Aryl hydrocarbon receptor is a proviral host factor and a candidate pan-SARS-CoV-2 therapeutic target

Abstract

The emergence of a series of SARS-CoV-2 variants has necessitated the search for broad-spectrum antiviral targets. The aryl hydrocarbon receptor (AhR) senses tryptophan metabolites and is an immune regulator. However, the role of AhR in SARS-CoV-2 infection and whether AhR can be used as the target of antiviral therapy against SARS-CoV-2 and its variants are yet unclear. Here, we show that infection with SARS-CoV-2 activates AhR signaling and facilitates viral replication by interfering with IFN-I-driven antiviral immunity and up-regulating ACE2 receptor expression. The pharmacological AhR blockade or AhR knockout reduces SARS-CoV-2 and its variants' replication in vitro. Drug targeting of AhR with AhR antagonists markedly reduced SARS-CoV-2 and its variants' replication in vivo and ameliorated lung inflammation caused by SARS-CoV-2 infection in hamsters. Overall, AhR was a SARS-CoV-2 proviral host factor and a candidate host-directed broad-spectrum target for antiviral therapy against SARS-CoV-2 and its variants, including Delta and Omicron, and potentially other variants in the future.

Fig. 1.. SARS-CoV-2 infection activates AhR signaling in an IFN-IDO-Kyn–dependent pathway.

(A) Visualization of single cells across different cell types in monkeys. tSNE plots show the cell type annotation of single cells. (B) Scatter plot of IDO1 and CYP1A1 mRNA expression in lung tissues of macaques infected with SARS-CoV-2. Upstream gene IDO1 and downstream gene CYP1A1 mRNA expression of AhR was determined by scRNA-seq. Monkey numbers 16001 and 16149 are SARS-CoV-2–infected monkeys, and monkey number 16271 is SARS-CoV-2–uninfected monkey. (C) mRNA expression of genes in the AhR pathway determined by quantitative polymerase chain reaction (qPCR) in H1299 cells at 24 hours after SARS-CoV-2 infection [multiplicity of infection (MOI) of 0.1]. Expression values are relative to mock-infected cells. (D) mRNA expression of genes in the AhR pathway determined by qPCR in Calu-3 cells at 24 hours after SARS-CoV-2 infection (MOI of 0.1). Expression values are relative to mock-infected cells. (E) Relative expression of IFN-β and ISGs IFIT2 and OAS1 in H1299 cells upon SARS-CoV-2 infection (MOI of 0.1). Expression values are relative to mock-infected cells. (F) Relative expression of IFN-β and ISGs IFIT2 and OAS1 in Calu-3 cells at 24 hours after SARS-CoV-2 infection (MOI of 0.1). Expression values are relative to mock-infected cells. (G) mRNA expression of IDO1, AhR, and CYP1A1 was determined by qPCR in IFN-β–treated and IFN-β–untreated H1299 cells. (H) Enzyme-linked immunosorbent assay (ELISA) quantification of Trp and Kyn in culture supernatants 48 hours after infection of H1299 cells with SARS-CoV-2 (MOI of 0.1).

Fig. 2.. SARS-CoV-2 infection promotes AhR entry into the nucleus.

(A) H1299 cells and HepG2 cells (B) were treated with dimethyl sulfoxide (DMSO) or indoxyl-3-sulfate (I3S) (200 μM) or infected with SARS-CoV-2 (MOI of 0.1) infection for 48 hours. Cells were immunostained with an anti-AhR antibody or anti–SARS-CoV-2 NP antibody and observed under a confocal microscope. Scale bars, 20 μm. Representative images are from three independent experiments. DAPI, 4′,6-diamidino-2-phenylindole.

Fig. 3.. AhR acts as a proviral host factor for SARS-CoV-2 infection.

(A) AhR^−/− HepG2 cell line was generated by CRISPR-Cas9–mediated genome engineering. (B) Confirmation of AhR deletion in AhR^−/− HepG2 cells at the mRNA and protein levels. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C) Immunoblot analysis of SARS-CoV-2 NP protein expression in wild-type (Wt) and AhR^−/− HepG2 cells. (D) Immunofluorescence assay of SARS-CoV-2 NP protein and cellular AhR protein expression in wild-type and AhR^−/− HepG2 cells by microscopy (scale bars, 100 μm). (E) Viral titer was determined by CCID₅₀ in wild-type and AhR^−/− HepG2 cells at 24 hours postinfection (hpi) with 0.1 MOI. (F) Immunoblot analysis of SARS-CoV-2 NP protein expression in I3S- or DMSO-treated Huh7 cells. Huh7 cells were pretreated with different concentrations of I3S or DMSO and infected with SARS-CoV-2 (MOI of 0. 1) for 24 hours (G) Viral titer was determined by CCID₅₀ in I3S- or DMSO-treated Huh7 cells. Data from at least three independent experiments (means ± SD). P values were determined using a two-tailed, unpaired Student’s t test. (H) Viral titer was determined by CCID₅₀ in AhR^−/− HepG2 cells treated with CH223191 or I3S at 24 hpi with MOI of 0.1.

