INTRODUCTION
Coronaviruses (CoVs), belonging to the
Coronaviridae family within the
Nidovirales order, have a broad host range from birds to mammals (
1–4), but have not yet been found in reptiles and amphibians. The seven strains of human CoVs identified to date fall within two genera. The
Alphacoronavirus genus includes HCoV-229E (
5) and HCoV-NL63 (
6,
7), whereas the
Betacoronavirus genus includes HCoV-OC43 (
8), HCoV-HKU1 (
9), severe acute respiratory syndrome (SARS)-CoV (
10,
11), Middle East respiratory syndrome (MERS)-CoV (
12), and the newly discovered SARS-CoV-2 (
13,
14).
The novel coronavirus SARS-CoV-2 emerged in December 2019 and rapidly swept across the globe, causing a severe respiratory illness, COVID-19, in millions of people (
15–20). SARS-CoV-2 has infected over 121 million people worldwide and has claimed 2.7 million lives in the last few months (
16,
20). While SARS-CoV-2 infection poses an unprecedented threat to public health, there are only very limited therapeutic options for treating COVID-19 (
17,
18). While three vaccines are being rolled out, the virus continues to evolve new variants that are not only more infectious but, in some cases, may have the potential to escape current vaccines. In addition, these vaccines are not likely to be effective against a new CoV. On the other hand, a drug can achieve much quicker responses to new viruses until their specific vaccines can be developed. Therefore, novel broad-spectrum antiviral therapeutics are needed to control future coronavirus outbreaks.
Like all coronaviruses, SARS-CoV-2 is an enveloped virus, with a single-stranded, positive-sense RNA genome (
18,
19,
21,
22). It shares 77.2% amino acid sequence identity with SARS-CoV, which was responsible for the SARS epidemic in 2002 to 2003 (
13). SARS-CoV-2 is also closely related to MERS-CoV, the agent of Middle East Respiratory Syndrome. SARS-CoV-2 is thought to have jumped from its animal hosts to humans and, subsequently, developed person-to-person transmission, leading to a once-in-a-century pandemic (
19,
21,
23). Since SARS-CoV-2 is being introduced to humans for the first time, much remains to be learned about its virology and pathogenic mechanisms (
18).
Infection by RNA viruses produces double-stranded RNA (dsRNA) as an intermediate in replication; the dsRNA is sensed by host RNA pattern recognition receptors (PRRs) to stimulate a cascade of signaling pathways that induce transcription of type I/III interferons (IFNs) and other innate proinflammatory cytokines, as well as the oligoadenylate synthetases (OAS)-RNase L and protein kinase R (PKR) pathways (
24). IFNs can induce a large number of downstream IFN-stimulated genes (ISGs) to mount an “antiviral state” in the host cell to clear viral infection (
23,
25–27). In addition, proinflammatory cytokines and chemokines potentiate the adaptive immune response and recruit a variety of immune cells to further combat virus infection and limit virus spread (
23,
25). However, SARS-CoV-2 and many other coronaviruses have evolved multiple mechanisms to evade innate immune responses (
15,
23,
25,
26,
28–38). For example, these coronaviruses can encode proteins to target host antiviral pathways, including the IFN signaling, OAS-RNase L, and PKR pathways (
24). In addition, coronaviruses replicate inside virus-induced cytosolic double-membrane vesicles (
39), which protect the dsRNA from recognition by cytosolic receptors. Furthermore, the viral genomic RNA, with a 5′ cap and 3′ poly(A) sequence, functions as messenger RNA (mRNA) that is translated into 16 nonstructural proteins by the host ribosome machinery to initiate infection immediately after the viral genome reaches the cytoplasm (
40). Together, these tactics allow the virus to escape immune destruction and establish rapid and unhindered viral replication in primary infected cells, producing large copy numbers of the virus (
23,
25,
26,
28–34). Some of these nonstructural proteins, as well as viral accessory proteins, also serve to dampen the host innate immune response (
2,
24).
