ICC-MS defines an influenza polymerase interactome.

(A) Schematic diagram to identify PB2 interactome. Lysate fractions from human A549 cells infected with PB2-FLAG-tagged influenza virus encoding avian RNP or the “humanized” PB2-E627K RNP were incubated with competing soluble anti-FLAG antibody followed by capture with resin-bound anti-FLAG and LC–MS/MS. (B-C) Immunocompetitive capture of the viral RNP. (B) Detection of PB2 627E and 627K (top: western blot) or PB2 627K with coprecipitating RNP components (bottom: silver stain). *, IgG heavy chain. (C) Relative protein abundance of PB1 in PB2 ICC-MS samples shows decreasing capture with increasing competition antibody. Data shown are in biological triplicate. (D) Minimum-cost flow simulations connect top PB2 interactors identified by ICC-MS (red) to previously identified influenza host factors (gray), in some cases through other host proteins (white). Modules comprising different PB2 interactors were enriched for GO terms, the most significant of which is enlarged. Node sizes indicate empirical P values derived from the control flow simulations. Individual quantitative observations that underlie the data summarized here can be located under the Supporting information file as S1 Data. Uncropped images can be found in the Supporting information file as S1 Raw Images. Ab, antibody; ICC-MS, immunocompetitive capture-mass spectrometry; IgG, immunoglobulin G; LC-MS/MS, liquid chromatography-tandem mass spectrometry; NP, nucleoprotein; RNP, ribonucleoprotein complex.