Research ArticleIMMUNOLOGY

RNF39 mediates K48-linked ubiquitination of DDX3X and inhibits RLR-dependent antiviral immunity

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Science Advances  05 Mar 2021:
Vol. 7, no. 10, eabe5877
DOI: 10.1126/sciadv.abe5877
  • Fig. 1 RNF39 inhibits RNA-mediated innate immune response.

    (A and B) Enzyme-linked immunosorbent assay (ELISA) (A) and reverse transcription polymerase chain reaction (RT-PCR) (B) analysis of IFN-β, TNF-α, and IL-6 expression in PMs from Rnf39+/+ or Rnf39−/− mice infected with SeV and VSV. (C) Luciferase activity analysis of IFN-β promoter activity in human embryonic kidney (HEK) 293T cells transfected with IFN-β reporter plasmid, together with empty vector control (Ctrl) plasmid, RNF39, or its mutant C108S after being infected with VSV. Plasmid expression was confirmed by immunoblot analysis (below blots). (D) RT-PCR analysis of Ifnb mRNA expression in Rnf39+/+ MEFs, Rnf39−/− MEFs, or Rnf39−/− MEFs transfected with RNF39 or its mutant C108S plasmid, followed by transfection with low–molecular weight (LMW) or high–molecular weight (HMW) polyinosinic:polycytidylic acid [poly(I:C)] for 4 hours. Plasmid expression was confirmed by immunoblot analysis (right blots). (E) ELISA analysis of IFN-β expression in mouse PMs transfected with Ctrl siRNA and Rnf39 siRNA 1 or 2 for 48 hours and then infected with VSV. All data are represented as means ± SD. Statistical significance was determined by unpaired two-tailed Student’s t tests: *P < 0.05 and **P < 0.01; NS, not significant. All experiments were repeated at a minimum of three times.

  • Fig. 2 RNF39 attenuates RLR-induced IRF3 activation.

    (A and B) Immunoblot analysis of p-TBK1, p-IRF3, and IRF3 in PMs from Rnf39+/+ or Rnf39−/− mice after being infected with SeV and VSV for the indicated time periods. (C) Immunoblot analysis of p-TBK1, p-IRF3, and IRF3 in mouse PMs transfected with Ctrl siRNA or Rnf39 siRNA 2 for 48 hours and then infected with VSV for the indicated time periods. (D) Luciferase activity analysis of IRF3 promoter activity in HEK293T cells transfected with IRF3 reporter plasmid and the indicated adaptor plasmids, together with RNF39 or empty vector control plasmid. Plasmid expression was confirmed by immunoblot analysis (right blots). All data are represented as means ± SD. Significance was determined by unpaired two-tailed Student’s t test: **P < 0.01. All experiments were repeated at a minimum of three times.

  • Fig. 3 Rnf39 deficiency enhances anti-RNA viral responses.

    (A and F) Immunoblot analysis of p-STAT1 and STAT1 in Rnf39+/+ or Rnf39−/− PMs infected with VSV [1 multiplicity of infection (MOI)] (A) or stimulated with IFN-β (20 ng/ml) (F) for the indicated time periods. (B and G) RT-PCR analysis of ISG mRNA expression in Rn39f +/+ or Rnf39−/− PMs infected with VSV (B) or stimulated with IFN-β (G). (C) RT-PCR analysis of Rantes mRNA expression in PMs from Rnf39+/+ or Rnf39−/− mice infected with SeV or VSV. (D and E) Microscopy (D) and RT-PCR (E) analysis of VSV replication in Rnf39+/+ or Rnf39−/− PMs infected with VSV-GFP (1 MOI) for 12 hours. Scale bars, 100 μm. All data are represented as means ± SD. Significance was determined by unpaired two-tailed Student’s t test: *P < 0.05 and **P < 0.01. All experiments were repeated at a minimum of three times.

  • Fig. 4 Rnf39 deficiency enhances innate immune responses against RNA viral infection in vivo.

    (A) ELISA analysis of IFN-β, TNF-α, and IL-6 production in serum from Rnf39+/+ or Rnf39−/− mice infected with VSV by intraperitoneal injection [phosphate-buffered saline (PBS), n = 2; VSV, n = 6 per condition]. (B) RT-PCR analysis of VSV replication in lung and spleen from Rnf39+/+ or Rnf39−/− mice infected with VSV by intraperitoneal injection (PBS, n = 2; VSV, n = 5 per condition). (C) Hematoxylin and eosin staining of lung tissue sections from Rnf39+/+ or Rnf39−/− mice infected with VSV by intraperitoneal injection (PBS, n = 4; VSV, n = 4 per condition). Scale bars, 100 μm. (D) Survival of Rnf39+/+ or Rnf39−/− mice infected with VSV by intraperitoneal injection (n = 11 per condition). The data are represented as means ± SD. Significance was determined by unpaired two-tailed Student’s t test: *P < 0.05 and **P < 0.01. The data of TNF-α and IL-6 in (C) are represented as medians ± SD. Significance was determined by nonparametric tests: *P < 0.05 and **P < 0.01. All experiments were repeated at a minimum of three times.

  • Fig. 5 RNF39 targets DDX3X.

