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Targeting a key disulfide linkage to regulate RIG-I condensation and cytosolic RNA-sensing

Nature Cell Biology volume 27, pages 817–834 (2025)Cite this article

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Abstract

Maintaining innate immune homeostasis is critical for preventing infections and autoimmune diseases but effective interventions are lacking. Here we identified C864–C869-mediated intermolecular disulfide-linkage formation as a critical step for human RIG-I activation that can be bidirectionally regulated to control innate immune homeostasis. The viral-stimulated C864–C869 disulfide linkage mediates conjugation of an SDS-resistant RIG-I oligomer, which prevents RIG-I degradation by E3 ubiquitin-ligase MIB2 and is necessary for RIG-I to perform liquid–liquid phase separation to compartmentalize downstream signalsome, thereby stimulating type I interferon signalling. The corresponding C865S ‘knock-in’ caused an oligomerization defect and liquid–liquid phase separation in mouse RIG-I, which inhibited innate immunity, resulting in increased viral load and mortality in mice. Using unnatural amino acids to generate covalent C864–C869 linkage and the development of an interfering peptide to block C864–C869 residues, we bidirectionally regulated RIG-I activities in human diseases. These findings provide in-depth insights on mechanism of RIG-I activation, allowing for the development of methodologies that hold promising implications in clinics.

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Fig. 1: RIG-I undergoes LLPS in vitro and forms cytoplasmic condensates after viral infection.
Fig. 2: R3 is necessary for RIG-I LLPS.
Fig. 3: The C864–C869 disulfide bond in R3 is essential for RIG-I oligomerization and phase separation.
Fig. 4: Both C864 and C869 are critical for human RIG-I activation.
Fig. 5: C865S knock-in impairs mouse RIG-I LLPS and leads to compromised innate antiviral immunity in mice.
Fig. 6: RIG-I monomer, but not the LLPS-forming oligomer, is targeted for poly-ubiquitination and degradation by E3 ligase MIB2.
Fig. 7: Covalent crosslinking of C864–C869 by unnatural amino acids promotes LLPS and activation of RIG-I both in cells and mice.
Fig. 8: RIPs developed to prevent C864–C869 intermolecular disulfide linkage were able disrupt RIG-I LLPS, thereby reducing RIG-I-mediated autoimmune responses.

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Data availability

RNA-sequencing data that support the findings of this study have been deposited in the NCBI Gene Expression Omnibus (accession number GSE283490). Mass spectrometry data have been deposited into the OMIX, China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (accession number OMIX008113). All information supporting the conclusions are provided with the paper. Source data are provided with this paper.

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Acknowledgements

This work was supported by Chinese National Natural Science Funds (grant numbers 31925013, U20A20393 and W2411011 to L. Zhang; 32125016, U24A20371 to F.Z.; and 22374128 and 22074132 to B.Y.), program from the Ministry of Science and Technology of China (grant numbers 2021YFA1101000 and 2024YFC2707400 to L. Zhang, 2022YFA1105200 and 2023YFA1800200 to F.Z., and 2022YFF0608402 to B.Y.), a Key R&D Program of Zhejiang Province (grant number 2024C03142 to F.Z.), Suzhou Innovation and Entrepreneurship Leading Talent Program (ZXL2022505 to F.Z.), Suzhou Medical College Basic Frontier Innovation Cross Project (grant number YXY2303027 to F.Z.), Jiangsu National Science Foundation (grant number 19KJA550003 to F.Z.), Bo Xi Clinical Research Project of the First Affiliated Hospital of Soochow University (grant number BXLC007 to F.Z.), Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and the Joint Project of Pinnacle Disciplinary Group from the Second Affiliated Hospital of Chongqing Medical. We thank J. Guo, W. Yin and S. Liu from the core facility platform of Zhejiang University School of Medicine for their technical support.

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  1. These authors contributed equally: Bin Wang, Yongqiang Wang, Ting Pan.

