Experimental and natural evidence of SARS-CoV-2-infection-induced activation of type I interferon responses

Abstract

Type I interferons (IFNs) are our first line of defense against virus infection. Recent studies have suggested the ability of SARS-CoV-2 proteins to inhibit IFN responses. Emerging data also suggest that timing and extent of IFN production is associated with manifestation of COVID-19 severity. In spite of progress in understanding how SARS-CoV-2 activates antiviral responses, mechanistic studies into wild-type SARS-CoV-2-mediated induction and inhibition of human type I IFN responses are scarce. Here we demonstrate that SARS-CoV-2 infection induces a type I IFN response in vitro and in moderate cases of COVID-19. In vitro stimulation of type I IFN expression and signaling in human airway epithelial cells is associated with activation of canonical transcriptions factors, and SARS-CoV-2 is unable to inhibit exogenous induction of these responses. Furthermore, we show that physiological levels of IFNα detected in patients with moderate COVID-19 is sufficient to suppress SARS-CoV-2 replication in human airway cells.

Keywords: Immunology; Virology.

Graphical abstract

Figure 1

Global response in SARS-CoV-2-infected human airway epithelial cells Calu-3 cells were infected with SARS-CoV-2 at an MOI of 1 or 2. RNA was extracted at different times post incubation. Viral and cellular gene expression was determined using time-series RNA-seq analysis or qPCR. (A) SARS-CoV-2 gene expression over 12 h (n = 3/time point). The genome organization of SARS-CoV-2 is indicated above in pink. (B) Major SARS-CoV-2 gene expression levels at different times post incubation (n = 3/time point). (C) Cellular genes (n = 124) that are significantly up- or downregulated (FDR-adjusted p < 0.05; |log2FC| > 1) in SARS-CoV-2-infected cells, relative to mock-infected cells at different times post incubation. Transcript levels are shown as Z score normalized expression (scaled by gene). See Figure S1E for a larger figure. (D) Cellular processes that are down- or upregulated at different times post incubation. The size of the circles represents the number of genes that are down- or upregulated at different times after incubation (n = 3/time point). (E) Transcript abundance of type I and III interferon (IFN) genes (IFNβ and IFNλ1-3) in mock-infected and SARS-CoV-2-infected Calu-3 cells at different times (n = 3). (F) Transcript abundance of representative interferon-stimulated genes (ISGs) in mock-infected and SARS-CoV-2-infected Calu-3 cells at different times (n = 3). (G) IFNβ transcript levels in Calu-3 cells infected with SARS-CoV-2 or mock infected for 12 h, normalized to GAPDH (n = 6). Transcript levels were determined by qPCR. (H) IRF7 transcript levels in Calu-3 cells infected with SARS-CoV-2 or mock infected for 12 h, normalized to GAPDH (n = 6). Transcript levels were determined by qPCR. (I) IFIT1 transcript levels in Calu-3 cells infected with SARS-CoV-2 or mock infected for 12 h, normalized to GAPDH (n = 6). Transcript levels were determined by qPCR. Data are represented as mean ± SD, n = 3 or 6, p∗<0.05, ∗∗<0.01, ∗∗∗<0.001, and ∗∗∗∗<0.0001 (Student's t test). See also Star methods for details on statistical analyses performed using R. See also Figures S1–S3, and Tables S1–S3. H and hpi, hours post incubation.