Although coronaviruses can evade host antiviral pathways, preactivation of these pathways can inhibit coronavirus infection and replication to some degree (
32,
41–43). For example, early evidence demonstrated that type I/III IFN pretreatment could inhibit SARS-CoV-2 infection, although the efficiencies vary across studies (
25,
38,
44,
45). The fact that IFNs are protective early after infection but later become pathological suggests that eliciting an IFN response at the early stages of infection is critical for blocking SARS-CoV-2 infection, spread, and associated pathogenesis. This notion is supported by SARS-CoV studies showing that failure to elicit an early IFN response correlates with the severity of disease (
25). Recently, deficiency in IFN signaling pathways has also been linked to severe COVID-19 (
46,
47).
STING is a key mediator of a host antiviral defense pathway (
48–50). The canonical role of the STING pathway is to sense damaged DNA and infection by DNA viruses. Recent studies showed that the STING pathway is also involved in sensing RNA virus infection (
51). After activation by signals from its upstream nucleic acid sensors, STING recruits TANK binding kinase 1 (TBK1) to phosphorylate itself. The STING-TBK1 complex translocates through the Golgi complex to the perinuclear lysosomal compartments, where it phosphorylates IRF3. Active STING can also stimulate IKK to phosphorylate IκBαB, causing its degradation and NF-κB activation. Activated IRF3 and NF-κB can then translocate into the nucleus to induce transcription of type I IFNs and other inflammatory cytokines, establishing an antiviral state (
48,
52,
53). As discussed above, a timely activation of host IFNs and antiviral ISG production is needed to overcome coronavirus immune evasion and inhibit viral infection. We therefore investigated the impact of the STING signaling pathway on coronavirus infection.
In the current study, we discovered that activation of the STING innate immune signaling pathway effectively blocks infection by human coronavirus OC43 (HCoV-OC43) and SARS-CoV-2. We also demonstrated that transcription factor IRF3, the STING downstream innate immune effector, is essential for this anticoronavirus activity. Furthermore, we found that the human STING agonist diABZI (
54) can robustly activate human STING to block infection of both HCoV-OC43 and SARS-CoV-2. Therefore, our study identifies the STING signaling pathway as an important therapeutic target that could be specifically activated for hampering infection of SARS-CoV-2 and other coronaviruses.
DISCUSSION
Abundant evidence suggests that SARS-CoV-2 and many other coronaviruses have evolved complex molecular mechanisms to suppress host innate immune responses and escape immune eradication (
15,
23,
25,
26,
28–39). A lack of an effective antiviral innate immune response during the early phase of infection allows the virus to establish rapid and robust replication in primary infected cells (
23,
25,
26,
28–34). However, unbridled viral replication could eventually incite a delayed massive innate inflammatory activation response that can overstimulate pathogenic immune cells to cause life-threatening tissue damage and multiorgan failure (
17,
23,
25,
26,
67). Therefore, stimulation of host IFNs and antiviral ISG production during the early phase of viral infection is needed to circumvent the immune evasion mechanism of coronaviruses and inhibit viral infection.
In this current study, we first demonstrated that activating STING signaling by DMXAA and STING
AII dual treatment effectively blocks HCoV-OC43 infection (
Fig. 1,
Fig. 2). We found that CRISPR KO of the STING downstream effector IRF3 almost completely abolished STING/DMXAA antiviral activity, whereas p65 KO and TBK1 KO only mildly affected the ability of STING
AII/DMXAA to block HCoV-OC43 infection (
Fig. 3). This study thus identified IRF3 as a key mediator for blocking HCoV-OC43 infection after STING activation. While the majority of the STING/DMXAA antiviral activity is mediated by IRF3, a small part of this activity is also dependent on NF-κB. TBK1 KO did not completely ablate STING/DMXAA antiviral activity, suggesting that other kinase(s) may be involved. IKKε, a kinase that has been shown to act redundantly with TBK1 to elicit STING-induced NF-κB activation (
68), is actively expressed in A549 cells (
Fig. 1A,
Fig. 3C), suggesting that it may contribute to the TBK1-independent STING antiviral activity.
