Journal: EMBO Reports
Article Title: Nsp16 shields SARS–CoV ‐2 from efficient MDA5 sensing and IFIT1 ‐mediated restriction
doi: 10.15252/embr.202255648
Figure Lengend Snippet: A549 luciferase KO (Ctrl) or IFIT1 KO cells were transduced with lentiviral particles encoding ACE2 followed by pretreatment with increasing amounts of type I IFN (IFNα2a) as indicated (10, 50, or 100 U/ml). At 24 h postinterferon treatment, cells were infected with SARS‐CoV‐2 wt or Nsp16mut at 0.025 RNA copies/cell. Virus release was quantified at 3 days postinfection by RT–qPCR targeting RdRp. Results are plotted as mean of triplicate infections with error bars representing the SD (biological repliactes). One out of three independent experiments is shown. Three days after SARS–CoV‐2 Nsp16mut infection of A549 cells described in (A), expression of IFIT1 and viral nucleocapsid protein N was analyzed by immunoblot. Membranes were probed with antibodies targeting IFIT1, SARS–CoV‐2 N protein, and the housekeeping gene HSP90. Calu‐3 control cells (Ctrl) or two different IFIT1 KO cell lines (IFIT1 KO #1, #2) were incubated with increasing amounts of type I IFN (10, 50, or 100 U/ml) 18 h prior to infection with SARS–CoV‐2 wt and Nsp16mut at 0.015 RNA copies/ml. Virus release was quantified at 3 dpi by RT–qPCR targeting RdRp. Results are plotted as mean of triplicate infections with error bars representing SD (biological replicates). One out of three independent experiments is shown. IFIT1 expression levels in Calu‐3 Ctrl, IFIT1 KO #1 and #2 lines treated with type I IFN (24 h; 100 U/ml) were analyzed by immunoblot. Membranes were probed with antibodies targeting IFIT1 or the housekeeping gene HSP90. A549 cells lacking IFIT1 (IFIT1 KO), the cellular methyltransferase MTR1 (MTR1 KO), both proteins (IFIT1/MTR1 KO), or control cells (Ctrl) were transduced with lentiviral particles encoding ACE2 and infected with SARS–CoV‐2 wt (WT) or Nsp16mut at 0.025 RNA copies/cell the next day. Virus release into the supernatant was quantified at the indicated time points by RT–qPCR. Results are plotted as mean of triplicate infections with error bars representing the SD. One out of three independent experiments is shown. Three days postinfection, A549 cells described in (E) were analyzed by immunoblot. Membranes were probed with antibodies targeting IFIT1, SARS–CoV‐2 N protein, or the housekeeping gene HSP90. A549 Ctrl and MTR1 KO cells were infected with SARS–CoV‐2 wt or Nsp16 mut virus at 0.015 RNA copies/cell or mock treated (triplicates, biological repeats). At 24 hpi, total RNA was extracted from lysed cells and expression of MxA and IFNβ transcripts was quantified using RT–qPCR. ISG levels were normalized on GAPDH transcripts. Data are plotted as mean of triplicate infections with error bars representing the SD. IFIT1 expression was analyzed in mock treated, SARS–CoV‐2 wt (WT) or Nsp16mut infected cells at 48 hpi by immunoblot. Membranes were probed with antibodies targeting IFIT1 and the housekeeping gene HSP90. Data information: Statistical analysis was performed using two‐way ANOVA followed by Bonferroni's multiple comparisons test. ns, not significant ( P > 0.05). Source data are available online for this figure.
Article Snippet: Samples were separated by SDS–PAGE, transferred onto Immobilon‐P PVDF membranes (Merck), and probed with primary antibodies targeting endogenous IFIT‐1 (Cell Signaling, 14769), SARS–CoV2 Nucleocapsid protein (novubio, NB100‐56576), and the house keeping genes HSP90 α/β (Santa Cruz, sc‐13119) or GAPDH (Cell Signaling, 2118).
Techniques: Luciferase, Transduction, Infection, Virus, Quantitative RT-PCR, Expressing, Western Blot, Control, Incubation
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