Activation of STING Signaling Pathway Effectively Blocks Human Coronavirus Infection

Abstract

The COVID-19 pandemic poses a serious global health threat. The rapid global spread of SARS-CoV-2 highlights an urgent need to develop effective therapeutics for blocking SARS-CoV-2 infection and spread. Stimulator of Interferon Genes (STING) is a chief element in host antiviral defense pathways. In this study, we examined the impact of the STING signaling pathway on coronavirus infection using the human coronavirus OC43 (HCoV-OC43) model. We found that HCoV-OC43 infection did not stimulate the STING signaling pathway, but the activation of STING signaling effectively inhibits HCoV-OC43 infection to a much greater extent than that of type I interferons (IFNs). We also discovered that IRF3, the key STING downstream innate immune effector, is essential for this anticoronavirus activity. In addition, we found that the amidobenzimidazole (ABZI)-based human STING agonist diABZI robustly blocks the infection of not only HCoV-OC43 but also SARS-CoV-2. Therefore, our study identifies the STING signaling pathway as a potential therapeutic target that could be exploited for developing broad-spectrum antiviral therapeutics against multiple coronavirus strains in order to face the challenge of future coronavirus outbreaks.IMPORTANCE The highly infectious and lethal SARS-CoV-2 is posing an unprecedented threat to public health. Other coronaviruses are likely to jump from a nonhuman animal to humans in the future. Novel broad-spectrum antiviral therapeutics are therefore needed to control known pathogenic coronaviruses such as SARS-CoV-2 and its newly mutated variants, as well as future coronavirus outbreaks. STING signaling is a well-established host defense pathway, but its role in coronavirus infection remains unclear. In the present study, we found that activation of the STING signaling pathway robustly inhibits infection of HCoV-OC43 and SARS-CoV-2. These results identified the STING pathway as a novel target for controlling the spread of known pathogenic coronaviruses, as well as emerging coronavirus outbreaks.

Keywords: COVID-19; HCoV-OC43; SARS-CoV-2; STING; antiviral therapy; coronavirus.

FIG 1

DMXAA activation of AAV-encoded human STING^AII inhibits HCoV-OC43 infection. (A) STING is silenced in A549 cells. Whole-cell lysates harvested from A549 and primary human dermal fibroblasts (HDFs) were immunoblotted using the indicated antibodies. GAPDH was used as a loading control. (B) AAV virions encoding STING^WT or STING^AII were used to infect A549 cells. At 2 days postinfection, the cells were treated with DMSO or DMXAA for 3 h and then washed with PBS prior to HCoV-OC43 infection. At 24 h postinfection, cells were immunostained with antibodies against the HCoV-OC43 N protein (OC43 N) and STING and counterstained with DAPI. (C and D) Treatment with IFNs does not significantly inhibit HCoV-OC43 infection but does inhibit NDV-GFP infection in A549 cells. (C) A549 cells were treated with PBS or IFNs for 3 h. The cells were washed with PBS prior to HCoV-OC43 infection. In one of the groups, the infected cells were treated with IFNs for an additional 23 h. At 24 h postinfection, cells were immunostained as in (B). (D) A549 cells were treated with PBS or IFNs for 3 h. The cells were washed with PBS prior to NDV-GFP infection. The IFN treatment was continued for an additional 23 h. At 24 h postinfection, cells were stained with Hoechst 33342. Bar: 50 μm.

FIG 2

DMXAA activation of stably expressed human STING^AII impedes HCoV-OC43 infection. (A) A549 cells stably expressing STING^WT or STING^AII were treated with PBS, IFNs, DMSO, or DMXAA for 3 h. The cells were washed with PBS prior to HCoV-OC43 infection. In one of the groups, the infected cells were treated with IFNs for an additional 23 h. At 24 h postinfection, cells were fixed and immunostained with antibodies against the HCoV-OC43 N protein (OC43 N) and STING and counterstained with DAPI. Bar: 50 μm. (B) Whole-cell lysates harvested from the cells treated as in (A) were immunoblotted using the indicated antibodies. GAPDH was used as a loading control. (C) DMXAA does not show significant cytotoxicity in A549 cells. A549 cells stably expressing STING^WT or STING^AII were treated with the indicated doses of DMSO or DMXAA (stock concentration: 10 mg/ml) for 3 h. The cells were washed with PBS and fed with fresh culture medium. At 27 h posttreatment, cell viability was measured using the CellTiter-GLO 3D cell viability assay.

