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. 2023 May 5;43(5):BSR20222152.
doi: 10.1042/BSR20222152.

Differential regulation of ATP hydrolysis of RIG-I-like receptors by transactivation response RNA-binding protein

Benyapa Chunhaphinyokul  1 , Emi Hosokai  1 , Masahiko Miyamoto  1 , Akihiko Komuro  1
Affiliations

Differential regulation of ATP hydrolysis of RIG-I-like receptors by transactivation response RNA-binding protein

Benyapa Chunhaphinyokul et al. Biosci Rep. .

Abstract

Retinoic acid inducible gene (RIG)-I-like receptors (RLRs), including RIG-I, melanoma differentiation associated-5 (MDA5), and laboratory of genetics and physiology 2 (LGP2), play pivotal roles in viral RNA sensing to initiate antiviral interferon (IFN) responses. We previously reported that an RNA-silencing regulator, transactivation response RNA-binding protein (TRBP), up-regulates MDA5/LGP2-mediated IFN responses through interaction with LGP2. Here, we aimed to investigate the mechanism underlying the TRBP-mediated up-regulation of IFN response. Data indicated that phosphomimetic TRBP showed a modest effect, whereas the nonphosphorylated form exhibited hyperactivity in enhancing Cardiovirus-triggered IFN responses. These results suggest that encephalomyocarditis virus (EMCV) attenuates the TRBP-mediated IFN response via TRBP phosphorylation, since EMCV infection activates the kinase responsible for TRBP phosphorylation for virus replication. Furthermore, we found that TRBP-mediated up-regulation of IFN response required the ATP hydrolysis and RNA binding of LGP2. TRBP enhanced RNA-dependent ATP hydrolysis by LGP2 but not that by RIG-I or MDA5. Nonphosphorylated TRBP exhibited higher levels of activity than phosphomimetic TRBP did, suggesting its possible involvement in the mechanism underlying the up-regulation of IFN response. TRBP activated the ATP hydrolysis of LGP2 and RIG-I, but not that of MDA5, in the absence of RNA. Collectively, we showed that TRBP differentially regulated RLR-mediated ATP hydrolysis. Further elucidation of the mechanism underlying the regulation of ATP hydrolysis leading to IFN response and self- and non-self-RNA discrimination could advance the development of effective therapeutic agents against autoimmune diseases.

Keywords: ATPase; RNA-binding proteins; host-pathogen interactions; innate immunity; interferons.

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Conflict of interest statement

The authors declare that there are no competing interests associated with the manuscript.

