Genistein Targets STING-Driven Antiviral Responses

Abstract

Cytoplasmic detection of DNA by cyclic GMP-AMP (cGAMP) synthase (cGAS) is an essential component of antiviral responses. Upon synthesis, cGAMP binds to the stimulator of interferon (IFN) genes (STING) in infected and adjacent cells through intercellular transfer by connexins forming gap-junctions, eliciting a strong IFN-β-driven antiviral response. We demonstrate here that Genistein, a flavonoid compound naturally occurring in soy-based foods, inhibits cGAS-STING antiviral signaling at two levels. First, Genistein pretreatment of cGAMP-producing cells inhibited gap-junction intercellular communication, resulting in reduced STING responses in adjacent cells. In addition, Genistein directly blocked STING activation by the murine agonist DMXAA, by decreasing the interaction of STING with TBK1 and IKKε. As a result, Genistein attenuated STING signaling in human and mouse cells, dampening antiviral activity against Semliki Forest Virus infection. Collectively, our findings identify a previously unrecognized proviral activity of Genistein mediated via its inhibitory effects at two levels of cGAS-STING signaling. IMPORTANCE Several reports suggest that Genistein exhibits antiviral activities against DNA viruses. Our work uncovers a previously unrecognized proviral effect of Genistein, through inhibition of the cGAS-STING pathway at the level of cGAMP transfer and its sensing by STING. This suggests that the use of Genistein as an antiviral should be taken with caution as it may reduce the protective antiviral effects elicited by host STING activation.

Keywords: Genistein; STING inhibitor; cGAMP; gap junction.

FIG 1

Genistein inhibits transactivation of neighboring cells. (A) HEK-cGAS^low cells pretreated with indicated compounds (Genistein [Gen], Resveratrol [Res], Quercetin [Quer], Apigenin [Api], and Kaempferol [Kaemp] at 50, 25, and 12.5 μM; EGCG at 25, 12.5, and 6.25 μM) for 48 h were cocultured overnight with LL171 cells. ISRE-Luciferase levels were measured from the cell lysates the next day. The data are shown relative to nontreated (NT) LL171 cells (n = 3). (B) LL171 cells were treated with indicated compounds for 48 h and ISRE-luciferase levels were analyzed. Data are shown relative to NT LL171 cells (n = 3). (C) HEK-cGAS^low cells pretreated with 50 or 30 μM Genistein for 48 h were collected and counted. ~20,000 HEK-cGAS^low cells were subsequently cocultured overnight with LL171 cells. ISRE-Luciferase levels are shown relative to NT LL171 cells (n = 3). (D) MG-63 cells pretreated for 48 h with 30 or 50 μM Genistein were transfected with 2.5 μg/mL of ISD70 for 4 h (after wash to remove ISD70 [7]) or untransfected (UT), and cocultured overnight with the same amount of recipient LL171 cells. ISRE-Luciferase levels are shown relative to the ISD70-treated cells (n ≥ 2). (E) HEK-cGAS^low cells were treated for 48 h with 50 μM Genistein prior to lysis and cGAMP-specific ELISA. Data are shown relative to NT HEK-cGAS^low cells (n = 3). (F) HEK-STING (HEK) and HEK-STING CX KO cells (HEK CXKO) were pretreated with 50 μM Genistein or not (NT) for ~40 h followed by treatment with 2 μg/mL Calcein-AM solution for 1 h. The donor cells (in Bold) were washed, counted, and 7,000 donor cells were cocultured (indicated with “>” sign) with ~40,000 recipient confluent cells of the same genotype, in the presence or not of CBX (100 μM). Calcein dye transfer was quantified after 4 h by counting the number of Calcein positive cells per fields. Data are shown reported to NT condition [Calcein positive cells only] (n = 2 for HEK CXKO and n = 3 for HEK). (G) Western blot analysis of CX43 levels from the lysates of MEFs treated with 50 μM Genistein for 48 h (representative of n = 2). (H) MEFs pretreated with or without 50 μM Genistein for ~40 h were cocultured overnight with HEK-cGAS^low (cGAS^low) cells. Supernatants were collected, and mouse IP-10 levels measured by specific ELISA (n = 3). (I) MEFs were pretreated for 48 h with 50 μM Genistein prior to the overnight transfection with 1 μg/mL ISD45 and mouse IP-10 levels measurement by specific ELISA (n = 2). (J) LL171 cells were pretreated for 1 h with 40 μM Genistein, prior to transfection with 2.5 μg/mL ISD45 for 6 h. ISRE-Luciferase levels are shown relative to the ISD-treated cells (n = 3). (A, B, C, D, E, F, H, I, J) Each point represents the mean data for each independent experiment. Unpaired t-tests (E, J), two-way (D, F) or one-way (A, B, C, H, I) ANOVA comparisons are shown. Data are mean ± s.e.m.; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, nonsignificant.