Authors and Affiliations

  1. Department of Radiation Oncology and the State Key Laboratory of Transvascular Implantation Devices, The Second Affiliated Hospital of Zhejiang University School of Medicine, Life Sciences Institute, Zhejiang University, Hangzhou, China

    Bin Wang, Yu Ran, Jing Zou, Shixian Lin, Aiming Ren, Fangwei Wang, Huasong Lu, Bing Yang & Long Zhang

  2. The MOE Basic Research and Innovation Center for the Targeted Therapeutics of Solid Tumors, The First Affiliated Hospital, Jiangxi Medical College Nanchang University, Nanchang, China

    Bin Wang, Xiaohua Yan & Long Zhang

  3. Institutes of Biology and Medical Sciences, The First Affiliated Hospital, Suzhou Medical College, Soochow University, Suzhou, China

    Yongqiang Wang, Lili Zhou, Yu Ran, Zhenke Wen & Fangfang Zhou

  4. Shenzhen Key Laboratory of Systems Medicine for Inflammatory Diseases, School of Medicine, Sun Yat-sen University, Shenzhen, China

    Ting Pan

  5. Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Institute of Functional Nano and Soft Materials (FUNSOM), Soochow University, Suzhou, China

    Zhuang Liu

  6. Departments of Cell Biology and General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Zhejiang University, Hangzhou, China

    Ting Liu

  7. Frontiers Medical Center, Tianfu Jincheng Laboratory, Chengdu, China

    Long Zhang

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Contributions

B.W. designed and performed the experiments, and analysed the data. T.P. and Y.W. performed the experiments related to SARS-CoV-2 infections. L. Zhou, Z.W., Z.L. and T.L. developed the mtRNA-LNPs-induced autoimmune disease mouse model. J.Z. analysed the RNA-sequencing data. Y.R. and B.Y. performed the mass spectrometry experiments. H.L., X.Y., F.W., T.L., A.R. and S.L. provided advice. F.Z. and L. Zhang provided funds and guided this work. B.W. and L. Zhang. wrote the paper.

Corresponding authors

Correspondence to Bing Yang, Fangfang Zhou or Long Zhang.

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Nature Cell Biology thanks Jonathon Ditlev, Jian Ma and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 RIG-I undergoes LLPS in response to infection of RNA virus.

a, Schematics illustrate the RIG-I-MAVS signalling pathway. b, Left: Domain structure and the low-complexity sequence containing region (R) of RIG-I. Right: Bacterially purified GFP-RIG-I-Strep and RIG-I-Strep proteins were analysed by SDS–PAGE and detected by Coomasssie blue staining. c–e, 20 μM GFP-RIG-I were treated with 5% Hex (c), 100 μg/ml Proteinase K for 30 min at 40 °C (d), or treated with heated-inactivated (5 min at 95 °C and immediately put on ice for 5 min) (e), and then subjected to droplet formation assay in vitro (150 mM NaCl, pH 7.5, room temperature); n = 8 for each experiment. f, Related to Fig. 1g, percentage of cells with puncta was shown; n = 3. g, In vivo fusion of Cy3-labelled 5′ triphosphate double-stranded RNA (5′ppp-dsRNA, hereinafter called dsRNA) and GFP-RIG-I condensate. After 6 h of SeV stimulation, Cy3–dsRNA was transfected into HeLa cells for 6 h. h, Immunofluorescence microscopy and DAPI staining of HeLa cells showed puncta of RIG-I after transfection of Cy3-SARS-CoV-2 RNA for 12 h. i, Left: immunofluorescence microscopy and DAPI staining of GFP-RIG-I with Cy3- dsRNA in HeLa and U2OS cells. Middle and Right: quantitative line profile of co-localization along a white arrow of the left image. j, Representative micrographs of mCherry–MAVS recruited to GFP-RIG-I puncta in HeLa cell infected with SeV for 6 h. Quantitative line profile of colocalization along a white arrow of the right image (left). k, Representative micrographs (middle), and quantification (right) of mouse lung tissue showed puncta of endogenous RIG-I after infection with VSV (2 × 109 p.f.u. per mouse, nasal inhalation); n = 3. Data are representative of at least three independent experiments. Scale bar, 20 μm (c–e), 10 µm (g–j), 50 µm (k). Mean ± s.d., statistical analysis was performed using two-tailed Student’s t-test (c–f,k); ****P < 0.0001. Exact P values, source numerical data are provided.

