Fig. 2.

RIG-I signalling pathway and its interaction with the influenza A virus polymerase subunits. Centre left to centre bottom: the RIG-I signalling pathway is activated upon binding of an RNA ligand, such as the 5′ppp-dsRNA region of the influenza virus promoter, by the CTD of RIG-I. A subsequent ATP-dependent conformational change exposes two N-terminal CARDs of RIG-I and promotes RIG-I translocation along the RNA and formation of a RIG-I oligomer. One of the host proteins that potentiates RIG-I signalling is PKR activating protein (PACT), which binds to the CTD domain of RIG-I and enhances ATPase activity. RIG-I filament formation brings RIG-I CARDs into close proximity, facilitating formation of a CARD tetramer. The tetramer is stabilised by ubiquitin chains that are added by TRIM25. The RIG-I complex then migrates towards the mitochondrial outer membrane, where it associates with MAVS. The interaction between the CARD domains of RIG-I and MAVS nucleates MAVS filament formation. MAVS aggregation induces binding of TRAF family E3 ubiquitin ligases to MAVS, which potentiate recruitment of IKKs. Activated IKKε and TBK1 phosphorylate transcriptional factors IRF3 and IRF7, while transcriptional factor NF-κB is activated by the IKKα/β/γ complex. The activated IRF3, IRF7 and NF-κB translocate into the nucleus where they induce transcriptional activation of IFN and pro-inflammatory cytokine genes. Top left: the influenza virus RNA polymerase shields viral promoter from RIG-I recognition. Polymerase subunits also bind RIG-I in an ‘ESIE’-motif-dependent manner and interact with PACT. Middle: PB2 binding to MAVS is associated with inhibition of IFN expression and is attributed either to its N-terminal domain or to PB2 amino acids 588 and 292. PB1 induces degradation of MAVS by forming a complex with MAVS, RNF5 and NBR1. PB1 induces Lys27-linked ubiquitination of MAVS by RNF5, which is then recognised by NBR1 that targets ubiquitinated MAVS for autophagic degradation. Bottom right: PB2 (residues 490–759) binds to TRAF3 and prevents its Lys63-linked polyubiquitination by TRIM35, disrupting the formation of TRAF3-MAVS complex and preventing activation of TBK1 and IKKε kinases. TRIM35, in turn, induces Lys48-linked polyubiquitination of PB2, targeting it for proteasomal degradation. PB2 is also targeted to mitochondria via residues L7, L10 and N9. Bottom left: PA inhibits IFN production by binding to IRF3 and precluding its phosphorylation and nuclear translocation