MINT MINT, Dpt of Biology, University of Rome Tor Vergata MINT http://mint.bio.uniroma2.it/mint url>http://mint.bio.uniroma2.it/mint/ https://mint.bio.uniroma2.it MINT-[0-9]+ https://mint.bio.uniroma2.it/index.php/results-interactions/?id=${ac} The Molecular INTeraction database (MINT) is a relational database designed to store interactions between biological molecules. http://mint.bio.uniroma2.it Acylation and cholesterol binding are not required for targeting of influenza A virus M2 protein to the hemagglutinin-defined budozone. Acylation and cholesterol binding are not required for targeting of influenza A virus M2 protein to the hemagglutinin-defined budozone. FEBS Lett. (0014-5793) 2014 imex curation Thaa B., Siche S., Herrmann A., Veit M. mveit@zedat.fu-berlin.de 02-Apr-2014: Contacted by IntAct-Help. Only protein-protein interactions crigr-cho chinese hamster ovary cell line cho chinese hamster ovary cell line Also called LR73 cells. fret fluorescent resonance energy transfer FRET RET FRET analysis FRET RET FRET analysis fret tag fluorescence tag visualisation by fluorescence tag fluorescence mveit@zedat.fu-berlin.de Accepted 2014-FEB-06 by LUANA 2014 imex curation hema_i34a0 Hemagglutinin HA protein protein i34a0 Influenza A virus (strain A/Fowl plague virus/Rostock/8/1934 H7N1) MNTQILVFALVAVIPTNADKICLGHHAVSNGTKVNTLTERGVEVVNATETVERTNIPKICSKGKRTTDLGQCGLLGTITGPPQCDQFLEFSADLIIERREGNDVCYPGKFVNEEALRQILRGSGGIDKETMGFTYSGIRTNGTTSACRRSGSSFYAEMEWLLSNTDNASFPQMTKSYKNTRRESALIVWGIHHSGSTTEQTKLYGSGNKLITVGSSKYHQSFVPSPGTRPQINGQSGRIDFHWLILDPNDTVTFSFNGAFIAPNRASFLRGKSMGIQSDVQVDANCEGECYHSGGTITSRLPFQNINSRAVGKCPRYVKQESLLLATGMKNVPEPSKKREKRGLFGAIAGFIENGWEGLVDGWYGFRHQNAQGEGTAADYKSTQSAIDQITGKLNRLIEKTNQQFELIDNEFTEVEKQIGNLINWTKDFITEVWSYNAELLVAMENQHTIDLADSEMNKLYERVRKQLRENAEEDGTGCFEIFHKCDDDCMASIRNNTYDHSKYREEAMQNRIQIDPVKLSSGYKDVILWFSFGASCFLLLAIAVGLVFICVKNGNMRCTICI 544C0520B83714E2 m2_i80a6 Matrix protein 2 M Proton channel protein M2 protein protein i80a6 Influenza A virus (strain A/Duck/Hokkaido/8/1980 H3N8) MSLLTEVETPTRNGWECKCSDSSDPLVIAASIIGILHLILWILDRLFFKCIYRRLKYGLKRGPSTEGVPESMREEYRQEQQNAVDVDDGHFVNIELE C522EFFCB4370382 ha-m 1 M2 3 tag fluorescence tag visualisation by fluorescence tag fluorescence tag fluorescence tag visualisation by fluorescence tag fluorescence unspecified role unspecified role fluorescence accept fluorescence acceptor fluorescence accept over-expressed over expressed level over-expressed living cell living cell yfp tag yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined HA 2 tag fluorescence tag visualisation by fluorescence tag fluorescence tag fluorescence tag visualisation by fluorescence tag fluorescence unspecified role unspecified role fluorescence donor fluorescence donor over-expressed over expressed level over-expressed living cell living cell cer tag fluorescent prot tag fluorescent protein tag fluorescent prot tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined P03459:p.[Cys551Ser;Cys559Ser;Cys562Ser] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain C S certain certain sequence position certain certain certain sequence position certain C S certain certain sequence position certain certain certain sequence position certain C S Wed May 02 16:58:06 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'cys551ser,cys559ser,cys562ser' Tue Jun 30 20:08:52 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Cys551Ser;Cys559Ser;Cys562Ser]' proximity proximity close-contact colocalization neighbourhood interaction f2 f3a When HA–Cer-C3S (lacking all three acylation sites) was employed for FLIM-FRET a relative KD value of 0.72 was determined (Fig. 3A), clearly higher than in the experiments with HA–Cer-wt (KD = 0.13, Fig. 2C). Also, the fitting curve appears quasi-linear rather than distinctly leveling off as in the case of clustering. The FLIM-FRET measurement with HA–Cer-VIL3A and M2–YFP-wt yielded a relative KD of 0.29 (Fig. 3B). Thus, removing the raft-targeting features of HA clearly reduced clustering with M2, more pronounced with non-acylated HA. Acylation of HA may thus be more important for budozone organization than the VIL motif. Accordingly, lack of HA’s VIL motif in recombinant viruses is less dramatic than mutating individual HA acylation sites (viruses with non-acylated HA cannot be rescued; virus titers of virus with HA-VIL3A are reduced by 2–4 logs [14]). The evaluation of clustering between HA–Cer-wt and