We also discovered that treatment with IFNs had very little effect on HCoV-OC43 infection, but could moderately repress SARS-CoV-2 infection (
Fig. 2,
Fig. 6). In contrast, the human STING agonist diABZI could almost completely block the infection of HCoV-OC43 and SARS-CoV-2 both
in vitro and
ex vivo (
Fig. 4,
Fig. 7). Therefore, compared to type I IFN pretreatment, temporary stimulation of the STING signaling pathway has demonstrated a much greater potential in blocking coronavirus infection. The fact that diABZI can act directly on endogenous human STING to inhibit HCoV-OC43 and SARS-CoV-2 replication makes it a great therapeutic candidate for impeding the infection of currently known and future emerging coronaviruses.
Our finding that STING activation effectively blocks HCoV-OC43 and SARS-CoV-2 infection suggests that inducing STING downstream IFN- and ISG-mediated antiviral activity can be exploited as a novel strategy to counteract the immune escape mechanism of coronaviruses. However, IFNs and cytokines are protective early in infection but destructive at the later stage of infection (
17,
23,
25,
26,
32,
38,
41–47,
67). Therefore, it is important to maintain a delicate balance between antiviral and inflammatory innate immune programs in order to successfully control coronavirus infection without causing inflammatory damage. Although IRF3 is essential for STING downstream antiviral activity (
Fig. 3), it is almost completely depleted after STING is activated by either DMXAA or diABZI (
Fig. 2,
Fig. 4,
Fig. 6). A similar observation was made in a previous study, in which STING activation by cyclic dinucleotides also led to IRF3 suppression (
69). While the mechanism underlying this phenomenon remains to be investigated, these findings reveal an intrinsic negative-feedback control mechanism of the STING signaling pathway. Our data showed that treating the cells with STING agonists for 3 h was sufficient to cause almost complete inhibition of HCoV-OC43 and SARS-CoV-2 infection. The subsequent shutdown of IRF3 ensures that the activation of STING by its agonists is temporary and does not cause prolonged stimulation of the inflammatory antiviral response. This mechanism therefore affords an opportunity for using STING agonists to achieve disease-stage-specific activation of IFNs, ISGs, and/or cytokines for maximizing anticoronavirus innate immunity while minimizing inflammatory damages.
Taken together, our findings suggest that stimulating STING and downstream innate immune signaling can be exploited as a novel strategy to activate an early and effective host innate immune response for blocking coronavirus infection. Our study also unveils the possibility of applying diABZI as a potential drug for treating infection of HCoV-OC43 and SARS-CoV-2.
In the last two decades, three novel betacoronaviruses, SARS-CoV, MERS-CoV, and SARS-CoV-2, have crossed the species barrier and spilled over to humans to cause highly fatal outbreaks (
23). Bats alone harbor more than 400 coronaviruses. Therefore, the spillover is likely to happen again in the future. What we learn from human betacoronaviruses HCoV-OC43 and SARS-CoV-2 will have general implication for developing innovative broad-spectrum antiviral therapeutics against multiple coronavirus strains, allowing us to face the challenge of future coronavirus outbreaks.
MATERIALS AND METHODS
Cell culture.
Vero E6, HEK293T, A549, A549
ACE2, and primary human dermal fibroblasts (HDF) (
70) were grown in Dulbecco’s modified Eagle medium (DMEM) (Gibco, 11965084) supplemented with 10% fetal calf serum (FCS) (HyClone, SH30071.03) at 37°C in humidified air containing 5% CO
2.
Compounds and reagents.
DMXAA (Sigma, D5817, CAS no. 117570-53-3) was purchased from Sigma. Human STING agonist 3 diABZI (Cayman chemical, 28054, CAS no. 2138299-34-8) and CAY10748 (Cayman chemical, 30022, CAS no. 2412902-55-5) were purchased from Cayman Chemical. Human IFN Alpha Hybrid (Universal Type I IFN, PBL, 11200-2) was purchased from PBL Assay Science.
Recombinant plasmid construction.
Adenoviral-associated vector (AAV) production was performed as previously described (
55). sgRNAs targeting firefly luciferase (sgLuc), IRF3 (sgIRF3) (
71), TBK1 (sgTBK1), and p65 (sgp65) were cloned into the LentiCRISPR-v2 plasmid (addgene, number 52961). The sequence information for sgRNAs is listed in
Table 1.
