Fig 1: SARS-CoV-2 ORF9b antagonizes the antiviral IFN response in a variety of human cells(A) ORF9b-mediated suppression of IFN-β production in human airway epithelial cells. At 24 h post-transfection of empty vector or FLAG-ORF9b-expressing plasmid, various human cells were uninfected (mock) or infected with VSV for 24 h. ELISA was conducted to measure the IFN-β production in BEAS-2B, Calu-3, and HEK293T cells. Data are represented as means ± SDs calculated from three independent experiments (∗p < 0.05, ∗∗p < 0.01; t test). See also Figure S2A.(B) Inhibition of virally induced cytokine and chemokine expression by ORF9b. Experiments were conducted as described in (A). qPCR was conducted to determine the induction of IFNB1, IL-6, TNF, ISG15, IP-10, and MCP-1. Data are represented as means ± SDs calculated from three independent experiments (∗p < 0.05, ∗∗p < 0.01; t test).(C and D) BEAS-2B cells were transfected with empty vector and FLAG-ORF9b-expressing plasmid for 24 h and then infected with VSV-GFP for another 24 h. Fluorescent images were taken to examine VSV proliferation (C). Plaque assay was conducted to quantitate VSV titers (D). Scale bar, 50 μm. Data are represented as means ± SDs calculated from three independent experiments (∗∗p < 0.01; t test). See also Figures S2B and S2C.
Fig 2: ORF9b inhibits the canonical NF-κB signaling pathway by interrupting the K63-linked polyubiquitination of NEMO(A) Inhibitory effects of ORF9b on the ubiquitination of NEMO under viral stimulation. Expressing vectors for HA-ubiquitin (HA-Ub), FLAG-NEMO, and V5-tagged ORF9b were transfected into HEK293T cells as indicated for 24 h. Cells were then infected with or without VSV for 12 h and subjected to immunoprecipitation using anti-FLAG beads.(B) Dose-dependent inhibition of virally induced Ub conjugation to NEMO by ORF9b. Similar to (A), except that an increasing dose of the V5-ORF9b-expressing vector was transfected into HEK293T cells. See also Figure S4A.(C) Effects of ORF9b on the conjugation of diverse polyubiquitin linkages to NEMO under viral stimulation. Plasmids encoding various HA-Ub (WT, KallR, K48 only, K63 only, K48R, and K63R as indicated), together with expressing vectors for FLAG-NEMO and V5-ORF9b, were co-transfected into HEK293T cells. Infection and immunoprecipitation were conducted as described in (A).(D) Interruption of the IKKα/β/γ-NF-κB signaling by ORF9b. HEK293T cells were transfected for 24 h with empty vector or with V5-ORF9b- and V5-ORF9bΔN30-expressing vectors and were then infected with VSV for the indicated time (0, 4, 8, or 12 h). Cells were collected and subjected to immunoblotting analysis by using indicated antibodies. β-actin was immunoblotted as loading control.(E) Inhibitory effects of ORF9b on the translocation of NF-κB/p65 into the nucleus. Transfection was performed as described in (D). HEK293T cells were then mock infected or infected with VSV for 12 h. Cytoplasmic (cytoplasm) and nuclear (nucleus) factions of cells were obtained using commercial reagents. Immunoblotting analysis was conducted using indicated antibodies.(F and G) At 24 h post-transfection of empty vector or V5-ORF9b- and V5-ORF9bΔN30-expressing plasmids, HEK293T cells were uninfected (mock) or infected with VSV for 12 h. qPCR was conducted to determine the expression of IFNB1 (F) and IL-6 (G). Data are represented as means ± SDs calculated from three independent experiments (∗∗p < 0.01, ∗∗∗p < 0.001, N.S., non-significant; t test). See also Figure S4B.
Fig 3: SARS-CoV-2 ORF9b suppresses viral-RNA-induced IFN production through RIG-I-MAVS signaling(A) Induction of IFNB1 by SARS-CoV-2 RNA relies on RIG-I and MAVS. HEK293T wild-type (WT), DDX58−/− (RIG-I−/−), IFIH1−/− (MDA5−/−), and MAVS−/− cells were transfected for 12 h with indicated amounts of RNA from mock-infected Vero E6 cells; and viral RNA was isolated from either SARS-CoV-2- or VSV-infected cells. qPCR was conducted to determine the induction of IFNB1 mRNA. See also Figure S1A. Data are represented as means ± SDs calculated from three independent experiments.(B–D) Induction of IFNB1 and accumulation of ORF9b during a 24-h period of SARS-CoV-2 infection. As shown in the experimental scheme, HPAEpiC and Caco-2 cells were either mock infected or infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 1 for the indicated time and were then collected for subsequent measurement (B). SARS-CoV-2-induced IFNB1 mRNA levels were measured by qPCR, with the IFNB1 levels of VSV infection shown as a control (C). Cell lysates were subjected to fluorescence quantification immunoblotting for measuring the ORF9b protein levels in individual sample, which were converted into concentrations (ng/1 × 106 cells) (D). Data are represented as means ± SDs calculated from three biological replicates in the same experiment. See also Figures S1B and S1C.(E–G) Inhibition of SARS-CoV-2 RNA-induced type I IFN response by ectopically expressed ORF9b under near-physiological levels. HPAEpiC were transfected with empty vector or increasing doses of expressing vector for FLAG-tagged ORF9b. At 24 h post-transfection, cells were transfected with 100 ng mock RNA or SARS-CoV-2 RNA as described in (A) for another 12 h. PCR was conducted to determine the expression of IFNB1 (E), ISG15 (F), and TNF (G). See also Figure S1D. Data are represented as means ± SDs calculated from three independent experiments (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; t test).(H) Dose-dependent inhibition of viral-RNA-induced IFNB1 activation by ORF9b in HEK293T cells. Similar to (A), except that HEK293T cells were transfected with increasing doses of empty vectors or FLAG-ORF9b expression vectors for 24 h before being stimulated with viral RNA. See also Figure S1E. Data are represented as means ± SDs calculated from three independent experiments (∗p < 0.05; t test).(I) Inhibitory effects of ORF9b on SeV-, VSV-, or poly(I:C)-induced IFN-β promoter activation. HEK293T cells were co-transfected with luciferase reporter plasmids plus empty vector or FLAG-ORF9b-expressing plasmid for 24 h and were non-stimulated (mock) or stimulated with SeV, VSV, or poly(I:C) for another 12 h. IFN-β luciferase (IFN-β-Luc) reporter activity is normalized to that of Renilla luciferase and shown as fold induction. Data are represented as means ± SDs calculated from three independent experiments (∗p < 0.05, ∗∗p < 0.01; t test).
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