.lastname: Lotte
authors.affiliation: Mechnikov Research Institute for Vaccines and Sera, Russian Academy of Medical Sciences, Mal. Kazenij per. Moscow, Russia.
authors.forename: Irina
authors.initials: I
authors.lastname: Koptiaeva
authors.affiliation: Semenov Institute of Chemical Physics, Russian Academy of Sciences, Kosygina Ul., 4, 119991 Moscow, Russia.
authors.forename: Elena
authors.initials: E
authors.lastname: Bogacheva
authors.affiliation: Belozersky Institute of Physico-Chemical Biology, Moscow State University, Leninskie Gory, 119991 Moscow, Russia.
authors.forename: Ludmila
authors.initials: L
authors.lastname: Baratova
authors.affiliation: Belozersky Institute of Physico-Chemical Biology, Moscow State University, Leninskie Gory, 119991 Moscow, Russia. Electronic address: varvic@belozersky.msu.ru.
authors.forename: Victor
authors.initials: V
authors.lastname: Radyukhin
mesh_terms: D000818:Animals; D001665:Binding Sites; D002784:Cholesterol / Q000378:metabolism*; D004252:DNA Mutational Analysis; D009980:Influenza A virus / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure*; D008958:Models, Molecular; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D059386:Reverse Genetics; D014763:Viral Matrix Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000648:ultrastructure
chemical_list: C488936:M1 protein, Influenza A virus; D014763:Viral Matrix Proteins; D002784:Cholesterol
keywords: Cholesterol binding (CRAC) domain; Influenza virus; M1 protein structure