Source data

Extended Data Fig. 2 R3 is necessary for RIG-I LLPS.

a, The pi–pi contacts of R1, R2 and R3 were determined using PScore (https://pound.med.utoronto.ca/~JFKlab/Software/psp.htm). The assessment of NCPR (net charge per residue), FCR (fraction of charged residue), hydrophobicity, and Shannon Entropy of R1, R2 and R3 was conducted with localCIDER (https://pappulab.github.io/localCIDER/). The displayed score is the phase separation prediction score. Features, from top to bottom, are pi interaction, NCPR, FCR, hydrophobicity, and Shannon Entropy. b, Related to Fig. 2a, immunoblot (IB) analysis of the indicated proteins; FL, full length. c, Related to Fig. 2f, percentage of cells with puncta was shown; n = 3. d, Representative micrographs of droplet formation (left) and quantification (right) of 5′ triphosphate double-stranded RNA (5′ppp-dsRNA, 1 μM) with purified GFP-RIG-I WT (3 μM) or GFP-RIG-I R3 where R3 was replaced by GGS linker (3 μM); n = 3. e, Immunofluorescence and DAPI staining of HeLa cells transfected with GFP-RIG-I WT and R3GGS followed by stimulated with SeV for 12 h. Quantification of cells with GFP-RIG-I puncta was shown (right); n = 3. Data are representative of at least three independent experiments. Scale bar, 10 μm (e), 20 μm (d). Mean ± s.d., statistical analysis was performed using two-tailed Student’s t-test (c–e); ****P < 0.0001. Exact P values, source numerical data and unprocessed blots are provided.

Source data

Extended Data Fig. 3 The Cys864-Cys869 intermolecular disulfide bond located in R3 is required for an efficient RIG-I LLPS.

a, Representative confocal microscopy images showing the effects of DTT treatments on droplet formation of GFP-RIG-I proteins with or without 2 μM dsRNA incubation; n = 3. b, Representative confocal microscopy images and quantitative data showing the effects of DTT treatments on droplet formation of GFP-SARS2-NP proteins (20 μM); n = 3; P = 0.4874 for DTT vs. DMSO. Right: Coomassie blue staining of purified SARS2-NP. c, Immunofluorescence microscopy and DAPI staining of HeLa cells transfected with GFP-SARS2-NP followed by treatment with DMSO or 0.2 mM DTT for 1 h (left). Quantification of cells with GFP-SARS2-NP puncta was shown (right); n = 3; P = 0.8142 for DTT vs. DMSO. d, Representative images and quantitative data showing the effects of PDI treatments on droplet formation of normal GFP-RIG-I proteins; n = 3; P = 0.5886 for GSH + GSSG vs. untreated, P = 0.9482 for PDI + GSH + GSSG vs. untreated. e, Representative images and quantitative data showing the effects of PDI treatments on droplet formation of reduced GFP-RIG-I proteins (20 μM); n = 3; P = 0.0980 for GSH + GSSG vs. pretreated. f, SDD–AGE analysis of RIG-I aggregation (top) and SDS–PAGE (bottom) of SeV-stimulated RIG-I knockout cells transfected with indicated plasmids. g, Coomassie blue staining of indicated proteins. h, Sedimentation analysis of GFP-RIG-I WT, C864S and C869S proteins after they were incubated with PBS or dsRNA (top). Quantified band intensity were shown (bottom); n = 3; P = 0.5201 for dsRNA vs. PBS in C864S, P = 0.0815 for dsRNA vs. PBS in C869S. i, Immunofluorescence microscopy and DAPI staining of HeLa cells transfected to express GFP-RIG-I WT, C864S or C869S proteins followed by stimulation with SeV for 12 h (left). Quantification of cells with GFP-RIG-I puncta was shown (right); n = 3; P = 0.0023 for C864S vs. WT, P = 0.0027 for C869S vs. WT. Data are representative of at least three independent experiments. Scale bar, 20 μm (a,b,d,e), 10 μm (c,i). Mean ± s.d., statistical analysis was performed using two-tailed Student’s t-test (b–e,h,i); ****P < 0.0001; ns, not significant. Exact P values, source numerical data and unprocessed blots are provided.