Fig. 4.. Pharmacological AhR inhibition limits replication of SARS-CoV-2 and its variants in vitro.

(A) Immunoblot analysis of SARS-CoV-2 NP protein expression in CH223191- or DMSO-treated Huh7 cells. Huh7 cells were pretreated with different concentrations of CH223191 or DMSO and infected with SARS-CoV-2 (MOI of 0.1) for 24 hours. (B) Immunofluorescence assay of SARS-CoV-2 NP protein expression in CH223191- or DMSO-treated Huh7 cells by microscopy (scale bars, 200 μm). (C) Viral titer was determined by CCID₅₀ assay in the supernatants of Huh7 cells pretreated with CH2213191 or DMSO and infected with SARS-CoV-2 (MOI of 0.1) for 24 hours. (D) Immunoblot analysis of SARS-CoV-2 NP protein expression in CH223191- or DMSO-treated Calu-3 cells. Calu-3 cells were pretreated with different concentrations of CH223191 or DMSO and infected with SARS-CoV-2 (MOI of 0. 05) for 24 hours. (E) Viral titer was determined by CCID₅₀ assay in the supernatants of Calu-3 cells pretreated with CH2213191 or DMSO and infected with SARS-CoV-2 (MOI of 0.05) for 24 hours. Data are presented from at least three independent experiments (means ± SD). P values were determined using a two-tailed, unpaired Student’s t test. (F) qPCR quantification of SARS-CoV-2 RNA in Huh7 cells treated with CH223191 or DMSO preinfection, during adsorption or internalization of SARS-CoV-2 (MOI of 2). The cells were harvested at 2 hpi for qPCR to determine viral RNA level. Data from at least three independent experiments (means ± SD). P values were determined by using a two-tailed, unpaired Student’s t test.

Fig. 5.. AhR boosts SARS-CoV-2 replication by limiting IFN-I response and up-regulating ACE2 transcription.

Relative expression of cellular genes and viral RNA levels was determined by qPCR in SARS-CoV-2–infected (MOI of 0.1) H1299 cells (A) or HepG2 cells (B) pretreated with CH223191 or DMSO. (C) Viral titer was determined by CCID₅₀ assay in the supernatants of Vero cells pretreated with CH2213191 or DMSO and infected with SARS-CoV-2 (MOI of 0.05) for 24 hours. (D) Effect of inhibitors of IFN signaling on SARS-CoV-2 replication determined 24 hpi by CCID₅₀ assay. HepG2 cells pretreated with DMSO, Janus kinase 1 (JAK1) inhibitor, or trichostatin A (TSA) and infected with SARS-CoV-2 (MOI of 0.1), and, 24 hpi, supernatants were harvested for CCID₅₀ assay. (E) Effect of CH223191 on HepG2 cells treated with JAK1 inhibitor. HepG2 cells were treated with JAK1 inhibitor and CH223191 as indicated and infected with SARS-CoV-2 (MOI of 0.1), and, 24 hpi, supernatants were harvested for CCID₅₀ assay. (F) Immunoblot analysis of ACE2 protein in HepG2 cells treated with CH223191 for 12 hours. (G) Immunoblot analysis of ACE2 and SARS-CoV-2 NP protein in AhR^−/− HepG2 cells infected with SARS-CoV-2 (MOI of 0.01 or 1) for 24 hours. (H) Immunoblot analysis of ACE2 and SARS-CoV-2 NP protein in H1299 or HepG2 cells infected with SARS-CoV-2 (MOI of 0.1) for 24 and 48 hours. (I) Effect of CH223191 on ACE2 mRNA and protein or SARS-CoV-2 NP protein in HepG2 cells or (J) H1299 cells. HepG2 or H1299 cells were pretreated with CH223191 and infected with SARS-CoV-2 (MOI of 0.1), and, 24 hpi, RNA and protein were harvested for qPCR and immunoblot analysis, respectively.

Fig. 6.. AhR antagonist limits SARS-CoV-2 replication and ameliorates pneumonia inflammation in hamsters.

(A) Schematic of SARS-CoV-2 infection and animal operations. Hamsters were infected with SARS-CoV-2 and treated with control DMSO or CH223191 10 mg/kg, intraperitoneally (i.p.) for 5 days. (B) Relative viral load of SARS-CoV-2. The hamster lung tissues were used for quantifying viral load by real-time RT-PCR. (C) Comprehensive pathological scores for lung sections. The data represent means ± SD. P values were determined by using a two-tailed, unpaired Student’s t test. (D) Representative lung histopathological images [hematoxylin and eosin (H&E) staining] for lung lobe sections collected from SARS-CoV-2–infected hamsters at 5 days postinfection (dpi). Scale bars, 50 μm.

Fig. 7.. Schematic diagram of AhR promoting SARS-CoV-2 replication and acting as a therapeutic target.

AhR was activated by SARS-CoV-2 infection by Trp-Kyn metabolism pathway. The activated AhR inhibits the host antiviral responses mediated by IFN-I and up-regulates ACE2 expression, thus promoting viral replication. AhR antagonists block the activation of AhR, enhance the host antiviral response, and reduce the expression of ACE2 receptor, thus reducing viral replication.