IncRNA IPAN antagonizes RIG-I/TRIM25-mediated degradation of influenza A virus PB1 to promote viral replication

Abstract

The productive infection of influenza A virus (IAV) requires the functional involvement of host long noncoding ribonucleic acids (lncRNAs). Identification of key cellular lncRNAs and elucidation of their molecular mechanisms in IAV replication are expected to expand our understanding of virus-host interactions and develop antiviral therapeutics. Our previous work has identified that influenza virus polymerase basic protein 1 (PB1)-associated long noncoding RNA (IPAN) associates with and stabilizes viral RNA-dependent RNA polymerase PB1 of IAV, warranting efficient viral RNA synthesis. This provides a unique viral strategy of co-opting host lncRNA for replication, whereas the molecular pathways exploited by the virus are unknown. Here, we aim to further investigate the detailed mechanisms underlying IPAN-mediated PB1 stabilization. We employed cellular-level molecular interaction techniques to demonstrate that both retinoic acid-inducible gene I (RIG-I) and tripartite motif-containing protein 25 (TRIM25) interacted with PB1 and co-operated to induce its degradation triggered by viral RNA synthesis. The experimental data obtained from RIG-I knockout cell lines and mutational analyses demonstrated RIG-I promoted PB1 degradation independently of its canonical signaling pathway, suggesting an "effector-like" antiviral activity of RIG-I. Furthermore, IPAN knockdown enhanced the association of PB1 with both RIG-I and TRIM25 to restore PB1 stability. These results collectively demonstrated that IAV hijacked host IPAN to protect PB1 from RIG-I/TRIM25-mediated antiviral degradation. Thus, our data reveal a mechanism of RIG-I and TRIM25 against IAV infection by degrading PB1 and highlight how IAV exploits host lncRNAs to evade immune surveillance.

Keywords: Degradation; Influenza A virus (IAV); Influenza virus PB1-associated long noncoding RNA (IPAN); Innate immunity; Long noncoding ribonucleic acids (lncRNAs); Retinoic acid-inducible gene I (RIG-I); Tripartite motif-containing protein 25 (TRIM25).

Fig. 1

Viral RNA-triggered PB1 degradation with IPAN depletion. A) The knockdown efficiency of IPAN by transfecting with esiIPAN was determined by RT-qPCR. B) The levels of PB1 protein upon IPAN knockdown were measured by western blot in the cells transfected with PB1 plasmid or RNP reconstitution plasmids (PB1, PB2, PA, NP), along with or without the reporter plasmid used to transcribe an IAV-like RNA (PolI-luc). C) The band intensity of the blot (B) was quantified by the software Image J. β-actin was used as an internal control. D) The levels of PB1 protein were measured by western blot in IAV replicon cells that were transfected with esiIPAN or esiEGFP, followed by treatment with increasing concentrations of RdRp inhibitor T705. E) The band intensity of the blot (D) was quantified by the software Image J. β-actin was used as an internal control. F) The levels of the PB1 protein were measured by western blot. HEK293T cells were transfected with esiIPAN or esiEGFP for 24 h before co-transfection of PB1 plasmid DNA together with vRNA or dsRNA for an additional 24 h. vRNA and control RNA represent natural RNA extracted from IAV-infected cells and uninfected cells, respectively. dsRNA is a synthetic 5′-triphosphate-containing double strand RNA (5′ppp-dsRNA). G) The band intensity of the blot (F) was quantified by the software Image J. β-actin was used as an internal control. H) The levels of PA protein were measured by western blot. HEK293T cells were transfected with esiIPAN or esiEGFP for 24 h before co-transfection of PA plasmid DNA together with vRNA for an additional 24 h. I) The band intensity of the blot (H) was quantified by the software Image J. β-actin was used as an internal control. Panels A, C, E, G, and I display the mean ± SD of at least three independent experiments. Statistical significance was analyzed by two-tailed Student’s t test (*, P ≤ 0.05; **, P ≤ 0.01; ns, not significant). Abbreviations: IPAN, Influenza virus PB1-associated long noncoding RNA; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; PB1, polymerase basic protein 1; PB2, polymerase basic protein 2; PA, polymerase acidic protein; NP, nucleoprotein; RNP, ribonucleoprotein; IAV, Influenza A virus; RdRp, RNA-dependent RNA polymerase; vRNA, viral RNA; dsRNA, double-stranded RNA; SD, standard deviation; RNA, ribonucleic acid; DNA, deoxyribonucleic acid; μM, μmol/L; contrl, control.