    (A) Coimmunoprecipitation of RNF39 with the indicated adaptors from HEK293T cells transfected with Flag-RNF39 and the indicated Myc-tagged plasmids. IP, immunoprecipitation; IB, immunoblot. (B) Coimmunoprecipitation of endogenous RNF39 with endogenous DDX3X from mouse PMs infected with SeV for the indicated time periods. (C) Colocalization between RNF39 and DDX3X in MEFs was examined by confocal microscopy infected with VSV. Scale bars, 10 μm. (D) HA-DDX3X was obtained by in vitro transcription and translation. Interaction between RNF39 and DDX3X was assayed by mixing recombinant protein of human RING finger protein 39 (RNF39) transcript and DDX3X-HA together, followed by immunoprecipitation with Flag antibody and immunoblot analysis with HA antibody. (E) Schematic diagram of RNF39 and its truncation mutants. RING, RING domain; PRY/SPRY, PRY/SPRY domain. (F) Flag-tagged RNF39 mutants and Myc-DDX3X were individually transfected into HEK293T cells. The cell lysates were immunoprecipitated with an anti-Myc antibody and then immunoblotted with the indicated antibodies. The specific bands were marked by asterisk. (G) Schematic diagram of DDX3X and its truncation mutants. RecA-1, RecA-like domains-1; RecA-2, RecA-like domains-2; RS, arginine-serine-rich domain. (H) Myc-tagged DDX3X or its mutants and Flag-RNF39 were individually transfected into HEK293T cells. The cell lysates were immunoprecipitated with Flag antibody and then immunoblotted with the indicated antibodies. The specific bands were marked by asterisk. All experiments were repeated at a minimum of three times.

  • Fig. 6 RNF39 promotes proteasomal degradation of DDX3X.

    (A and B) Immunoblot analysis of extracts (A) or RT-PCR analysis (B) of mouse PMs from Rnf39+/+ or Rnf39−/− mice infected with VSV for the indicated time points. (C) Immunoblot analysis of extracts from HEK293T cells transfected with Myc-DDX3X and increasing amount of Flag-RNF39 (0, 0.5, 1, or 2 μg) expression plasmids. (D and E) Immunoblot analysis of extracts (D) from Rnf39+/+ or Rnf39−/− mouse PMs infected with VSV for 4 hours and then treated with cycloheximide (CHX) for various times. DDX3X expression level was quantitated by measuring band intensities using “ImageJ” software (E). The values were normalized to actin. (F) Immunoblot analysis of extracts from HEK293T cells transfected with Myc-DDX3X and Flag-RNF39 expression plasmid and then treated with MG132, chloroquine, or 3-MA for 4 hours. (G) Immunoblot analysis of extracts from HEK293T cells transfected with Myc-DDX3X, together with Flag-tagged RNF39 or RNF39 C108S expression plasmids. All data are represented as means ± SD. Significance was determined by unpaired two-tailed Student’s t test: **P < 0.01. All experiments were repeated at a minimum of three times.

  • Fig. 7 RNF39 promotes K48-linked ubiquitination of DDX3X.

    (A) Lysates from HEK293T cells transiently cotransfected with HA-Ub, Myc-DDX3X along with RNF39-Flag or RNF39 C108S-Flag, were subjected to immunoprecipitation with Myc antibody followed by immunoblot analysis with HA antibody. (B) Lysates from HEK293T cells transiently cotransfected with HA-Ub, Myc-DDX3X along with RNF39-Flag or RNF39 C108S-Flag, were subjected to immunoprecipitation with Myc antibody. The immunoprecipitates were denatured and re-immunoprecipitated with Myc antibody (two-step immunoprecipitation, Re-IP) and then analyzed by immunoblot analysis. (C) Re-IP analysis lysates from HEK293T cells transiently cotransfected with HA-Ub (WT and its mutants), Flag-RNF39, and Myc-DDX3X. (D) Immunoprecipitation analysis lysates from HEK293T cells transiently cotransfected with K48-Ub or K63-Ub mutant, Flag-RNF39, and Myc-DDX3X. (E and F) Lysates from Rnf39+/+ or Rnf39−/− mouse PMs infected with SeV for 8 hours were immunoprecipitated with DDX3X antibody, followed by immunoblot analysis with indicated antibodies. (G and H) Re-IP analysis lysates from HEK293T cells transiently cotransfected with HA-Ub, Flag-RNF39, along with Myc-DDX3X (WT and its point mutants). (I) Immunoblot analysis of extracts from HEK293T cells transfected with Myc-DDX3X or Myc-DDX3X K55/138/162R mutant, together with Flag-tagged RNF39 or RNF39 C108S expression plasmid. (J) Luciferase activity analysis of IFN-β promoter activity in HEK293T cells transfected with IFN-β reporter plasmid, together with empty vector control plasmid, Myc-DDX3X, or Myc-DDX3X K55/138/162R mutant after being infected with VSV. Plasmid expression in the HEK293T cells was confirmed by immunoblot analysis. All data are represented as means ± SD. Significance was determined by unpaired two-tailed Student’s t test: **P < 0.01. All experiments were repeated at a minimum of three times.

Supplementary Materials

  • Supplementary Materials

    RNF39 mediates K48-linked ubiquitination of DDX3X and inhibits RLR-dependent antiviral immunity

    Wenwen Wang, Mutian Jia, Chunyuan Zhao, Zhongxia Yu, Hui Song, Ying Qin, Wei Zhao

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