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Extended Data Fig. 4 C865S ‘knock-in’ leads to comprised innate antiviral immunity in mice.

a, Immunoblot (IB) analysis of the endogenous RIG-I protein levels in WT and RIG-I knockout (KO) HEK293T cells. b, Schematic diagram of Rig-IC865S/C865 knock-in strategy (upper). Rig-IC865S/C865 mice in C57BL/6N background was generated by Cyagen Biosciences Inc. by targeting of exon 16 of Rig-I using CRISPR–Cas9. Sequencing verification of the codon replacement by CRISPR–Cas9 resulting in the RIG-I C865S mutant (bottom). c, The Rig-IC865S/C865 mice were largely normal in appearance and weight and were fertile. d,e, qPCR analysis of Ifnb1, Cxcl10 or Ccl5 mRNA in wild-type and Rig-IC865S/C865 MEFs (d) and BMDMs (e) infected with SeV (top) or VSV (bottom), or transfected with 5′-ppp RNA (middle) or poly (I:C) (bottom), for indicated time periods. All results are presented relative to those of 18S; n = 3 for each experiment. Data are representative of at least three independent experiments. Mean ± s.d., statistical analysis was performed using a two-tailed Student’s t-test (d-e); **P < 0.01, ***P < 0.001, ****P < 0.0001. Exact P values, source numerical data and unprocessed blots are provided.

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Extended Data Fig. 5 Cells isolated from C865S ‘knock-in’ mice showed defect of innate immune responses to infection with RNA virus.

a, Heatmap showing colour-coded intensity levels of IFN target genes in SeV-stimulated Rig-IWT/WT and Rig-IC865S/C865S MEFs. n = 2 biologically independent samples. b, GSEA showing the most significant downregulated pathways in SeV-stimulated Rig-IC865S/C865S MEFs, as compared with the control wild-type MEFs. The data are derived from two repeated experiments, and the P values were calculated based on 1,000 permutations of the GSEA algorithm and were not adjusted for multiple comparisons. NES, normalized enrichment scores.

Extended Data Fig. 6 RIG-I monomer, but not the LLPS-forming oligomer, is targeted for poly-ubiquitination and degradation by E3 ligase MIB2.

a, Normalized IFNB1 mRNA expression (determined by qPCR analysis) in RIG-I knockout HEK293T cells transfected with the indicated plasmids, followed by SeV infection for 12 h; n = 3. b, Microscale thermophoresis (MST) binding affinity between dsRNA and prokaryotic expressed GFP-RIG-I-WT, C810S or C813S mutant as indicated; n = 3. c, ATPase activities of RIG-I-WT, C810S, or C813S mutants were assessed in the presence of dsRNA; n = 3; P = 0.0015 for C810S vs. WT, P = 0.0012 for C813S vs. WT. d, Immunoblot (IB) analysis of HEK293T cells stably expressed with RIG-I-Strep WT, C810S, C813S, C864S or C869S and treated with Cycloheximide (CHX) (5 μM) for the indicated time periods (top). Quantified RIG-I-Strep band intensity was shown (bottom). n = 3. e, The representative RIG-I peptide carrying ubiquitin-conjugated Lys169, Lys256 and Lys652 were identified by mass spectrometry. f, Coomassie blue staining of purified Strep-tagged RIG-I WT and C864S. g, Fold change of IFN-β-luciferase (luc) activity in RIG-I knockout HEK293T cells transfected with control vector and the indicated expression plasmids, followed by SeV stimulation for 12 h (left). Immunoblot (IB) analysis of the RIG-I expression was shown (right); n = 3. h, Normalized IFNB1 and ISG56 mRNA expression (determined by qPCR analysis) in RIG-I knockout HEK293T cells transfected with the indicated plasmids, followed by SeV infection for 12 h; n = 3. i, IB analysis of HEK293T cells stably expressed with Flag-RIG-I-C864S and transfected with increased dosage of Myc-MIB2 WT or Myc-MIB2 C977S as indicated. Quantified Flag-RIG-I-C864S band intensity was shown (bottom). n = 3. j, In vitro binding between Flag-MIB2 and the purified RIG-I monomer or oligomer from Fig. 6n. k, Coomassie blue staining of Strep-tagged mCherry–MIB2. Data are representative of at least three independent experiments. Mean ± s.d., statistical analysis was performed using a two-tailed Student’s t-test (a,c,g,h), **P < 0.01, ****P < 0.0001. Exact P values, source numerical data and unprocessed blots are provided.