Figure 2

SARS-CoV-2 infection does not inhibit type I IFN expression To determine if SARS-CoV-2 can modulate IFNβ gene expression and downstream stimulation of ISGs, Calu-3 cells were infected with SARS-CoV-2 for varying times, following which cells were mock transfected or transfected with poly(I:C). Mock-infected and mock-transfected cells served as controls. Transcript levels were quantified using qPCR. Protein expression was observed and quantified using immunoblot analysis. (A) Calu-3 cells were infected with SARS-CoV-2 (MOI 1) for 0, 24, 48, and 72 h. Cells were fixed and stained to visualize the nucleus and SARS-CoV-2 nucleocapsid (N) protein. Scale bar indicates 300 μm. (B) SARS-CoV-2 genome (UpE) levels in Calu-3 cells infected with SARS-CoV-2 (MOI 1) or mock infected for 12 h and transfected with 100 ng of poly(I:C) or mock transfected for 6 h (n = 6). Primers for the UpE region were designed to quantify SARS-CoV-2 genome levels (see methods). 1/dCT values are represented after normalizing Ct values for SARS-CoV-2 genome levels at 18 hpi with Ct values observed at 0 hpi (immediately after removal of virus inoculum). Gel (below): UpE qPCR amplicons were visualized on an agarose gel. (C) IFNβ transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 12 h. Twelve hpi, cells were either transfected with 100 ng of poly(I:C) or mock transfected for 6 h. IFNβ transcript levels were normalized to GAPDH transcript levels (n = 6). (D) IFIT1 transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 12 h. Twelve hpi, cells were either transfected with 100 ng of poly(I:C) or mock transfected for 6 h. IFIT1 transcript levels were normalized to GAPDH transcript levels (n = 6). (E) IRF7 transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 12 h. Twelve hpi, cells were either transfected with 100 ng of poly(I:C) or mock transfected for 6 h. IRF7 transcript levels were normalized to GAPDH transcript levels (n = 6). (F) IFIT1, SARS-CoV-2 N, and ACTB protein expression in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 24 h. Twenty-four hpi, cells were either transfected with 1,000 ng of poly(I:C) or mock transfected for 24 h (n = 3). (G) IFNβ transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 0.1 or 1) or mock infected for 24 h. Twenty-four hpi, cells were transfected with 10 ng of poly(I:C) or mock transfected for 12 h. IFNβ transcript levels were normalized to GAPDH transcript levels (n = 3). (H) IFIT1 transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 0.1 or 1) or mock infected for 24 h. Twenty-four hpi, cells were transfected with 10 ng of poly(I:C) or mock transfected for 12 h. IFIT1 transcript levels were normalized to GAPDH transcript levels (n = 3). (I) IFNβ transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 24 h. Twenty-four hpi, cells were either transfected with varying concentrations of poly(I:C) or mock transfected for 12 h. IFNβ transcript levels were normalized to GAPDH transcript levels (n = 3). (J) IFIT1 transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 24 h. Twenty-four hpi, cells were either transfected with varying concentrations of poly(I:C) or mock transfected for 12 h. IFIT1 transcript levels were normalized to GAPDH transcript levels (n = 3). (K) IRF7 transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 24 h. Twenty-four hpi, cells were either transfected with varying concentrations of poly(I:C) or mock transfected for 12 h. IRF7 transcript levels were normalized to GAPDH transcript levels (n = 3). (L) pTBK1-S172, TBK1, pIRF3-S396, IRF3, SARS-CoV-2 N, and ACTB protein expression in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 24 h. Twenty-four hpi, cells were either transfected with 1,000 ng of poly(I:C) or mock transfected for an additional 24 h (n = 3). (M) Calu-3 cells were infected with SARS-CoV-2 (MOI 1) or mock infected for 24 h, followed by transfection with 1,000 ng of rhodamine-labeled poly(I:C) or mock transfection for 3 h. Cells were fixed and stained to visualize SARS-CoV-2 nucleocapsid (N) protein and rhodamine-labeled poly(I:C). SARS-CoV-2 N and poly(I:C)-rhodamine containing cells are indicated by arrows. Cells that only contained detectable levels of poly(I:C)-rhodamine are indicated by arrow heads. Scale bar indicates 150 μm. Data are represented as mean ± SD, n = 3 or 6, p∗∗<0.01, ∗∗∗<0.001, and ∗∗∗∗<0.0001 (Student's t test and Tukey's multiple comparisons test). pTBK1-S172 and pIRF3-S396 protein expression levels are expressed as ratios of pTBK1-S172/TBK1 and pIRF3-S396/IRF3 levels, respectively. Blots were quantified using Image Studio (Li-COR) (n = 3). Ct, cycle threshold. See also Figure S3.