Generation of A549 stable cell lines.
To generate A549 cells stably expressing STINGWT or STINGAII, lentivirus was produced by transfecting pLenti-MCPyVEP-STINGWT-IRES-Puro and pLenti-MCPyVEP-STINGAII-IRES-Puro plasmids into HEK293T cells together with psPAX2 and pMD2.G using Lipofectamine 2000 (Invitrogen). A549 cells were transduced with the purified lentiviruses supplemented with polybrene. Starting on day 2 after transduction, cells were selected using 5 μg/ml puromycin for at least 7 days.
To generate A549 cells stably expressing STINGAII and either sgLuc, sgIRF3, sgTBK1, or sgP65, the plasmid pLenti-MCPyVEP-STINGAII-IRES-Zeocin, pLenti-CRISPR-sgLuc, pLenti-CRISPR-sgIRF3, pLenti-CRISPR-sgTBK1, or pLenti-CRISPR-sgp65 was transfected into HEK293T cells together with psPAX2 and pMD2.G using Lipofectamine 2000 (Invitrogen). A549 cells were cotransduced with pLenti-MCPyVEP-STINGAII-IRES-Zeocin lentivirus and either pLenti-CRISPR-sgLuc, pLenti-CRISPR-sgIRF3, pLenti-CRISPR-sgTBK1, or pLenti-CRISPR-sgP65 lentivirus supplemented with polybrene. Starting on day 2 after transduction, cells were selected using 10 μg/ml puromycin for 3 weeks and then maintained in culture medium containing 100 μg/ml zeocin.
Cell proliferation assay.
A549 parental cells or A549 stably expressing STINGWT or STINGAII were seeded at a density of 5,000 cells in 100 μl of medium per well in a 96-well plate. The cells were incubated in humidified air containing 5% CO2 with or without drug using the amount of time as indicated in the figure legends. Cell viability was measured using CellTiter-Glo 3D (Promega) following the manufacturer’s instructions.
Western blot analysis.
The protein samples were resolved on SDS-PAGE gels, transferred onto polyvinylidene difluoride (PVDF) membranes, and immunoblotted using specific primary antibodies as indicated in the figure legends. The primary antibodies used in this study includes anticoronavirus OC43 nucleoprotein protein (OC43 N) (
72) (1:4,000, MAB9012, Millipore), anti-SARS-CoV-2 nucleoprotein (SARS-CoV-2 N) (1:4,000, GTX635712, GenTex), anti-STING (1:1,000, 13647S, Cell Signaling Technology), anti-phospho-STING (Ser366) (1:1,000, 50907S, Cell Signaling Technology), anti- IKKε (1:1,000, 3416S, Cell Signaling Technology), anti-GAPDH (1:2,000, 5174S, Cell Signaling Technology), anti-p65 (1:1,000, sc-8008, Santa Cruz Biotechnology), anti-TBK1 (1:1,000, 3013S, Cell Signaling Technology), anti-ACE2 (1:1,000, 21115-1-AP, Proteintech), and anti-IRF3 (1:250, sc-33641, Santa Cruz Biotechnology). The secondary antibodies used were horseradish peroxidase (HRP)-linked anti-rabbit IgG (1:3,000, 7074S, Cell Signaling Technology) and HRP-linked anti-mouse IgG (1:3,000, 7076S, Cell Signaling Technology). Western blots were developed using Western Lightning ECL solution (PerkinElmer) and the images were captured using a Fuji imaging system.
Adenoviral-associated vector production.
Adenoviral-associated vector (AAV) production was performed as previously described (
55). HEK293T cells were cultured in 100-mm dishes. The cells were transfected with 5 μg of serotype packaging plasmid, 10 μg of pAdDeltaF6 helper plasmid (University of Pennsylvania Vector Core), and 5 μg of pscAAV carrying the construct of interest. The cell lysates were prepared with three successive freeze-thaw cycles (−80°C/37°C). The cell lysates were then purified using OptiPrep centrifugation at 300,000 ×
g for 3.5 h at 16°C. Pure gradient fractions were concentrated and desalted using an Amicon Ultra-15 centrifugal concentrator.
