STING exerts antiviral innate immune response by activating pentose phosphate pathway

Abstract

Background: The innate immune system serves as the host's first line of defense against invading pathogens. Stimulator of interferon genes (STING) is a key component of this system, yet its relationship with glucose metabolism, particularly in antiviral immunity, remains underexplored.

Methods: Metabolomics analysis was used for detecting metabolic alterations in spleens from STING knockout (KO) and wild-type (WT) mice. Co-immunoprecipitation was employed for determining ubiquitination of TKT. Mass spectrometry was used for detecting interaction proteins of STING. Enzyme activity kits were used for detecting the activities of TKT and G6PD.

Results: In this study, we demonstrate that herpes simplex virus (HSV) infection activates the pentose phosphate pathway (PPP) in host cells, thereby initiating an antiviral immune response. Using STING-manipulated cells and systemic knockout mice, we show that STING positively regulates PPP, which, in turn, limits HSV infection. Inhibition of the PPP significantly reduced the production of antiviral immune factors and dampened STING-induced innate immune responses. Mechanistically, we discovered that STING interacts with transketolase (TKT), a key enzyme in the non-oxidative branch of the PPP, and reduces its ubiquitination via the E3 ubiquitin ligase UBE3A, stabilizing TKT. Silencing TKT or inhibiting its activity with oxythiamine diminished antiviral immune factor production.

Conclusion: Our findings reveal that the PPP plays a synergistic role in generating antiviral immune factors during viral infection and suggest that PPP activation could serve as an adjunct strategy for antiviral therapy.

Keywords: Antiviral immune response; Inflammatory factors; PPP; STING; Transketolase.

Fig. 1

STING upregulates the pentose phosphate pathway (PPP). A Analysis of metabolomics in spleens of wild-type and STING knockout mice. B Metabolic pathway enrichment analysis in spleens of wild-type and STING knockout mice. C Levels of R-5P, 6-PG, and F-6P in spleens of wild-type and STING knockout mice. D E Whole cell extracts (WCEs) from wild-type and STING-overexpressing KYSE-30 cells were analyzed for PPP metabolites (NADPH, GSH, R5P) and key enzymes (TKT, G6PD) using ELISA kits. F, G WCEs from wild-type and STING knockdown KYSE-30 cells were similarly analyzed. Data are expressed as mean ± SEM from three biological replicates. **p < 0.01, *** p < 0.001

Fig. 2

PPP participates in the antiviral immune response. A, B Analysis of metabolomics and metabolic pathway enrichment in spleens of control (PBS) and HSV-treated mice (HSV-1, 5 × 10^6 PFU/mouse, tail vein injection). Spleens were collected six days post-injection for analysis. C ELISA detection of PPP pathway metabolites (TKT, R-5P, GSH) in KYSE-30 cells transfected with poly(dA:dT). D qPCR detection of inflammatory factors (IL-6, CCL5, IFN-β) and PPP enzymes (TKT, G6PD) mRNA in poly(dA:dT)-transfected KYSE-30 cells. E Levels of IL-6, CCL5, IFN-β, and TKT in KYSE-30 cells treated with poly(dA:dT) and/or the PPP inhibitor 6-AN (10 µM, 8 h). F Levels of IL-6, CCL5, IFN-β, and TKT in KYSE-30 cells treated with HSV (MOI = 1, 8 h) and/or 6-AN (10 µM, 8 h). Data are mean ± SEM from three biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001

Fig. 3

PPP mediates STING-facilitated antiviral immune response. A Analysis of glucose metabolomics in spleens from wild-type and STING knockout mice injected with HSV via tail vein. B mRNA expression of inflammatory factors (IFN-β, IL-6, CCL5) in STING-overexpressed KYSE-30 cells transfected with poly(dA:dT). C Levels of TKT, G6PD, R-5P, and GSH in STING-overexpressed KYSE-30 cells treated with poly(dA:dT) and 6-AN or OT (10 mM, TKT inhibitor) for 10 h. D Levels of STING and inflammatory cytokines (IFN-β, CCL5, IL-6) in STING-overexpressed KYSE-30 cells treated with poly(dA:dT) and 6-AN. E mRNA expression of TKT and inflammatory cytokines (IFN-β, CCL5, IL-6) in STING-overexpressed KYSE-30 cells treated with ISD (DNA virus analogue) and OT. Data are mean ± SEM from three biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001

Fig. 4

STING upregulates TKT. A Transcriptome sequencing analysis revealed mRNA expression of key PPP enzymes (TKT, G6PD) in wild-type and STING knockout mice. B mRNA expression of TKT and G6PD in wild-type and STING-overexpressed cells detected by qPCR. C Co-IP using an anti-STING antibody, followed by mass spectrometry to identify proteins interacting with STING. D Interaction between TKT and STING detected by anti-TKT and anti-STING antibodies. E Expression of G6PD and TKT in STING-overexpressing KYSE-30 cells and HSV-infected cells. F IHC staining of STING and TKT in liver tissues from wild-type and STING knockout mice. G TKT enzyme activity and mRNA expression of STING in KYSE-30 cells treated with poly(dA:dT). Data are mean ± SEM from three biological replicates. *** p < 0.001; ns, no significance

Fig. 5

STING stabilizes TKT by inhibiting UBE3A-mediated TKT ubiquitination. A Pan-phosphorylation antibody of serine and threonine was used to detect TKT phosphorylation. B, C Identification of K48-linked ubiquitination of TKT in 293 T cells transfected with His-Ub and treated with MG132. D, E STING downregulates K48-linked ubiquitination of TKT. F, G Detection of interactions among STING, TKT, and UBE3A using specific antibodies. H Detection of the correlation between STING and UBE3A by western blots in KYSE-30 cells with STING knockdown and overexpression. I Effect of UBE3A on the ubiquitination of TKT was detected by immunoprecipitation in KYSE-30 cells transfected with His-Ub and shUBE3A. J K48-linked ubiquitination of TKT was detected by immunoprecipitation in STING-knocked down KYSE-30 cells transfected with His-Ub and shUBE3A. Data are mean ± SEM from three biological replicates

Fig. 6

TKT mediates the antiviral immune response of STING. A Expression and enzyme activity of TKT in control and TKT siRNA KYSE-30 cells. B ELISA detection of antiviral immune factors (IL-6, IFN-β) in control and TKT siRNA KYSE-30 cells with or without poly(dA:dT). C qPCR detection of inflammatory factors (CCL-5, IL-6, IFN-β) in control and TKT siRNA KYSE-30 cells with or without poly(dA:dT). D qPCR detection of inflammatory factors in control and TKT overexpressing KYSE-30 cells with or without poly(dA:dT). E qPCR detection of inflammatory factors in control and STING-knocked down KYSE-30 cells transfected with TKT expressing vector and/or poly(dA:dT). Data are mean ± SEM from three replicates in each group. ** p < 0.01; *** p < 0.001

Fig. 7

Schematic diagram depicting that STING exerts antiviral innate immune response by activating pentose phosphate pathway. DNA virus infection activates the PPP in host cells, thereby initiating STING-mediated antiviral immune response. STING interacts with TKT, a key enzyme in the non-oxidative branch of the PPP, and reduces its ubiquitination via the E3 ubiquitin ligase UBE3A, stabilizing TKT and upregulating PPP