RESEARCH ARTICLE TRIM32 Senses and Restricts Influenza A Virus by Ubiquitination of PB1 Polymerase

Polymerase basic protein 1 (PB1) is the catalytic core of the influenza A virus (IAV) RNA po-lymerase complex essential for viral transcription and replication. Understanding the intrin-sic mechanisms which block PB1 function could stimulate development of new anti-influenza therapeutics. Affinity purification coupled with mass spectrometry (AP-MS) was used to identify host factors interacting with PB1. Among PB1 interactors, the E3 ubiquitin li-gase TRIM32 interacts with PB1 proteins derived from multiple IAV strains. TRIM32 senses IAV infection by interacting with PB1 and translocates with PB1 to the nucleus following in-fluenza infection. Ectopic TRIM32 expression attenuates IAV infection. Conversely, RNAi depletion and knockout of TRIM32 increase susceptibility of tracheal and lung epithelial cells to IAV infection. Reconstitution of trim32-/-mouse embryonic fibroblasts with TRIM32, but not a catalytically inactive mutant, restores viral restriction. Furthermore, TRIM32 direct-ly ubiquitinates PB1, leading to PB1 protein degradation and subsequent reduction of poly-merase activity. Thus, TRIM32 is an intrinsic IAV restriction factor which senses and targets the PB1 polymerase for ubiquitination and protein degradation. TRIM32 represents a mode