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

Bishi Fu,Hao Ding,Shitao Li,Martin E Dorf,Jens C Schwamborn,Lingyan Wang,Christopher F Basler

doi:10.1371/journal.ppat.1004960

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.

Highlights

Influenza A virus A (IAV) is a human respiratory pathogen that causes seasonal epidemics and occasional global pandemics with devastating levels of morbidity and mortality
We describe how the E3 ubiquitin ligase, tripartite motif-containing protein 32 (TRIM32), inhibits the activity of the influenza RNA polymerase and defends respiratory epithelial cells against infection with influenza A viruses
Replication and transcription of these IAV segments is catalyzed by a heterotrimeric RNA-dependent RNA polymerase complex, which consists of an acidic subunit (PA) and two basic subunits, PB1 and PB2 [1,2]

Summary

Introduction

Influenza A virus A (IAV) is a human respiratory pathogen that causes seasonal epidemics and occasional global pandemics with devastating levels of morbidity and mortality. IAV is a member of the Orthomyxoviridae family and possesses eight segments of negative-sense singlestranded RNA genome. Replication and transcription of these IAV segments is catalyzed by a heterotrimeric RNA-dependent RNA polymerase complex, which consists of an acidic subunit (PA) and two basic subunits, PB1 and PB2 [1,2]. Since the activity of RNA-dependent polymerases is distinct from enzymes found in host cells, these viral proteins are promising drug targets for interfering with viral replication [7,8]. Little is understood about the natural defenses employed by host cells to defend against the IAV polymerase. We analyze PB1 protein complexes and find a host interactor, tripartite motif-containing protein 32 (TRIM32), which directly targets PB1 proteins to restrict influenza virus replication

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