LUBAC is required for RIG-I sensing of RNA viruses

Abstract

The ability of cells to mount an interferon response to virus infections depends on intracellular nucleic acid sensing pattern recognition receptors (PRRs). RIG-I is an intracellular PRR that binds short double-stranded viral RNAs to trigger MAVS-dependent signalling. The RIG-I/MAVS signalling complex requires the coordinated activity of multiple kinases and E3 ubiquitin ligases to activate the transcription factors that drive type I and type III interferon production from infected cells. The linear ubiquitin chain assembly complex (LUBAC) regulates the activity of multiple receptor signalling pathways in both ligase-dependent and -independent ways. Here, we show that the three proteins that constitute LUBAC have separate functions in regulating RIG-I signalling. Both HOIP, the E3 ligase capable of generating M1-ubiquitin chains, and LUBAC accessory protein HOIL-1 are required for viral RNA sensing by RIG-I. The third LUBAC component, SHARPIN, is not required for RIG-I signalling. These data cement the role of LUBAC as a positive regulator of RIG-I signalling and as an important component of antiviral innate immune responses.

Fig. 1. RIG-I dependent RNA and RNA virus sensing in A549 cells.

A Western blotting analysis of A549 WT and RIG-I−/− cells with and without stimulation with IFNα for 24 h. qPCR to measure transcription of indicated genes in A549 WT and RIG-I−/− cells stimulated by B SeV infection at 1:300 dilution, C Zika virus infection at MOI 3, D transfection with 1 μg 3p-hpRNA and E transfection with 1 μg poly(I:C).

Fig. 2. HOIP is required for RIG-I-driven transcription, chemokine secretion and signalling pathway activation.

A A549 WT and HOIP−/− were infected with SeV at 1:300 dilution and qPCR used to measure transcription of indicated genes. B qPCR to measure transcription of indicated genes in A549 WT and HOIP−/− cells transfected with 1 μg 3p-hpRNA or poly(I:C). C Western blotting analysis of signalling protein activation in the presence and absence of 10 µM MG-132 ('4+'). D ELISA to measure CXCL10 secretion in A549 WT and HOIP−/− cells infected with SeV at 1:300 dilution or transfected with 1 μg 3p-hpRNA or 1 μg poly(I:C).

Fig. 3. HOIP is required for ZIKV-driven interferon responses.

A549 WT and HOIP−/− cells infected with ZIKV at MOI 3 and A qPCR to measure transcription of indicated genes, B ZIKV replication measured by plaque assay on Vero cells and C Western blotting analysis. Quantification of nuclear translocation of D NF-κB P65 and E IRF3 in A549 WT and HOIP−/− cells infected with ZIKV at MOI 1 for 24 h or stimulated with 100 ng/mL TNF, analysed by immunofluorescence (left panels) and quantified by scoring cells with nuclear staining (right panels).

Fig. 4. HOIL-1 is required for RIG-I immune response to SeV and synthetic RNAs.

MEF Tnf−/− Rbck+/− and Tnf−/− Rbck−/− cells infected with SeV at a 1:300 dilution and A qPCR to measure transcription of indicated genes and B Western blotting analysis of signalling protein activation in the presence and absence of 10 µM MG-132 (‘6+’). qPCR to measure transcription of indicated genes in MEF TNF−/− HOIL+/− and TNF−/− HOIL−/− cells transfected with C 1 μg 3p-hpRNA and D 1 μg Poly(I:C). E Western blot of WT and HOIL-1−/− HCT116 cells indicating successful generation of two knockout clones. qPCR to measure transcription of indicated genes in HCT116 WT and HOIL−/− cells F infected with SeV or G transfected with 1 μg poly(I:C).

Fig. 5. SHARPIN is not required for RIG-I immune response to SeV and synthetic RNAs in A549 cells.

A549 WT and SHARPIN−/− cells A infected with SeV at a 1:300 dilution or B stimulated with 1 μg 3p-hpRNA were analysed by qPCR to measure transcription of indicated genes, C Cells were infected with SeV and analysed by Western blotting for signalling protein activation in the presence and absence of 10 µM MG-132 (‘4+’) and D CXCL10 secretion measured by ELISA.

Fig. 6. The E3 ligase activity of LUBAC is partially required for its function in RIG-I signalling.

A Western blotting analysis of A549 WT, HOIP−/−, TAP-HOIP-WT and TAP-HOIP-C885S cells. A549 WT, HOIP−/−, TAP-HOIP-WT and TAP-HOIP-C885S cells infected with SeV at a 1:300 dilution and B qPCR to measure transcription of indicated genes. C Western blotting analysis of signalling protein activation in A549 TAP-HOIP-WT and TAP-HOIP-C885S cells infected with SeV at a 1:300 dilution in the presence and absence of 10 µM MG-132 (labelled as ‘4+’). D ELISA to measure CXCL10 secretion following infection with SeV. E qPCR to measure transcription of indicated genes in A549 WT, HOIP−/−, TAP-HOIP-WT and TAP-HOIP-C885S cells transfected with 1 μg 3p-hpRNA.

Fig. 7. LUBAC interacts with TBK1 and NEMO downstream of RIG-I activation.

qPCR to measure transcription of indicated genes during SeV infection at 1:300 dilution in A A549 WT, NEMO−/−, NEMO−/− + TAP-NEMO cells and B A549 WT, TBK1−/−, IKKε−/− and TBK1/IKKε−/− cells. Western blotting analysis of signalling protein phosphorylation during SeV infection at 1:300 dilution in the presence and absence of 10 µM MG-132 (‘4+’) in C A549 WT, NEMO−/−, NEMO−/− + TAP-NEMO cells and D A549 WT, TBK1−/−, IKKε−/− and TBK1/IKKε−/− cells. Western blotting analysis of Flag-M2 IP in E A549 TAP-NEMO and F A549 TAP-HOIP-WT G A549 TAP-HOIP-WT or A549 TAP-HOIP-C885S cells infected with SeV at a 1:300 dilution. H Western blotting analysis of affinity-purified (AP) biotin-poly(I:C) from A549 WT, HOIP−/− or SHARPIN−/− cells.