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Extended Data Fig. 7 Covalent crosslinking of Cys864-Cys869 by unnatural amino acid (UAA) promotes LLPS and activation of RIG-I both in cells and in mice.

a, Immunoblot (IB) analysis of in vitro crosslinking specificity of RIG-IWT to RIG-I C864BprY in presence of 1 μM dsRNA. b, WT or the crosslinked GFP-RIG-I were mixed with Cy3–dsRNA at the indicated module concentration and were imaged for fluorescence (left). Statistical analysis of the droplet formation was shown (right). c, Immunofluorescence microscopy and DAPI staining of HeLa cells pretreated with TAT-Flag-RIG-I-Strep or TAT-Flag-RIG-I-C864BprY-Strep (3.6 μg/mL) for 4 h and stimulated for 12 h with SeV (left). Quantified percentage of cells with puncta and the average puncta per cell were shown (right); n = 3; *P = 0.0147, **P = 0.0038. d, Related to Fig. 7d, normalized CXCL10 (left) and ISG56 (right) mRNA expression (determined by qPCR analysis) was shown; n = 3; **P = 0.0038, ***P = 0.0009. e, IB analysis of the TAT-Flag-RIG-I-C864BprY-Strep expression in E. coli cells. f, Coomassie blue staining of purified TAT-Flag-RIG-I-Strep and TAT-Flag-RIG-I-C864BprY-Strep proteins. g, Related to Fig. 7m, left: IB analysis of the endogenous RIG-I protein crosslinking in lungs from hACE2 transgenic mice; n = 6. Middle and right: representative micrographs and quantification of indicated mouse lung tissue showed puncta of endogenous RIG-I; n = 3; *P = 0.0252. h,i, Normalized Cxcl10 mRNA (h) and Ccl5 mRNA (i) expression in the lungs (left), liver (middle) and spleen (right; determined by qPCR analysis) of hACE2 transgenic mice in (g); n = 6; P = 0.0049 (left), P = 0.0024 (middle), P = 0.0044 (right) (h); P = 0.0010 (left), P = 0.0020 (middle), P = 0.0020 (right) (i). j, Immunofluorescence microscopy and DAPI staining of the SARS-CoV-2 S protein antigen in lung sections of hACE2 transgenic mice in (g); n = 6. The fold change in fluorescence intensity (S protein) was quantified by ImageJ (right). Data are representative of at least three independent experiments and shown as mean ± s.d. Scale bar, 10 μm (c), 20 μm (b), 500 μm (g,j). Statistical analysis was performed using a two-tailed Student’s t-test (c,d,g–j); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Exact P values, source numerical data and unprocessed blots are provided.

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Extended Data Fig. 8 Developing RIG-I interfering peptides (RIPs) to prevent Cys864-Cys869 disulfide linkage can disrupt RIG-I LLPS thereby reducing RIG-I-mediated autoimmune responses.