FIG 3

The STING-IRF3 signaling axis is critical for blocking HCoV-OC43 infection. (A) CRISPR/Cas9-mediated knockout of p65, IRF3, and TBK1 in A549 cells. A549 cells stably expressing human STING^AII were treated with LentiCRISPR v2 constructs encoding gRNAs targeting the indicated immune effector genes. Whole-cell lysates of A549 parental and the CRIPSR KO cells were immunoblotted using the indicated antibodies to validate the gene KO effect. GAPDH was used as a loading control. (B) A549 cells stably expressing STING^AII and a guide RNA targeting firefly luciferase (sgLuc), IRF3 (sgIRF3), TBK1 (sgTBK1), or p65 (sgp65) were treated with DMSO or DMXAA for 3 h. The cells were washed with PBS prior to HCoV-OC43 infection. At 24 h postinfection, cells were immunostained with antibodies against the HCoV-OC43 N protein (OC43 N) and STING and counterstained with DAPI. Bar: 50 μm. (C) Whole-cell lysates extracted from the cells treated as in (B) were immunoblotted using the indicated antibodies. GAPDH was used as a loading control.

FIG 4

Treatment with STING agonist diABZI effectively blocks HCoV-OC43 infection. (A and B) Human STING agonist diABZI is much less toxic than another human STING agonist CAY10748. (A) A549 cells were treated with DMSO or increasing doses of diABZI or CAY10748. At 72 h posttreatment, cell viability was measured using the CellTiter-GLO 3D cell viability assay. (B) A549 cells stably expressing STING^WT or STING^AII were treated with DMSO or increasing doses of diABZI for 3 h. The cells were washed with PBS and fed with fresh culture medium. At 27 h posttreatment, cell viability was measured using the CellTiter-GLO 3D cell viability assay. (C) A549 cells stably expressing STING^WT were treated with DMSO or diABZI for 3 h. The cells were washed with PBS before infection with HCoV-OC43. At 24 h postinfection, cells were immunostained with antibodies against HCoV-OC43 N protein (OC43 N) and STING and counterstained with DAPI. Bar: 50 μm. (D) Whole-cell lysates of the cells treated as in (C) were immunoblotted using the indicated antibodies. GAPDH was used as a loading control.

FIG 5

Human STING agonist diABZI can efficiently block HCoV-OC43 infection in human lung tissue slices. Human lung tissue slices were pretreated with DMSO or 1 μM human STING agonist diABZI for 3 h in 200 μl keratinocyte serum-free medium. The slices were treated with HCoV-OC43 virions diluted in 1 ml keratinocyte serum-free medium at 0.5 × 10e6 PFU/ml. The slices were incubated at 33°C in 5% CO₂. At 6 days postinfection, slices were immunostained using the antibody against the HCoV-OC43 N protein (OC43 N) and counterstained with DAPI. OC43 N⁺ bright red dots in the tissues are infected cells. Bar: 50 μm.

FIG 6

Human STING agonist diABZI can efficiently block SARS-CoV-2 infection. (A) Introducing ACE2 stable expression in A549 cells. Whole-cell lysates harvested from A549 parental or ACE2 stable cells were immunoblotted using the indicated antibodies. GAPDH was used as a loading control. (B) A549/ACE2 cells stably expressing STING^WT were incubated with DMSO or diABZI for 3 h before treatment with SARS-CoV-2. At 24 h postinfection, cells were immunostained with antibodies against SARS-CoV-2 N protein (SARS-CoV-2 N) and STING and counterstained with DAPI. Bar: 50 μm. (C) Whole-cell lysates of the cells treated as in (B) were immunoblotted using the indicated antibodies. GAPDH was used as a loading control.

FIG 7

Human STING agonist diABZI can efficiently block SARS-CoV-2 infection in human lung tissue slices. Human lung tissue slices were pretreated with DMSO or 1 μM human STING agonist diABZI for 3 h in 200 μl keratinocyte serum-free medium. The slices were then treated with SARS-CoV-2 virions diluted in 1 ml keratinocyte serum-free medium at 10e6 PFU/ml. The slices were incubated at 37°C in 5% CO₂. After 10 days, slices were immunostained using an antibody against the SARS-CoV-2 N protein (SARS-CoV-2 N) and counterstained with DAPI. SARS-CoV-2 N⁺ bright red dots in the tissues represent infected cells. Bar: 50 μm.