key: cord-0001438-60j1dm90 authors: Beale, Rupert; Wise, Helen; Stuart, Amanda; Ravenhill, Benjamin J.; Digard, Paul; Randow, Felix title: A LC3-Interacting Motif in the Influenza A Virus M2 Protein Is Required to Subvert Autophagy and Maintain Virion Stability date: 2014-02-12 journal: Cell Host Microbe DOI: 10.1016/j.chom.2014.01.006 sha: 0af7ad44d9fe38134408a1374103ac32ccad1491 doc_id: 1438 cord_uid: 60j1dm90 Autophagy recycles cellular components and defends cells against intracellular pathogens. While viruses must evade autophagocytic destruction, some viruses can also subvert autophagy for their own benefit. The ability of influenza A virus (IAV) to evade autophagy depends on the Matrix 2 (M2) ion-channel protein. We show that the cytoplasmic tail of IAV M2 interacts directly with the essential autophagy protein LC3 and promotes LC3 relocalization to the unexpected destination of the plasma membrane. LC3 binding is mediated by a highly conserved LC3-interacting region (LIR) in M2. The M2 LIR is required for LC3 redistribution to the plasma membrane in virus-infected cells. Mutations in M2 that abolish LC3 binding interfere with filamentous budding and reduce virion stability. IAV therefore subverts autophagy by mimicking a host short linear protein-protein interaction motif. This strategy may facilitate transmission of infection between organisms by enhancing the stability of viral progeny. Human epithelial HCT116 cells expressing GFP-LC3 were infected with IAV at an MOI of 0.2, fixed at 16 hours p.i. and stained for M2. Scale bar 10µm. Recombinant viruses were rescued by 8 plasmid transfection into 293T cells followed by amplification in Madin-Darby Canine Kidney (MDCK) cells as previously described (Hutchinson et al., 2008) . PR8 was rescued using the system described and generously provided by (de Wit et al., 2004) . PR8-MUd has been previously described (Noton et al., 2007) . A549 human lung adenocarcinoma and HCT116 human colon adenocarcinoma cells were transduced with MLV-based retroviruses encoding GFP-LC3 (Randow and Sale, 2006) . Cells were cultured according to standard procedures, and were infected by allowing virus to adsorb for 30-60 min in serum free medium. Plaque assays were carried out in MDCK cells using an Avicel overlay followed by staining with toluidine blue (Hutchinson et al., 2008) . Growth kinetics were determined by performing a plaque-assay on the initial inoculum, which was then removed by acid washing. The medium was replaced by SFP supplemented with 0.14%BSA and 1µg/ml TPCK trypsin, and further plaque assays were performed at different time-points post infection. To detect IAV M2, mouse monoclonal antibody clone 14/C2 (Abcam) was used. To detect IAV NP, either monoclonal AA5H (Abcam) or a rabbit polyclonal serum raised against whole NP (A2915) (Noton et al., 2007) were used. To stain the surface of virus infected cells, a rabbit polyclonal serum raised against whole PR8 virions was used (Amorim et al., 2007) . A mouse monoclonal to GFP (clone JL/8) was sourced from Abcam. The JPRED algorithm was used to identify putative structural features in the C-terminus of M2 (Cole et al., 2008) . The sequence logo was compiled by identifying all unique M2 sequences in the database and the C-terminal 13 amino-acids were fed into the WebLogo 3 program (Schneider and Stephens, 1990 ). Immunoprecipitation, Western Blot, and LUMIER assays were performed as described previously (Barrios-Rodiles et al., 2005; Ryzhakov and Randow, 2007) . For fluorescence anisotropy, LC3 was expressed in E. coli and purified as described (Muhlinen et al., 2012) . GFP-trap experiments (Chromotek) were performed at 16 hours post infection according to the manufacturer's protocols (Amorim et al., 2011) . Fluorescence measurements of LC3, serially diluted and mixed with 100 nM hydroxycoumarin-labeled M2 LIR peptide, were performed on a Cary Eclipse fluorescence spectrophotometer (Agilent) in triplicate. Binding data was analyzed with GraphPad Prism 6. For fluorescence microscopy, cells were grown on glass coverslips and fixed in 4% paraformaldehyde or formaldehyde. They were incubated with primary, followed by secondary antibodies and/or Wheat-Germ agglutinin (Invitrogen) before being mounted. 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