Fig. 2

The involvement of RIG-I in PB1 degradation with IPAN depletion. A) The levels of RIG-I, TLR3, and MDA5 proteins were determined by western blot in HeLa cells that were transfected with siRNAs (100 nM) targeting RIG-I, TLR3 or MDA5. B) The knockdown efficiencies of RIG-I, TLR3, and MDA5 were determined by RT-qPCR in 293 T cells. C) The levels of PB1 protein were measured by western blot. HEK293T cells were transfected with esiIPAN and indicated siRNAs (100 nM) for 48 h before being infected with IAV for 24 h. D) The band intensity of the blot (C) was quantified and β-actin was used as an internal control. E) The levels of PB1 protein were measured by western blot. HEK293T cells were transfected with esiEGFP and esiIPAN before co-transfection of vRNA and plasmid DNA expressing PB1 and RIG-I WT or mutants. F) The band intensity of the blot (E) was quantified and β-actin was used as an internal control. G) The levels of PB1 protein were measured by western blot. RIG-I-KO HEK293T were transfected with esiEGFP and esiIPAN before co-transfection of vRNA and plasmid DNA expressing PB1 and RIG-I WT or mutants. H) The band intensity of the blot (G) was quantified and β-actin was used as an internal control. Graphs B, D, F, and H display the mean ± SD. Statistical significance was analyzed by two-tailed Student’s t-test (*, P ≤ 0.05; **, P ≤ 0.01; ns, not significant). Abbreviations: nM, nmol/L; vRNA, viral RNA; DNA, deoxyribonucleic acid; WT, wild type; RIG-I, retinoic acid-inducible gene I; RIG-I-KO, RIG-I knockout; TLR3, toll-Like receptor 3; MDA5, melanoma differentiation-associated protein 5; PB1, polymerase basic protein 1; siRNAs, small interfering RNAs; IAV, influenza A virus; SD, standard deviation; NC, negative control.

Fig. 3

The role of interferon pathway in RIG-I-mediated PB1 degradation. A) The levels of PB1 protein were determined by western blot. HEK293T cells were transfected with PB1 plasmid DNA and esiIPAN or esiEGFP prior to treatment with IFN-α (1,000 IU/mL). IAV infection of the transfected HEK293T cells (MOI = 0.5) was performed to show the decrease of PB1 protein upon IPAN knockdown. B) The band intensity of the blot (A) was quantified using the Image J program. Data are shown as mean ± SD. Statistical significance was analyzed by two-tailed Student’s t-test (*, P ≤ 0.05; ns, not significant). C) The levels of PB1 protein were determined after the treatment with increasing doses (500, 1,000, 2,000 IU/mL) of different types of IFNs (IFN-α, IFN-β, or IFN-γ). IAV infection of the transfected HEK293T cells (MOI = 1) was performed to show the decrease of PB1 protein upon IPAN knockdown. D) HEK293T cells were treated with different doses of indicated IFNs. Levels of SAMHD1 were detected as a control ISG. Abbreviations: RIG-I, retinoic acid-inducible gene I; PB1, polymerase basic protein 1; DNA, deoxyribonucleic acid; IFN, interferon; IAV, influenza A virus; IPAN, Influenza virus PB1-associated long noncoding RNA; MOI, multiplicity of infection; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ISG, IFN-stimulated genes; SD, standard deviation.

Fig. 4

The role of IFN-independent pathway on RIG-I-mediated PB1 degradation. A) IFN-β luciferase activity in poly (I:C) transfected cells w or w/o MAVS knockdown. B) The levels of PB1 protein were detected by western blot. HEK293T were transfected with esiIPAN and siMAVS for 24 h before co-transfection of PB1 plasmid DNA together with vRNA for an additional 24 h. C) The band intensity of the blot (B) was quantified using the Image J program. Levels of PB1 in the controls were arbitrarily set as 1. D) The levels of PB1 protein were detected by western blot. HEK293T cells were transfected with esiIPAN or esiEGFP for 24 h before co-transfection of PB1 plasmid DNA together with vRNA. NF-κB inhibitor BAY11-7082 (5 μM) was added 18 h before cells were harvested. E) 293 T cells were co-transfected with esiRNA together with plasmid DNA expressing PA, PB2, NP and polI-luc with or without PB1 for 24 h, followed by a transfection with IFN-β promoter renilla luciferase reporter. The dual luciferase reporter activity was measured. Poly (I: C) was used as a positive control. F) 293 T cells were co-transfected with plasmid DNA expressing IPAN, IFN-β firefly luciferase reporter and vRNA for 24 h. The IFN-β luciferase activity was measured and results were normalized to a renilla luciferase internal control. Graphs C, E, and F display the mean ± SD of at least three independent experiments. Statistical significance was analyzed by two-tailed Student’s t-test (*, P ≤ 0.05; **, P ≤ 0.01; ns, not significant). Abbreviations: RIG-I, retinoic acid-inducible gene I; PB1, polymerase basic protein 1; ISGs, IFN-stimulated genes; MAVS, mitochondrial antiviral signaling protein; DNA, deoxyribonucleic acid; vRNA, viral RNA; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; PA, polymerase acidic protein; PB2, polymerase basic protein 2; NP, nucleoprotein; IFN, interferon; RdRp, RNA-dependent RNA polymerase; IPAN, Influenza virus PB1-associated long noncoding RNA; vRNP, viral ribonucleoprotein complex; DMSO, dimethyl sulfoxide; NC, negative control; SD, standard deviation.