M2–YFP-wt yielded a relative KD value of 0.13 (Fig. 2C). This is very similar to the result obtained previously [25] for the same FRET pair (relative KD = 0.09). Thus, we corroborated that HA–Cer and M2–YFP cluster in the plasma membrane of live cells. ha-m-1 1 HA 2 tag fluorescence tag visualisation by fluorescence tag fluorescence tag fluorescence tag visualisation by fluorescence tag fluorescence unspecified role unspecified role fluorescence donor fluorescence donor over-expressed over expressed level over-expressed living cell living cell cer tag fluorescent prot tag fluorescent protein tag fluorescent prot tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined P03459:p.Val527_Leu529delinsAlaAlaAla mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain VIL AAA Thu Jul 07 12:07:00 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was val527ala_ile528ala_leu529ala Fri Jul 22 10:58:43 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'val527ala,ile528ala,leu529ala' Wed May 02 17:55:57 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'val_ile_leu527-529ala_ala_ala' Tue Jun 30 17:57:18 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Val527_Leu529delinsAlaAlaAla' M2 3 tag fluorescence tag visualisation by fluorescence tag fluorescence tag fluorescence tag visualisation by fluorescence tag fluorescence unspecified role unspecified role fluorescence accept fluorescence acceptor fluorescence accept over-expressed over expressed level over-expressed living cell living cell yfp tag yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined proximity proximity close-contact colocalization neighbourhood interaction f2 f3b When HA–Cer-C3S (lacking all three acylation sites) was employed for FLIM-FRET a relative KD value of 0.72 was determined (Fig. 3A), clearly higher than in the experiments with HA–Cer-wt (KD = 0.13, Fig. 2C). Also, the fitting curve appears quasi-linear rather than distinctly leveling off as in the case of clustering. The FLIM-FRET measurement with HA–Cer-VIL3A and M2–YFP-wt yielded a relative KD of 0.29 (Fig. 3B). Thus, removing the raft-targeting features of HA clearly reduced clustering with M2, more pronounced with non-acylated HA. Acylation of HA may thus be more important for budozone organization than the VIL motif. Accordingly, lack of HA’s VIL motif in recombinant viruses is less dramatic than mutating individual HA acylation sites (viruses with non-acylated HA cannot be rescued; virus titers of virus with HA-VIL3A are reduced by 2–4 logs [14]). The evaluation of clustering between HA–Cer-wt and M2–YFP-wt yielded a relative KD value of 0.13 (Fig. 2C). This is very similar to the result obtained previously [25] for the same FRET pair (relative KD = 0.09). Thus, we corroborated that HA–Cer and M2–YFP cluster in the plasma membrane of live cells. ha-m-2 1 HA 2 tag fluorescence tag visualisation by fluorescence tag fluorescence tag fluorescence tag visualisation by fluorescence tag fluorescence unspecified role unspecified role fluorescence donor fluorescence donor over-expressed over expressed level over-expressed living cell living cell cer tag fluorescent prot tag fluorescent protein tag fluorescent prot tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined M2 3 tag fluorescence tag visualisation by fluorescence tag fluorescence tag fluorescence tag visualisation by fluorescence tag fluorescence unspecified role unspecified role fluorescence accept fluorescence acceptor fluorescence accept over-expressed over expressed level over-expressed living cell living cell yfp tag yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined proximity proximity close-contact colocalization neighbourhood interaction f2 f4 Finally, we assessed whether the raft-association features of M2 play a role for the intrinsic clustering with HA. We performed FLIM-FRET experiments with HA–Cer-wt and mutants of M2– YFP. The results (Fig. 4) show that HA–M2 clustering was evident irrespective of the M2 version, relative KD values in the same range as for HA–Cer-wt and M2–YFP-wt were obtained: 0.04 for HA–Cer-wt and M2–YFP-C50S (lacking acylation; Fig. 4A), 0.11 with M2–YFP-Y52S, Y57S (cholesterol-interaction motifs disrupted; Fig. 4B), and 0.10 with the M2 mutant lacking both raft-targeting features (Fig. 4C). Surprisingly, M2–HA clustering was not even diminished in the absence of cholesterol-binding sites and/or acylation.