Activation of cGAS/STING pathway upon paramyxovirus infection

Abstract

During inflammatory diseases, cancer, and infection, the cGAS/STING pathway is known to recognize foreign or self-DNA in the cytosol and activate an innate immune response. Here, we report that negative-strand RNA paramyxoviruses, Nipah virus (NiV), and measles virus (MeV), can also trigger the cGAS/STING axis. Although mice deficient for MyD88, TRIF, and MAVS still moderately control NiV infection when compared with wild-type mice, additional STING deficiency resulted in 100% lethality, suggesting synergistic roles of these pathways in host protection. Moreover, deletion of cGAS or STING resulted in decreased type I interferon production with enhanced paramyxoviral infection in both human and murine cells. Finally, the phosphorylation and ubiquitination of STING, observed during viral infections, confirmed the activation of cGAS/STING pathway by NiV and MeV. Our data suggest that cGAS/STING activation is critical in controlling paramyxovirus infection and possibly represents attractive targets to develop countermeasures against severe disease induced by these pathogens.

Keywords: Virology; immune system; molecular biology.

Graphical abstract

Figure 1

STING plays a role in the control of NiV infection in mice Wild-type (WT) mice and mice deficient in indicated pathogen recognition signaling pathways were infected intraperitoneally with 10⁶ plaque-forming unit (PFU) of NiV (5 or 6 animals per group). (A) Survival of mice infected by NiV was followed up for 24 days. †p < 0.05 (MyD88/TRIF/MAVS/STING KO versus WT), ‡p < 0.05 (IFNAR KO versus WT), and ₸p < 0.01 (MyD88/TRIF/MAVS KO versus MyD88/TRIF/MAVS/STING KO) (Gehan-Breslow-Wilcoxon test). (B) Immunohistochemistry of murine brains following NiV infection. Brains of WT mouse, IFNAR KO mouse, MyD88/TRIF/MAVS KO, and MyD88/TRIF/MAVS/STING KO were collected on days 2, 6, 13, and 11, respectively. Scale bars, 100 μm. (C–E) Expression of NiV nucleoprotein (NiV-N) in the brain of NiV-infected mice, harvested on the day of death or euthanized at the end of protocol for different genotypes, was determined by RT-qPCR. Data are represented as mean ± SEM. Analysis of IFNβ and IFNα expression by RT-qPCR in organs harvested 2–13 days after infection. All samples were analyzed using one-way analysis of variance, followed by the Tukey multiple comparisons test, ∗p < 0.05 compared with WT condition.

Figure 2

STING controls NiV replication in primary murine embryonic fibroblasts (pMEFs) pMEFs obtained from mice deficient in the indicated signaling pathways were infected with rNiV-eGFP (MOI of 0.3) and cultured for 24 h. (A) Cells were analyzed for eGFP expression by fluorescence microscopy. Scale bars, 100 μm. (B–E) Cells and supernatants were harvested and analyzed by RT-qPCR for NiV-N (B and C), IFNβ (D), and IFNα (E) expression. Data are represented as mean ± SEM. The statistical significance of differences between infected wild-type (WT) cells and knockout (KO) cells was analyzed using one-way analysis of variance, followed by the Tukey multiple comparisons test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 compared with NiV-infected WT condition.

Figure 3

cGAS/STING pathway has a critical role in the control of paramyxovirus infection in human THP-1 cells THP-1 cells deficient in the indicated signaling pathways were infected with NiV-eGFP and MeV-eGFP (MOI of 0.1) for 48 and 24 h respectively. (A and B) Cells were analyzed for eGFP expression by fluorescence microscopy. (C–J) Cell lysates and/or supernatants were harvested and analyzed by RT-qPCR for the expression of NiV-N (C and D), MeV-N (G and H), IFNβ (E and I), and IFNα (F and J). Data are represented as mean ± SEM. THP-1 WT cells treated or not (NT) with the specific inhibitors for cGAS (RU.521) or STING (H151) were infected with NiV-eGFP and MeV-eGFP (MOI of 0.1) for 24 and 48 h (K and L) Cells were analyzed for eGFP expression by fluorescence microscopy. Scale bars, 100 μm. The statistical significance of differences between infected WT and KO cells was analyzed using one-way analysis of variance, followed by the Tukey multiple comparisons test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 compared with infected WT condition.

Figure 4

Paramyxovirus infection activates the cGAS/STING pathway in human cells Human pulmonary microvascular endothelial (HPMEC) cells and THP-1 cells deficient in the indicated signaling platforms were infected with either NiV or MeV (MOI of 1) for 6, 24, or 48 h (A–C) Cells were analyzed for phospho-STING (p-STING), STING, Caspase 3, cleaved Caspase 3, and GAPDH expression by western blot analysis. (D and E) HPMEC cells were infected with NiV (D) and MeV (E) (MOI of 1) for 48 h before cell lysis. Endogenous STING was immunoprecipitated using anti-STING antibodies followed by western blot analyses using anti-STING, anti-Ub-K63, anti p-STING, and anti-GAPDH antibodies. In parallel, endogenous expression levels of STING, p-STING, and Ub-K63 together as GAPDH as loading control in cell lysates were analyzed by western blot. Shown data are representative of three independent experiments showing similar results. See also Figure S3C for western blot showing multiple ubiquitination forms of STING.