Figures

Figure 1
Figure 1. ATP hydrolysis and RNA-binding activity of LGP2 are required for TRBP-mediated IFNβ promoter up-regulation
293FT cells seeded onto a 48-well plate were transfected with MDA5 with WT LGP2 (A), ATPase-deficient LGP2 (K30A) (B), or other LGP2 mutants (C), and TRBP expression vectors with the −110 luc IFNβ promoter vector and pRL-SV40 and finally infected (+) or mock infected (−) with EMCV (MOI = 1) for 14–17 h. Cell lysates were analyzed for dual-luciferase activity (n=3). Differences between groups were assessed using Student’s t-test, with significance set at P<0.05. (D) Schematic representation of LGP2 structure and mutants.
Figure 2
Figure 2. Phosphorylation status of TRBP impacts the LGP2/MDA5-dependent IFNβ promoter activity induced by EMCV infection
(A) (Left) L929 cells were mock-infected or EMCV-infected (MOI = 1) for the indicated time period. Cell lysates were subjected to western blot analysis using anti-TRBP and GAPDH antibodies. The control mock infected cell lysate (C) was treated with lambda phosphatase to assess the phosphorylation status (P). (Right) 293FT cells transfected with HA-tagged TRBP (WT) or nonphosphorylatable TRBP (SD) vector were mock- or EMCV infected (MOI = 0.5–5). Cell lysates were subjected to western blot analysis using anti-HA and GAPDH antibodies. The asterisk indicates phosphorylated TRBP. (B) Schematic representation of TRBP structure and mutants. (C,D) 293FT cells were transfected with an empty vector, MDA5 and LGP2 expression vectors, and WT, phosphomimetic (SD), or nonphosphorylatable TRBP (SA) expression vector with the 110 luc IFNβ promoter vector and pRL-SV40 and finally infected with EMCV (MOI = 1) for 14–17 h. Cell lysates were analyzed using a luciferase assay (n=3). (E) The dsRNA-TRBP complex was separated on a 5% acrylamide gel, followed by staining with SYBR-Gold. A portion of the complex was analyzed via western blot analysis with anti-TRBP antibody. Relative RNA binding was quantified (below) as the ratio of the shifted and original RNA band intensities.
Figure 3
Figure 3. Real-time measurement of the RLR-induced RNA-dependent ATP hydrolysis
(A) Recombinant LGP2, (B) RIG-I, or (C) MDA5 was incubated with the indicated amount of HMW pIC and 1 mM ATP in duplicate. The released Pi reacted with the substrate and was continuously monitored by measuring the absorbance at 360 nm for up to 7000 s using a spectrophotometer. (D) ATP hydrolysis velocity was calculated by determining the increased absorbance at the linear reaction phase (1500–4000 s); error bars indicate mean (n=2) ± standard deviation. (E) Recombinant proteins used in ATP hydrolysis and gel-shift assays; 200 ng LGP2, RIG-I, MDA5, nonphosphorylated TRBP (WT), and phosphomimetic TRBP (SD) were electrophoresed via SDS-PAGE and stained with Coomassie brilliant blue.
Figure 4
Figure 4. Regulation of LGP2-induced ATP hydrolysis by TRBP
Effect of increasing concentrations (0–400 ng) of (A) HMW pIC or (D) LMW pIC on the LGP2-induced ATP hydrolysis. Arrows indicate the amounts of pIC used in (B,C,E,F). LGP2 (0.66 pmol) was incubated with phosphomimetic (SD) or WT TRBP (0.6–15 pmol) with 10 or 200 ng (B,E) HMW or (C,F) LMW pIC, and ATP hydrolysis was measured. Buffer, pIC, LGP2, or TRBP-only controls (Lanes 1–4 and 7) and LGP2 or TRBP with pIC controls (Lanes 5, 6, and 8) were also included. To ensure equal loading of TRBP, assay samples were analyzed via western blotting with TRBP antibody. Error bars indicate mean (n=2) ± standard deviation.
Figure 5
Figure 5. Regulation of RIG-I -induced ATP hydrolysis by TRBP
Effect of increasing concentrations (0–400 ng) of (A) HMW pIC or (D) LMW pIC on the RIG-I-induced ATP hydrolysis. RIG-I (0.66 pmol) was incubated with phosphomimetic (SD) or WT TRBP (0.6–15 pmol) with (B,E) 10 ng or (C,F) 200 ng HMW or LMW pIC, and ATP hydrolysis was measured as in Figure 4.
Figure 6
Figure 6. Regulation of Regulation of MDA5-induced ATP hydrolysis by TRBP
Effect of increasing concentrations (0–400 ng) of (A) HMW pIC or (D) LMW pIC on the MDA5-induced ATP hydrolysis. MDA5 (1 pmol) was incubated with phosphomimetic (SD) or WT TRBP (0.6–15 pmol) with 10 or 200 ng (B,E) HMW or (C,F) LMW pIC, and ATP hydrolysis was measured as in Figure 4.
Figure 7
Figure 7. Regulation of RLR-induced ATP hydrolysis by TRBP without RNA
(A) LGP2 (0.66 pmol), MDA5 (1 pmol), or RIG-I (0.66 pmol) was incubated with phosphomimetic (SD) or WT TRBP (0.6–15 pmol) without pIC, and ATP hydrolysis was measured as in Figure 4. (B) LGP2 (150 ng), MDA5 (150 ng), or RIG-I (150 ng) was subjected to incubation in the presence of either phosphomimetic (SD) or WT TRBP (50 ng), utilizing a whole-cell extract buffer (50 mM Tris-HCl [pH 8.0]-280 mM NaCl-0.5% NP-40-0.2 mM EGTA-2 mM EDTA-1 mM dithio-threitol-1 mM MgCl2). TRBP in isolation, in the absence of any RLR protein, was utilized as a negative control (−). The FLAG-RLR proteins were isolated using FLAG agarose beads, and the captured RLRs were subsequently assessed alongside co-purified TRBP through western blot analysis, utilizing both FLAG and TRBP antibodies to evaluate both the input mixture prior to purification and the resulting output.
Figure 8
Figure 8. Model of TRBP-mediated differential regulation of the ATP hydrolysis activity of RLRs, IFN response, and RNA silencing
TRBP phosphorylated by MAPK/ERK (p-TRBP) has enhanced activity in RNA silencing [28], while nonphosphorylated TRBP (non-p-TRBP) has enhanced activity in MDA5/LGP2-mediated IFN response. Since EMCV activates a kinase responsible for TRBP phosphorylation, MAPK, for virus replication [29], EMCV may attenuate the TRBP-mediated IFN response via TRBP phosphorylation. TRBP up-regulates the ATP hydrolysis activity of LGP2 and down-regulates that of MDA5 and RIG-I in the presence of RNA (HMW pIC), wherein TRBP positively and negatively regulates the MDA5/LGP2- and RIG-I-mediated IFN response, respectively. In addition, TRBP up-regulates the ATP hydrolysis activity of LGP2 and RIG-I, but not that of MDA5, in the absence of RNA.
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