Fig. 5

A potential role of binding of RIG-I to PB1 in the degradation of PB1. A–B) HEK293T were transfected with esiIPAN (A) or IPAN-L (B) before co-transfection of vRNA together with plasmid DNA expressing PB1 and RIG-I-HA for an additional 24 h. The cell lysates were immunoprecipitated with anti-PB1 antibodies. The inputs and the immunoprecipitated samples were analyzed by western blots with the indicated antibodies. C) The interaction of HA-tagged RIG-I with PB1 was detected by PLA and confocal imaging in HEK293T cells. Representative data are shown. Nuclei were stained with DAPI (blue). All images were captured with a 100 × objective. Scale bars indicate 5 μm. D) Quantitative analysis of red fluorescent dot counts (C) in randomly selected cells, n = 12 cells per group. Data are shown as mean ± SD. Statistical significance was analyzed by two-tailed unpaired Student’s t-test between group esiEGFP and group esiIPAN (***, P ≤ 0.001). Abbreviations: RIG-I, retinoic acid-inducible gene I; PB1, polymerase basic protein 1; DNA, deoxyribonucleic acid; vRNA, viral RNA; IPAN, Influenza virus PB1-associated long noncoding RNA; NC, negative control; PLA, in situ proximity ligation assay; DAPI, 4′,6-diamidino-2-phenylindole; SD, standard deviation.

Fig. 6

The involvement of TRIM25 in the PB1 degradation induced by silencing IPAN. A) HEK293T were transfected with esiIPAN and siTRIM25 for 24 h before co-transfection of vRNP reconstitution plasmids (PB1, PB2, PA, NP, and reporter plasmid) for an additional 24 h. The proteins were detected by western blot. B) TRIM25 knockout cells were transfected with esiIPAN and TRIM25 or ΔRING mutant for 24 h before co-transfection of vRNP reconstitution plasmids (PB1, PB2, PA, NP, and reporter plasmid) for an additional 24 h. The proteins were detected by western blot. C–D)​​ esiEGFP or esiIPAN was transfected in HEK293T (C) or RIG-I-KO HEK293T cells (D) expressing PB1 and vRNA. Anti-PB1 antibody was used for Co-IP and the immunoprecipitates were analyzed by immunoblotting. E) The interaction of TRIM25 with PB1 was detected by PLA and confocal imaging in HEK293T and RIG-I-KO HEK293T cells. Representative data are shown. Nuclei were stained with DAPI (blue). All images were captured with a 100 × objective. Scale bars indicate 5 μm. F) Quantitative analysis of red fluorescent dot counts (E) in randomly selected cells, n = 14 cells per group in HEK293T and n = 10 cells per group in RIG-I-KO HEK293T. Data are shown as mean ± SD, and significance was analyzed by two-tailed unpaired Student’s t-test between group esiEGFP and group esiIPAN (***, P ≤ 0.001). Abbreviations: RIG-I, retinoic acid-inducible gene I; PB1, polymerase basic protein 1; vRNA, viral RNA; PA, polymerase acidic protein; PB2, polymerase basic protein 2; NP, nucleoprotein; IPAN, Influenza virus PB1-associated long noncoding RNA; vRNP, viral ribonucleoprotein complex; TRIM25, tripartite motif-containing protein 25; WT, wild type; RIG-I-KO, RIG-I knockout; NC, negative control; PLA, in situ proximity ligation assay; DAPI, 4′,6-diamidino-2-phenylindole; SD, standard deviation.