FIG 2

Genistein inhibits STING sensing of its ligands. (A) LL171 cells were treated for 1 h with indicated dose of Genistein followed by DMXAA stimulation (20 μg/mL) for ~8 h. ISRE-luciferase levels were normalized to the “DMXAA only” condition, after background correction with NT condition (n = 4). (B) HEK-STING cells transiently overexpressing cGAS-GFP, and an IFN-β-luciferase or an NF-κB-luciferase reporter were treated overnight with indicated dose of Genistein. The data are shown reported to the “NT” condition (n = 3 for IFN-β-luciferase and n = 2 for NF-κB-luciferase). (C) iBMDMs were pretreated or not for 1 h with 40 μM Genistein, washed and treated with 50 μg/mL DMXAA for indicated times before lysis and immunoblotting (n = 3). (D) iBMDM cells were pretreated or not for 1 h with 40 μM Genistein prior to overnight stimulation with 50 μg/mL DMXAA and mouse TNF-α levels measured by specific ELISA (n = 3). (E) Undifferentiated THP-1 cells were pretreated or not for 1 h with 40 μM Genistein before 6 h stimulation with 100 nM diABZI (left) or 30 μM ADU-S100 (right), and IP-10 levels were analyzed by specific ELISA (n = 3). (F) MG-63 cells were pretreated or not for 1 h with 40 μM Genistein, followed by 7 h stimulation with 100 nM diABZI, and IP-10 levels were analyzed by specific ELISA (n = 2). (G, H) HEK293T-GFP (HEK-GFP) transiently expressing an IFN-β-luciferase reporter with either Citrine (control), STING(R284S) (G), TBK1, or IKKε vectors (H), were treated overnight with indicated dose of Genistein or 3.6 μM H-151 (STING inhibitor). Data are shown relative to the “NT” condition (n = 3). (I) Immunoblot of Sting^KO iBMDMs expressing mCitrine-STING, after 1 h stimulation with 50 μg/mL DMXAA and 150 μM Genistein. Pink: pull-down of mCitrine-STING with anti-GFP antibody. Black: whole cell lysates (n = 3). (J) In silico docking of Genistein with a STING dimer—the model shown is that of pose 1, with the most favorable binding energy (see Text S1 and Fig. S3). Genistein is shown as sticks and 3 h-bonds are indicated as dashed lines. (K) LL171 cells were pretreated for 1 h with or without 40 μM Genistein followed by stimulation with 20 μg/mL DMXAA. After 4 h stimulation with DMXAA, the cells were washed and infected in biological triplicate with Semliki Forest virus (SFV) for 24 h (MOI 2). Viral titers were assayed with log₁₀ fold dilutions on confluent Vero cells as shown. NI: not infected (uninfected cells stain with crystal violet); NT: nontreated prior infection. Data shown are representative of three independent experiments. (D, E, F) Each point represents the mean data for each independent experiment. Two-way (G) or one-way (D, E, F) ANOVA comparisons are shown. Data are mean ± s.e.m.; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ns, is nonsignificant.