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Proteins congregate into complexes to perform diverse cellular functions. Protein complexes are remodeled by protein-coding mutations or cellular signaling changes, driving phenotypic outcomes in health and disease. We present an affinity purification-mass spectrometry (AP-MS) proteomics protocol to express affinity-tagged "bait" proteins in mammalian cells, identify and quantify purified protein interactors, and visualize differential protein-protein interaction networks between pairwise conditions. Our protocol possesses general applicability to various cell types and biological areas. For complete details on the use and execution of this protocol, please refer to Bouhaddou et al. 1 ., Competing Interests: Declaration of interests The Krogan Laboratory has received research support from Vir Biotechnology, F. Hoffmann-La Roche, and Rezo Therapeutics. N.J.K. has a financially compensated consulting agreement with Maze Therapeutics. N.J.K. is the President and is on the Board of Directors of Rezo Therapeutics, and he is a shareholder in Tenaya Therapeutics, Maze Therapeutics, Rezo Therapeutics, and Interline Therapeutics. M.B. is a scientific advisor for GEn1E LifeSciences., (Copyright © 2024. Published by Elsevier Inc.)

RNA viruses are a major human health threat. The life cycles of many highly pathogenic RNA viruses like influenza A virus (IAV) and Lassa virus depends on host mRNA, because viral polymerases cleave 5'-m7G-capped host transcripts to prime viral mRNA synthesis ("cap-snatching"). We hypothesized that start codons within cap-snatched host transcripts could generate chimeric human-viral mRNAs with coding potential. We report the existence of this mechanism of gene origination, which we named "start-snatching." Depending on the reading frame, start-snatching allows the translation of host and viral "untranslated regions" (UTRs) to create N-terminally extended viral proteins or entirely novel polypeptides by genetic overprinting. We show that both types of chimeric proteins are made in IAV-infected cells, generate T cell responses, and contribute to virulence. Our results indicate that during infection with IAV, and likely a multitude of other human, animal and plant viruses, a host-dependent mechanism allows the genesis of hybrid genes., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Ebola virus (EBOV) infection often results in fatal illness in humans, yet little is known about how EBOV usurps host pathways during infection. To address this, we used affinity tag-purification mass spectrometry (AP-MS) to generate an EBOV-host protein-protein interaction (PPI) map. We uncovered 194 high-confidence EBOV-human PPIs, including one between the viral transcription regulator VP30 and the host ubiquitin ligase RBBP6. Domain mapping identified a 23 amino acid region within RBBP6 that binds to VP30. A crystal structure of the VP30-RBBP6 peptide complex revealed that RBBP6 mimics the viral nucleoprotein (NP) binding to the same interface of VP30. Knockdown of endogenous RBBP6 stimulated viral transcription and increased EBOV replication, whereas overexpression of either RBBP6 or the peptide strongly inhibited both. These results demonstrate the therapeutic potential of biologics that target this interface and identify additional PPIs that may be leveraged for novel therapeutic strategies., (Copyright © 2018 Elsevier Inc. All rights reserved.)