Fig. 6: SARS-CoV-2 N suppresses 53BP1 activation and inhibits repair by NHEJ.
a, Immunofluorescence (IF) images of V+ or V− Huh7; nuclei were stained with DAPI. b, Quantification of 53BP1 foci shown in a. Each dot represents the number of 53BP1 foci per nucleus. c, IF images of infected HNEpC in which SARS-CoV-2 RNA was detected by FISH; nuclei were stained with Hoechst. d, Quantification of 53BP1 foci shown in c; the histograms show the percentage of nuclei with 53BP1 foci (>1) in cells expressing (+) or not (−) SARS-CoV-2 RNA; n = 3 independent infections. e, IF images of irradiated Huh7 transfected with N-protein or EV as control; nuclei were stained with DAPI. f, Quantification of DDR foci shown in e; the dot plots show the number of γH2AX and 53BP1 foci per nucleus in N-protein- or EV-expressing samples. Values are relative to irradiated cells transfected with EV; bars represent the mean ± 95% CI of three independent experiments. g, Quantification of 53BP1 foci per nucleus over time in irradiated cells injected with recombinant N-protein or BSA as control. Error bars represent s.e.m.; the experiment was repeated three times with similar results. h, NIH2/4 expressing (n = 3) or not (n = 2) I-SceI were transfected with N-protein. Cell lysates were incubated with anti-N-protein or normal rabbit IgG and co-precipitated RNA analysed by strand-specific RT–qPCR. H2AX mRNA was used as an unrelated transcript. Values are shown as percentage of input RNA. i, Endogenous 53BP1 was immunoprecipitated from I-SceI-expressing NIH2/4 transfected with N-protein or EV as control. 53BP1-bound transcripts were monitored as in h and shown as percentage of input RNA. Values are the average of two independent experiments. j, EJ5-GFP U2OS were transfected with N-protein or EV, ± I-SceI. DSB re-joining events were evaluated by qPCR on gDNA isolated at 72 h post-transfection. Values are relative to I-SceI-transfected cells not expressing N-protein. Scale bar, 10 μm (a, c and e). Source numerical data are available in source data.