Figure 3

SARS-CoV-2 is unable to inhibit type I IFN signaling To determine if SARS-CoV-2 can inhibit IFNβ-mediated stimulation of ISGs, such as IFIT1, Calu-3 cells were infected with SARS-CoV-2 for 12 or 24 h, following which cells were mock treated or treated with recombinant IFNβ. Mock-infected and mock-treated cells served as controls. Transcript levels were quantified using qPCR, and protein expression was observed using immunoblots. (A) SARS-CoV-2 genome (UpE) levels in Calu-3 cells infected with SARS-CoV-2 (MOI 1) or mock infected for 12 h and treated with recombinant IFNβ or mock treated for 6 h (n = 6). 1/dCT values are represented after normalizing Ct values for SARS-CoV-2 genome levels at 18 hpi with Ct values observed at 0 hpi (immediately after removal of virus inoculum). Gel (below): UpE qPCR amplicons were visualized on an agarose gel. (B) IRF7 transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 12 h. Twelve hpi, cells were either treated with recombinant IFNβ or mock treated for 6 h. IRF7 transcript levels were normalized to GAPDH transcript levels (n = 6). (C) IFIT1 transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 12 h. Twelve hpi, cells were either treated with recombinant IFNβ or mock treated for 6 h. IFIT1 transcript levels were normalized to GAPDH transcript levels (n = 6). (D) SARS-CoV-2 N, IFIT1, and GAPDH protein expression in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 12 h. Twelve hpi, cells were either treated with recombinant IFNβ or mock treated for 6 h (n = 3). (E) pSTAT1-Y701, STAT1, pSTAT2-Y690, STAT2, SARS-CoV-2 N, and ACTB protein expression in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 24 h. Twenty-four hpi, cells were either treated with recombinant IFNβ or mock treated for 30 min (n = 3). (F) IFIT1 transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 0.1 or 1) or mock infected for 24 h. Twenty-four hpi, cells were mock treated or treated with recombinant IFNβ containing media (20 μg/mL total protein) for 12 h. IFIT1 transcript levels were normalized to GAPDH transcript levels (n = 3). (G) IRF7 transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 0.1 or 1) or mock infected for 24 h. Twenty-four hpi, cells were mock treated or treated with recombinant IFNβ-containing media (20 μg/mL total protein) for 12 h. IRF7 transcript levels were normalized to GAPDH transcript levels (n = 3). (H) IFIT1 transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 24 h. Twenty-four hpi, cells were either treated with varying concentrations of recombinant IFNβ or mock treated for 12 h. IFIT1 transcript levels were normalized to GAPDH transcript levels (n = 3). (I) IRF7 transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 24 h. Twenty-four hpi, cells were either treated with varying concentrations of recombinant IFNβ or mock treated for 12 h. IRF7 transcript levels were normalized to GAPDH transcript levels (n = 3). Data are represented as mean ± SD, n = 3 or 6, ns: not significant, p∗<0.05, p∗∗<0.01, ∗∗∗<0.001, and ∗∗∗∗<0.0001 (Student's t test and Tukey's multiple comparison's test). Ct, cycle threshold. pSTAT1-Y701 and pSTAT2-Y690 protein expression levels are expressed as ratios of pSTAT1-Y701/STAT1 and pSTAT2-Y690/STAT2 levels, respectively. Blots were quantified using Image Studio (Li-COR) (n = 3). For IFNβ treatment, cell culture supernatant containing recombinant IFNβ was used. Cell culture supernatant containing 2 mg/mL of total protein, including IFNβ, was used in A-E. A range of concentrations was used for other figures as indicated. Ct, cycle threshold. See Star methods for recombinant IFNβ generation. See also Figures S3 and S4.

Figure 4

Cytokine protein levels in human sera from moderate and severe cases of COVID-19 relative to healthy controls and effect of type I IFNs on SARS-CoV-2 replication (A) To determine protein levels of cytokines in sera from moderate and severe cases of COVID-19 relative to healthy controls, we analyzed protein levels in sera using a 48-plex human cytokine and chemokine array. Mean log₂ fold-change in serum cytokine protein levels in patients with moderate (n = 10) or severe (n = 10) case of COVID-19, relative to levels in healthy donors (n = 5) are represented here. (B) SARS-CoV-2 genome (UpE) levels in Calu-3 cells infected with SARS-CoV-2 (MOI 1) or mock infected for 1 h followed by treatment with recombinant IFNβ or mock treatment for 72 h (n = 6). 1/dCT values are represented after normalizing Ct values for SARS-CoV-2 genome levels in infected cells (with or without recombinant IFNβ treatment) with Ct values observed in mock-infected cells. Blot (below): IFIT1, SARS-CoV-2 N, and ACTB protein expression in Calu-3 cells that were infected with SARS-CoV-2 or mock infected for 1 h, followed by treatment with recombinant IFNβ or mock treatment for 72 h (n = 3). For IFNβ treatment, cell culture supernatant containing 2 mg/mL of total protein, including IFNβ was used. (C) IFIT1 transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 24 h. Twenty-four hpi, cells were treated with varying concentrations of recombinant IFN-α2 or mock treated for 6 h. IFIT1 transcript levels were normalized to GAPDH transcript levels (n = 3). (D) IRF7 transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 24 h. Twenty-four hpi, cells were treated with varying concentrations of recombinant IFN-α2 or mock treated for 6 h. IRF7 transcript levels were normalized to GAPDH transcript levels (n = 3). (E) SARS-CoV-2 genome (UpE) levels in Calu-3 cells infected with SARS-CoV-2 (MOI 1) or mock infected for 1 h followed by treatment with recombinant IFN-α2 (1 ng/mL or 10 ng/mL) or mock treatment for 72 h (n = 3). 1/dCT values are represented after normalizing Ct values for SARS-CoV-2 genome levels in infected cells (with or without recombinant IFN-α2 treatment) with Ct values observed in mock-infected cells. (F) IFIT1, SARS-CoV-2 N, and ACTB protein expression in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 1 h, followed by treatment with recombinant IFN-α2 (1 ng/mL or 10 ng/mL) or mock treatment for 72 h (n = 3). Data are represented as mean ± SD, n = 5 for healthy human controls, and n = 10 each for moderate or severe cases of COVID-19, n = 3 or 6 for in vitro experiments. p∗<0.05, p∗∗<0.01, p∗∗∗<0.001, and p∗∗∗∗<0.0001 (Student's t tests with Benjamini-Hochberg multiple testing correction, Student's t tests and Tukey's multiple comparison test). See also Tables S4 and S5, and Figure S3.