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

Abstract

Polymerase basic protein 1 (PB1) is the catalytic core of the influenza A virus (IAV) RNA polymerase complex essential for viral transcription and replication. Understanding the intrinsic 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 ligase 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 influenza 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 directly ubiquitinates PB1, leading to PB1 protein degradation and subsequent reduction of polymerase activity. Thus, TRIM32 is an intrinsic IAV restriction factor which senses and targets the PB1 polymerase for ubiquitination and protein degradation. TRIM32 represents a model of intrinsic immunity, in which a host protein directly senses and counters viral infection in a species specific fashion by directly limiting viral replication.

Ubiquitination Upregulates Influenza Virus Polymerase Function

“…Ubiquitination mediates multiple cellular processes and is perhaps most commonly associated with proteasomal degradation (35). Ubiquitination had previously been associated with degradation of NP and free polymerase subunits (12,14,26). However, overexpression of WT or mutant forms of ubiquitin did not appreciably alter the steady-state levels of viral proteins (Fig.…”

“…The shift in molecular mass was consistent with posttranslational modification by the addition of an 8-kDa ubiquitin moiety. Since the ubiquitin proteasome pathway has been implicated in the regulation of the IAV life cycle, including the viral polymerase function (13,14,23,24), we sought to establish whether these new species were ubiquitinated forms and if the IAV RNP complex undergoes ubiquitination. The IAV RNP was reconstituted in cells expressing a green fluorescent protein (GFP)-based viral reporter gene, NP, and the polymerase subunits PB1, PB2, and PA. To detect ubiquitination, epitope tagged HA-ubiquitin or a control plasmid was included.…”

“…Posttranslational modifications (PTMs) of viral proteins by host enzymes have been suggested to impact this change in activity. A number of PTMs have been reported to occur on NP and subunits of the viral polymerase, including phosphorylation of PB1, PB2, PA, and NP (9), sumoylation of PB1 and NP (10,11), poly-ADP ribosylation of PB2 and PA (12), and ubiquitination of PB1 and NP (13,14). These modifications can impact multiple steps in the assembly and function of RNPs.…”

“…Multiple E3 ubiquitin ligases have been identified as key host factors regulating the IAV infectious cycle, and ubiquitination of viral proteins has been shown to regulate their stability and function (12-14, 22, 23, 26, 27). Indeed, ubiquitin-mediated proteasomal degradation of IAV protein has recently been shown to restrict infection (12,14).…”

Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity

“…This event is critical to promote interaction with the adaptor protein MAVS and the subsequent activation of a signalling pathway leading to type 1 interferon production (Gack et al , 2007). TRIM32 has a broader cellular role and has been shown to regulate neuronal development, the stability of sarcomeric architecture and has more recently been reported to restrict influenza A virus (Shieh et al , 2011; Zhang et al , 2012; Fu et al , 2015). Both TRIM ligases work with UBE2N/UBE2V1 (Ubc13/Uev1) to form K63‐linked chains which play a non‐degradative role, but are also active with UBE2D isoforms (Zeng et al , 2010; Napolitano et al , 2011; Zhang et al , 2012).…”