Fig. 1.

Azacytidine targets SARS-CoV-2 RNA to inhibit virus infection. Vero E6 and Calu-3 cells were infected with SARS-CoV-2 at a multiplicity of infection (moi) of 0.2 in the presence of different doses of azacytidine. At 24 h post-infection, cell supernatants were collected. (a, b) IC₅₀ and CC₅₀ were calculated by detecting viral RNA with quantitative real-time PCR (qRT-PCR) and CCK-8 assay, respectively. (c–e) The anti-SARS-CoV-2 effect of azacytidine in vivo. 8 weeks old female BALB/c mice were randomly divided into 3 groups with 10 mice per group. Mice were intranasally challenged with 4 × 10³ plaque forming unit (PFU) MA-SARS-CoV-2 in 50 μL Dulbecco’s modified Eagle’s medium (DMEM) or equal DMEM. At 1 dpi, mice were intraperitoneally injected with either 2 mg/kg 5Aza, 25 mg/kg Remdesivir, or an equivalent volume of sterile saline, once daily for seven consecutive doses. (c) Body weight was measured daily for 14 d; mice with 30% body weight loss were considered moribund and euthanized. (d) The survival rates of mice (n = 6). (e) At 4 dpi, 4 mice per group were euthanized to detect viral RNA copy numbers in lungs, and data are presented as values relative to the control. (f) The pseudo m⁵C locations indicated incorporated azacytidine. Vero E6 cells were infected with 1 moi SARS-CoV-2 in the presence of 10 μmol/L 5Aza or saline. After 12 h, total RNA was isolated and a comparison of 5Aza-treated viral RNA with the control using bisulfite sequencing was performed; the non-overlapping points are pseudo m⁵C locations that indicate where 5Aza was incorporated. (g) RNA-BSseq identified m⁵C locations in the SARS-CoV-2 genome. There were at least 5 methylation-supporting reads per point. Data are averaged from 3 technological repeats. (h) RNAs of cells infected with SARS-CoV-2 (treated with 5Aza or untreated) were subjected to nanopore direct RNA sequencing. (i) Vero E6 cells were transiently overexpressed with DNMT2, NSUN2, or GFP; 24 h later, cells were infected with 0.1 moi SARS-CoV-2. After 24 h, the supernatant was collected for viral RNA detection by qRT-PCR, and cells were lysed and subjected to western blotting. (j) Vero E6 cells were transfected with 100 μmol/L siRNA targeting NSUN2, DNMT2, or negative control siRNA; 48 h later, cells were infected with 0.1 moi SARS-CoV-2. After 30 h, viral RNA in the supernatant was detected by RT-PCR, and cells were lysed for western blotting. (k) Vero E6 cells transiently overexpressing DNMT2, NSUN2, or GFP with a hemagglutinin (HA) tag were infected with 0.2 moi SARS-CoV-2 for 20 h, either in the presence of 16 μmol/L 5Aza or not. Lysates were prepared and split for incubation with mouse anti-HA antibody. Co-precipitated RNA was analyzed by qRT-PCR. The level of viral RNA amplicon was determined as the percentage of input (1% of lysate) (left); the expression of indicated protein and products of immunoprecipitation (IP) was validated by western blotting (right). Data are presented as mean ± standard deviation and analyzed using Student’s t-test or one-way analysis of variance (body weight change); Log-rank test was used to analyze the significance of survival differences. * P < 0.05, ** P < 0.01, *** P < 0.001.