Select Paramyxoviral V Proteins Inhibit IRF3 Activation by Acting as Alternative Substrates for Inhibitor of κB Kinase ϵ (IKKe)/TBK1

Abstract

V accessory proteins from Paramyxoviruses are important in viral evasion of the innate immune response. Here, using a cell survival assay that identifies both inhibitors and activators of interferon regulatory factor 3 (IRF3)-mediated gene induction, we identified select paramyxoviral V proteins that inhibited double-stranded RNA-mediated signaling; these are encoded by mumps virus (MuV), human parainfluenza virus 2 (hPIV2), and parainfluenza virus 5 (PIV5), all members of the genus Rubulavirus. We showed that interaction between V and the IRF3/7 kinases, TRAF family member-associated NFB activator (TANK)-binding kinase 1 (TBK1)/inhibitor of B kinase ⑀ (IKKe), was essential for this inhibition. Indeed, V proteins were phosphorylated directly by TBK1/IKKe, and this, intriguingly, resulted in lowering of the cellular level of V. Thus, it appears that V mimics IRF3 in both its phosphorylation by TBK1/IKKe and its subsequent degradation. Finally, a PIV5 mutant encoding a V protein that could not inhibit IKKe was much more susceptible to the antiviral effects of double-stranded RNA than the wild-type virus. Because many innate immune response signaling pathways, including those initiated by TLR3, TLR4, RIG-I, MDA5, and DNA-dependent activator of IRFs (DAI), use TBK1/ IKKe as the terminal kinases to activate IRFs, rubulaviral V proteins have the potential to inhibit all of them.Innate immunity is stimulated by viruses in part via RNA. dsRNA 2 specifically is present in several forms: viral genomes, single-stranded RNA virus replication intermediates, DNA virus symmetric transcription products, defective viral particles, and debris from lysed cells (1). Although extracellular dsRNA is sensed by Toll-like receptor 3 (TLR3), intracellular dsRNA is detected in part by the RNA helicases retinoic acidinducible gene 1 (RIG-I) and melanoma differentiation-associated gene 5 (Mda-5). These receptors signal through Toll-IL-1R (TIR) domain containing adaptor-inducing IFN␤ (TRIF) and IFN␤ promoter stimulator 1 (IPS1), respectively, to activate the kinases TANK-binding kinase 1 (TBK1) and inhibitor of B kinase ⑀ (IKKe). They, in turn, phosphorylate IFN regulatory factor 3 (IRF3), promoting its nuclear translocation and subsequent IFN-stimulated regulatory element-mediated transcription of IFN-stimulated genes (ISG), such as ISG56, as well as IFN and other cytokines. IFN then, through Janus kinase (JAK)/ STAT activation of IRF9, modulates microRNAs in addition to up-regulating itself and more ISGs to heighten the antiviral state and also to initiate the adaptive immune response by promoting dendritic cell maturation, memory T cell proliferation, and B cell differentiation (2-4).To control this immune response, pathogens and hosts have developed methods of down-regulation and evasion at a variety of different points (5-7). At the level of sensing infection, NS1 from influenza virus binds to and sequesters dsRNA and also interacts with RIG-I (5, 6, 8). At the level of signal transduction, the NS3/4A protease from hepatitis C virus cle...

Mechanisms of Protein Kinase PKR-Mediated Amplification of Beta Interferon Induction by C Protein-Deficient Measles Virus

“…MV replicates in the cytoplasm and activates the RIG-I sensor due to the presence of cytosolic 5Ј-triphosphate-containing viral transcripts (24,25), but paradoxically, the V protein of MV blocks the mda-5 sensor of the RLR IFN-␤ induction pathway (6,18). Furthermore, TLR3 signaling through the adapter TRIF is impaired by V proteins of rubulaviruses but not by the MV V protein (14). Both the cytoplasmic RLRs and membrane-bound TLR3 act as sensors of viral dsRNA to initiate the innate immune response and IFN induction.…”

Mechanism of mda-5 Inhibition by Paramyxovirus V Proteins

“…This raises the possibility that paramyxoviruses have other mechanisms to block signaling responses through RIG-I. In this context, it has recently been reported that SeV-C can block RIG-I (28) and that some paramyxovirus V proteins, including PIV5-V, can block IRF-3 phosphorylation by interacting with IKKε/TBK1 and acting as alternative substrates (16). The latter phenomenon is presumably cell type specific, since we have failed to observe the expected inhibition of RIG-I by PIV5-V (2), and furthermore PIV5-V does not block induction by overexpression of the CARD domains of mda-5 or IPS-1 (Fig.…”

Paramyxovirus evasion of innate immunity: Diverse strategies for common targets

“…Rubulaviruses including MuV, hPIV2, and PIV5 use V protein as a decoy substrate for the IRF-3 kinases TANK-binding kinase 1 (TBK-1) and inhibitor of NF-κB kinase (IKK)e (Figure 3), both inhibiting phosphorylation of IRF-3 and facilitating IKKe/TBK-1 polyubiquitination and degradation to prevent further signalling [81] . Henipavirus V proteins do not cause IKKe/TBK-1 degradation [81] or block TLR-3/IRF-3 dependent signalling [76,81] . For henipaviruses, this appears to be a function of the W protein, as NiV W, although having no effect on MDA5 signalling, inhibited TLR-3-dependent phosphorylation of IRF-3 [82] .…”