UBXN3B positively regulates STING-mediated antiviral immune responses

Abstract

The ubiquitin regulatory X domain-containing proteins (UBXNs) are likely involved in diverse biological processes. Their physiological functions, however, remain largely unknown. Here we present physiological evidence that UBXN3B positively regulates stimulator-of-interferon genes (STING) signaling. We employ a tamoxifen-inducible Cre-LoxP approach to generate systemic Ubxn3b knockout in adult mice as the Ubxn3b-null mutation is embryonically lethal. Ubxn3b^-/-, like Sting^-/- mice, are highly susceptible to lethal herpes simplex virus 1 (HSV-1) and vesicular stomatitis virus (VSV) infection, which is correlated with deficient immune responses when compared to Ubxn3b^+/+ littermates. HSV-1 and STING agonist-induced immune responses are also reduced in several mouse and human Ubxn3b^-/- primary cells. Mechanistic studies demonstrate that UBXN3B interacts with both STING and its E3 ligase TRIM56, and facilitates STING ubiquitination, dimerization, trafficking, and consequent recruitment and phosphorylation of TBK1. These results provide physiological evidence that links the UBXN family with antiviral immune responses.

Fig. 1

UBXN3B is critical for IFN-I induction by and control of HSV-1 infection in vivo. a Immunoblots showing Ubxn3b knockout efficiency in various tissues without/with tamoxifen (TMX) treatment in Cre^+/− Ubxn3b^flox/flox mice. Tubulin is a housekeeping protein control. b, c The survival curves of tamoxifen (TMX)-treated Ubxn3b^flox/flox, TMX-treated Cre^+/−, mock-treated Cre^+/− Ubxn3b^flox/flox (designated Ubxn3b^+/+) and TMX-treated Cre^+/− Ubxn3b^flox/flox (designated Ubxn3b^−/−) littermate and Sting^−/− mice challenged with 1 × 10⁷ plaque-forming units (PFU) per mouse of HSV-1 i.v. N = 8–9 mice/genotype. ***P < 0.001 (log-rank test). d The serum IFN-I concentrations (mean ± s.e.m) of mice challenged with 2 × 10⁶ PFU per mouse of HSV-1 i.v. **P < 0.01; *P < 0.05 (non-parametric Mann–Whitney test), N = 10 (Ubxn3b^+/+) or N = 6 (Ubxn3b^−/−). e The viral titers (mean ± s.e.m) in the brain on day 3 after infection (PFU per gram tissue). **P < 0.01 (non-parametric Mann–Whitney test), N = 5 mice per genotype. The data are pooled from two independent experiments

Fig. 2

UBXN3B is critical for STING-dependent IFN-I induction in mouse primary cells. a ELISA of IFN-α in the cell culture supernatants of (Ubxn3b^+/+, Ubxn3b^−/−) bone marrow-derived macrophages (M^ϕ), Flt3-induced pDCs, and GM-CSF-induced cDCs (pooled from five littermates) 20 h after the indicated treatments. N = 3 per genotype. *P < 0.05 (unpaired Student’s t test). b Immunoblots of an interferon-stimulated gene Oas1a, Sting, and Ubxn3b expression in cDCs 20 h after the indicated treatments. Tubulin is a housekeeping protein control. qPCR analysis of (c) Ifnb1 and Tnfa mRNA expression and d cellular HSV-1 genome loads in cDCs infected with HSV-1 (MOI = 10) for the indicated time. e Immunoblots showing Ubxn3b and housekeeping Gapdh protein expression in mock (Ubxn3b^+/+) or 4-hydroxyl tamoxifen-treated Cre^+/− Ubxn3b^flox/flox (Ubxn3b^−/−) MEFs. f Viral titers (plaque-forming units/ml) in the supernatant of MEFs infected with HSV-1 (MOI = 0.1). N = 3 per genotype per time point. *P < 0.05; **P < 0.01 (unpaired Student’s t test). g qPCR analysis of selected immune gene mRNA expression in MEFs infected with HSV-1 as in g. h The immunoblots show knockout efficacy of STING and UBXN3B by CRISPR-Cas9 in human primary trophoblasts. Actin is a housekeeping protein control. i Fluorescent microscopic images of human primary trophoblasts infected with HSV-1-GFP (MOI = 0.3) for 18 h. Objective, ×5. Scale bar, 10 µm. j qPCR analysis of Ifnb1 mRNA expression in human primary trophoblasts infected with HSV-1-GFP for the indicated time. Bars/data points: mean ± s.e.m. Two biological replicates were pooled for qPCR (N = 2 per genotype per time point). *P < 0.05; **P < 0.01 (unpaired Student’s t test). The results are representative of two independent experiments

Fig. 3

UBXN3B is critical for type I IFN responses to SeV and VSV, but not EMCV infection. a The survival curves of Ubx3b^−/− (N = 6) and Ubxn3b^−/− (N = 7) mice infected with 1 × 10⁷ PFU of VSV i.v. The results are pooled from two experiments. *P < 0.05 (log-rank test). b, c qPCR analysis of Ifnb1 expression in cDCs infected with b VSV (MOI = 5) or c SeV (200 hemagglutination units per 10⁵ cells). d ELISA of IFN-α in the cell culture supernatants of cDCs infected with SeV and VSV as in b, c. N = 3 per genotype per time point. *P < 0.05 (unpaired Student’s t test). e The survival curves of Ubx3b^+/+ (N = 7) and Ubxn3b^−/− (N = 8) mice infected with 200 PFU of EMCV i.p. P = 0.53 (log-rank test). The results are pooled from two experiments. f qPCR analysis of Ifnb1 expression in cDCs infected with EMCV (MOI = 5) for the indicated time. Bars/data points: mean ± s.e.m. Two biological replicates were pooled for qPCR (N = 2 per genotype per time point). *P < 0.05 (unpaired Student’s t test). The results are representative of two independent experiments