Immunofluorescent staining.
Cells were fixed with 3% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min. IF staining was performed as described previously (
73). The following primary antibodies were used: anticoronavirus OC43 N protein (1:2,000, MAB9012, Millipore), anti-SARS-CoV-2 nucleoprotein (1:2,000, GTX635712, GenTex), and anti-STING (1:500, 19851-1-AP, Proteintech). The secondary antibodies used were Alexa Fluor 594 goat anti-mouse IgG (Thermo Fisher Scientific) and Alexa Fluor 488 goat anti-rabbit IgG (Thermo Fisher Scientific). All IF images were collected using an inverted fluorescence microscope (IX81; Olympus) connected to a high-resolution charge-coupled-device camera (FAST1394; QImaging). Images were analyzed and presented using SlideBook (version 5.0) software (Intelligent Imaging Innovations, Inc.). The scale bars were added using ImageJ software.
HCoV-OC43 infection.
HCoV-OC43 was purchased from ATCC and amplified in Vero E6 cells. A549 cells were treated with PBS, 500 units/ml of human IFN Alpha Hybrid universal type I IFN (PBL, 11200-2), AAV virions, DMSO, 10 μg/ml DMXAA, or 100 nM human STING agonists, as described in the figure legends. The cells were washed once with PBS and then treated with HCoV-OC43 virions (10e6 PFU/ml) diluted in serum-free DMEM serum-free (at an MOI of 1) for 1 h at 33°C. The cells were then overlaid with DMEM containing 2% FBS and cultured at 33°C for one more day.
Plaque assay.
Confluent Vero E6 cells cultured in 6-well plates were treated with 200 μl of DMEM containing a serial 10-fold dilution of HCoV-OC43 stock for 1 h at 33°C. Inoculum was overlaid with DMEM plus 0.7% agarose and incubated for 7 days at 33°C. Cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet before plaque counting (
24).
SARS-CoV-2 infection.
SARS-CoV-2 (USA-WA1/2020 strain) was obtained from BEI and propagated in Vero E6 cells. A549ACE2 cells stably expressing STINGWT were treated with IFNs, DMSO, or human STING agonist diABZI, as described in the figure legends. The cells were treated with SARS-CoV-2 virions diluted in serum-free DMEM (at an MOI of 5) for 1 h at 37°C. The cells were then overlaid with DMEM containing 2% FBS and cultured at 37°C for one more day.
NDV-GFP infection.
Newcastle disease virus (NDV)-GFP was obtained from Luis Martinez-Sobrido (University of Rochester School of Medicine). A549 cells were treated with PBS or 500 units/ml of human IFN Alpha Hybrid universal type I IFN (PBL, 11200-2). The cells were washed once with PBS and then treated with NDV-GFP virions (diluted in serum-free DMEM) at an MOI of 5 for 1 h at room temperature. The cells were then overlaid with DMEM containing 2% FBS and cultured at 37°C for one more day.
HCoV-OC43 and SARS-CoV-2 infection of ex vivo cultured human lung slices.
Normal human lung tissues were obtained from the Penn-CHOP Lung Biology Institute (LBI) at the University of Pennsylvania. Following our previous skin tissue
ex vivo culture protocol (
70), the tissues were cut into 700-μm-thick slices using a McIlwain tissue chopper. About 12 tissue slices were frozen in 90% FBS and 10% DMSO in each vial in liquid nitrogen for further experiments.
The slices were thawed and washed in PBS twice and then transferred to 200 μl keratinocyte serum-free medium (Gibco, catalog number 17005042) with 1% penicillin-streptomycin. The slices were treated with DMSO or 1 μM human STING agonist diABZI for 3 h. For HCoV-OC43 infection, 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 an antibody against the OC43 N protein (OC43 N) and counterstained with DAPI (4′,6-diamidino-2-phenylindole). For SARS-CoV-2 infection, the slices were 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. At 10 days postinfection, slices were immunostained using an antibody against the SARS-CoV-2 N protein (SARS-CoV-2 N) and counterstained with DAPI.