The RNA sensor MDA5 detects SARS-CoV-2 infection

Abstract

Human cells respond to infection by SARS-CoV-2, the virus that causes COVID-19, by producing cytokines including type I and III interferons (IFNs) and proinflammatory factors such as IL6 and TNF. IFNs can limit SARS-CoV-2 replication but cytokine imbalance contributes to severe COVID-19. We studied how cells detect SARS-CoV-2 infection. We report that the cytosolic RNA sensor MDA5 was required for type I and III IFN induction in the lung cancer cell line Calu-3 upon SARS-CoV-2 infection. Type I and III IFN induction further required MAVS and IRF3. In contrast, induction of IL6 and TNF was independent of the MDA5-MAVS-IRF3 axis in this setting. We further found that SARS-CoV-2 infection inhibited the ability of cells to respond to IFNs. In sum, we identified MDA5 as a cellular sensor for SARS-CoV-2 infection that induced type I and III IFNs.

Figure 1

Calu-3 cells respond to SARS-CoV-2 infection by upregulating type I and III IFNs, ISGs and cytokines. (A) The indicated cell lines were mock-infected or infected with SARS-CoV-2 (MOI = 0.1 or 0.5) for 24 h prior to RNA extraction and RT-qPCR for the indicated transcripts. Data are relative to GAPDH expression. (B) Calu-3 cells were stimulated with 100 U/ml IFN-A/D for 16 h. Cell lysates were analysed by western blot using the indicated antibodies. Individual membranes were probed for each protein. Membranes were subsequently re-probed for β-actin. See Supplementary Figure 2 for full blots. Data are from a single experiment. Data points in (A) are from technical duplicates and bars indicate the average.

Figure 2

MAVS is required for the type I and III IFN response to SARS-CoV-2. (A,B) Calu-3 cells were depleted of MAVS by lentiviral shRNA delivery using two independent shRNAs (shRNA-MAVS-06 and shRNA-MAVS-45). Knockdown efficiency was assessed by western blot (A) and RT-qPCR (B). The control shRNA targeted GFP, which is absent in Calu-3 cells. See Supplementary Figure 3 for full blots. (C,D) Calu-3 cells depleted of MAVS using shRNA-MAVS-45 were infected with SARS-CoV-2 (MOI = 0.1) for 48 h, followed by RNA extraction and RT-qPCR for the indicated transcripts. Data are relative to GAPDH expression. Data in (A,B) are representative of two independent biological repeats. Data in (C,D) are pooled from 3 independent biological repeats, with bars representing the average. Data were analysed by t-test and significant differences are indicated (**P < 0.01; *P < 0.05).

Figure 3

SARS-CoV-2 activates type I and III IFN responses via MDA5 and IRF3, and inhibits ISG induction in infected Calu-3 cells. (A) Calu-3 cells were depleted of MAVS, STING, IRF3, MDA5 or RIG-I using a lenti-CRISPR approach. Depletion of the targeted proteins was assessed by western blot. Control cells were targeted for GFP, which is absent in Calu-3 cells. See Supplementary Figure 4 for full blots. (B) Cells from (A) were infected and analysed as described in Fig. 2C. (C–F) Cells from (A) were infected as in Fig. 2C, stained for live cells, SARS-CoV-2N protein and MxA, and analysed by flow cytometry. Live cells were assessed for SARS-CoV-2 N protein expression (D) and MxA induction, shown as mean fluorescence intensity (MFI; E). (F) Cells in the SARS-CoV-2-infected samples were further subdivided into SARS-CoV-2 N positive (N+) and SARS-CoV-2 N negative (N−) cells, and MxA MFI was determined within these subpopulations. Data in (A,C) are representative of two independent biological repeats. Data in (B,D–F) are pooled from two independent biological repeats, with bars representing the average.