Fig. 4

UBXN3B regulates STING dimerization, phosphorylation, and degradation. a Immunoblots showing Sting dimerization in untreated (Ubxn3b^+/+) and 4-hydroxyl tamoxifen-induced Cre^+/− Ubxn3b^flox/flox (Ubxn3b^−/−) primary MEFs. The cells were infected without (mock) or with HSV-1 (MOI = 0.5) for 8 h. b Immunoblotting analysis of the whole-cell lysates of bone marrow-derived cDCs infected with HSV-1 (MOI = 5). Mono monomer, 2-ME β-mercaptoethanol, a chemical compound that reduces disulfide bonds. c qPCR quantification of Ifnb1 and Tnfa mRNA induction in cDCs by HSV-1 (MOI = 5). Bars: mean ± s.e.m. Two biological replicates were pooled for qPCR (N = 2 per genotype per time point). *P < 0.05 (unpaired Student’s t test). d–f Immunoblotting analysis of the whole-cell lysates of d MEFs infected with HSV-1 (MOI = 0.5), e MEFs transfected with ISD (8 µg/ml) in the absence or presence of 40 µM of chloroquine (+CQ) and f trophoblasts transfected with cGAMP (8 µg/ml). In e, f, the arrows indicate phosphorylated STING with long and short exposure. Actin, Tubulin (Tub), and GAPDH are housekeeping protein controls. g qPCR quantification of IFNB1 and TNFA mRNA induction in trophoblasts transfected with cGAMP (8 µg/ml). In c, g, the bars are: mean ± s.e.m. Two biological replicates were pooled for qPCR (N = 2 per genotype per time point). *P < 0.05 (unpaired Student’s t test). The results are representative of two to three independent experiments

Fig. 5

UBXN3B interacts with STING and TRIM56. a Co-immunoprecipitation (co-IP) of STING with UBXN3B from HEK293 cells transfected with FLAG-UBXNs and HA-STING plasmids using anti-FLAG magnetic beads, followed by immunoblotting (IB) with an anti-HA (STING) and FLAG (UBXN) antibody. b Immunofluorescence staining of STING and UBXN3B in H1975 cells treated without (Mock) or with ISD (8 µg/ml) for 3 h. The cells were stained with a mouse anti-human STING and rabbit anti-UBXN3B antibody followed by a secondary antibody conjugated with Alexa Fluor 594/488. The nuclei were stained with DAPI. Objective, ×40. Scale bar, 200 µm. c A schematic diagram of UBXN3B functional domains. d Co-IP of STING with the truncated forms of UBXN3B. The procedures are similar to b. e Co-IP of UBXN3B with TRIM from HEK293 cells transfected with FLAG-UBXN3B and Myc-TRIM plasmids using anti-Myc magnetic beads, followed by IB with an anti-Myc (TRIM) and FLAG (UBXN3B) antibody. f Co-IP of TRIM56 with the truncated forms of UBXN3B from HEK293 cells transfected with FLAG-UBXN3B truncates and Myc-TRIM56 plasmids using anti-FLAG magnetic beads, followed by IB with an anti-Myc (TRIM56) and FLAG (UBXN truncates) antibody. GAPDH is a housekeeping protein control. WCE whole-cell extract. The results are representative of two independent experiments

Fig. 6

UBXN3B mediates STING interaction with and ubiquitination by TRIM56. a WT and Sting^gt/gt MEFs were infected with or without HSV-1 (MOI = 0.5) for 8 h. Sting was immunoprecipitated (IP) from the MEF lysates using a rabbit anti-Sting antibody. The immunoblots indicate Sting co-immunoprecipitation with Ubxn3b and Trim56 after viral infection. WCE whole-cell extract. b WT and Ubxn3b^−/− MEFs were transfected with vector or FLAG-UBXN3B plasmids by electroporation, and then infected with HSV-1 (MOI = 0.5) for 8 h. Sting IP was performed as in a. Rabbit IgG was used as a negative control. c The immunoblots show knockout efficacy of UBXN3B in HEK293T-STING cell line. d WT and UBXN3B^−/− cells (parent cell line is HEK293T-FLAG-STING) were transfected with the indicated combinations of plasmids. FLAG-STING was immunoprecipitated (IP) with anti-FLAG magnetic beads. The proteins in IP and WCE were immunoblotted by an anti-K63-linked polyubiquitin, anti-Myc (UBXN3B and TRIM56) and anti-FLAG (STING) antibody, respectively. e Sting was precipitated with a rabbit anti-Sting antibody from cDCs infected without or with HSV-1 (MOI = 10) for 4 and 8 h. The Sting^−/− cDCs were used as an IP control. The proteins in IP and WCE were immunoblotted with an anti-K63 and Sting antibody, respectively. Tubulin and GAPDH are housekeeping controls. The results are representative of two independent experiments