a, Schematics show the C268F and E373A mutations within the RIG-I protein (left). Coomassie blue staining of GFP-RIG-I proteins (right). b, Representative images and quantification of the in vitro droplets formed by indicated protein; n = 3; P = 0.0022 (C268F vs. WT), P = 0.0078 (E373A vs. WT). c, SDD–AGE analysis of RIG-I aggregation and immunoblot (IB) analysis of RIG-I KO cells transfected with indicated plasmids. d, Immunofluorescence microscopy and DAPI staining of HeLa cells transfected with indicated plasmids. Quantified percentage of cells with RIG-I puncta was shown; n = 3. e, Normalized IFNB1 mRNA expression and IB analysis (right) of RIG-I KO HEK293T cells transfected with indicated plasmids; n = 3; ***P = 0.0002. f, Normalized CXCL10 and ISG56 mRNA expression in RIG-I KO HEK293T cells transfected with the indicated plasmids; n = 3. g, Related to Fig. 8d, normalized CXCL10 and ISG56 mRNA expression was shown; n = 3. h, Droplet formation (left) and quantification (right) of GFP-RIG (10 μM) treated with control BSA or RIPs (5 µM) for 30 min. n = 3. i, Related to Fig. 8h, normalized CXCL10 (left) and ISG56 (right) mRNA expression was shown; n = 3. j, Representative images (left) and quantification (right) of the in vitro droplet formation by GFP-RIG-I WT, C864S or C869S (10 μM) and mixed with control PBS or mtRNA (1 μg); n = 3. k, Related to Fig. 8i, normalized CXCL10 and ISG56 mRNA expression was shown; n = 3. l, IB analysis of p-IRF3, p-TBK1 and IRF3 dimerization and total IRF3, TBK1 in Rig-IWT/WT and Rig-IC865S/C865S MEFs transfected without (−) or with (+) mtRNA (2.5 μg/mL) for 12 h; n = 3. m, Related to Fig. 8l, normalized Cxcl10 and Ccl5 mRNA expression was shown; n = 3; ***P = 0.0001. Data are representative of at least three independent experiments and shown as mean ± s.d. Scale bar, 10 μm (d), 20 μm (b,h,j). Statistical analysis was performed using a two-tailed Student’s t-test (b,d–k,m); ****P < 0.0001; ns, not significant. Exact P values, source numerical data and unprocessed blots are provided.

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Extended Data Fig. 9 RIG-I inactivation by RIP-III ameliorates mtRNA-induced autoimmune responses in mice.

a, In situ PLA of RIG-I and MAVS in MEF cells stimulated of mtRNA (2.5 μg/mL, delivered by transfection) for 10 h, followed by treatment with RIP-III (50 µM) for 2 h (left); n = 3. The PLA-detected proximity (PROX) complexes (red dots) were quantified (right). b, Immunoblot (IB) of p-IRF3, p-TBK1 and IRF3 dimerization (native gel) and total IRF3, TBK1 and qPCR analysis of Ifnb1, Cxcl10 and Ccl5 mRNA expression in Rig-IWT/WT and Rig-IC865S/C865S MEFs transfected without (−) or with (+) mtRNA (2.5 μg/mL) for 10 h, followed by treatment with control BSA (−) or RIP-III (50 µM) for 2 h; n = 3; ***P = 0.0006. c,d, Related to Fig. 8o, qPCR analysis was performed to measure the mRNA levels of Il-1b (left), Il-6 (middle) and Tnf (right) in the spleen (c) and kidney (d) from the indicated groups; n = 6; ***P = 0.0006; (d) left: P = 0.0042, right: P = 0.0021. e,f, H&E staining of mice spleen tissues (e) and kidney tissues (f) from Fig. 8o. Data are representative of at least three independent experiments and shown as mean ± s.d. Scale bar, 10 μm (a), 100 μm (e,f). Statistical analysis was performed using a two-tailed Student’s t-test (a–d); **P < 0.01, ***P < 0.001, ****P < 0.0001. Exact P values, source numerical data and unprocessed blots are provided.

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Representative video of the still images in Fig. 1f.

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Representative video of the still images in Fig. 1j.

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Representative video of the still images in Fig. 2d (top).

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Representative video of the still images in Fig. 2d (bottom).

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Wang, B., Wang, Y., Pan, T. et al. Targeting a key disulfide linkage to regulate RIG-I condensation and cytosolic RNA-sensing. Nat Cell Biol 27, 817–834 (2025). https://doi.org/10.1038/s41556-025-01646-5

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