SARS-CoV-2 infection induces DNA damage, through CHK1 degradation and impaired 53BP1 recruitment, and cellular senescence

SARS-CoV-2 causes DNA damage and an altered DDR activation

We studied the engagement of the DDR pathways at different timepoints upon infection by SARS-CoV-2 of Huh7 cells, a human cell line naturally permissive to SARS-CoV-2 (refs. 25,26), by immunoblotting of whole cell lysates. As negative control we used mock-infected cells; as positive control we exposed cells to hydroxyurea (HU), which induces DNA replication stress and activates the ATR–CHK1 axis27,28, or ionizing radiation (IR) that causes DSBs and activates the ATM–CHK2 pathway11 (Fig. 1a). We observed that SARS-CoV-2 infection triggered the autophosphorylation, and thus activation, of the master kinases DNA-PK (pDNA-PKS2056, involved in DNA repair12) and ATM (pATMS1981) but not ATR (pATRT1989) (Fig. 1a,b). CHK2, the direct downstream target of ATM, was not detectably phosphorylated on its activating site (T68); similarly, CHK1, a target of ATR, was not phosphorylated on S317. Also P53 was not significantly phosphorylated on S15, an ATM/ATR target site (Fig. 1a,b). Differently, KAP1 (also known as TRIM28), a chromatin-bound ATM target12, was strongly phosphorylated (pKAP1S824) together with phosphorylated H2AX (γH2AX) and RPA (pRPAS4/8), markers of DSB and SSB, respectively11 (Fig. 1a,b). Similar results were generated in infected human lung epithelial Calu-3 cells29,30 (Extended Data Fig. 1a,b).

Fig. 1: SARS-CoV-2 infection causes DNA damage and altered DDR activation.figure 1

a, Immunoblotting of whole cell lysates of Huh7 cells infected, or not, with SARS-CoV-2 analysed at different timepoints post-infection for markers of DDR activation. Lysates from Huh7 cells not treated (NT) or treated with 6 mM HU or exposed to 2 Gy IR and collected at different timepoints were used as positive controls. Viral infection was monitored by probing for SARS-CoV-2 N-protein. Where present, dashed lines indicate where the blot was cropped. b, Quantification of activated protein levels shown in a. Values are normalized to mock-infected samples. c, Representative immunofluorescence (IF) images of SARS-CoV-2-infected (V+) or mock-infected (V−) Huh7 cells fixed at 48 h post-infection and stained for DDR markers. SARS-CoV-2 N-protein was used to label infected cells. Nuclei were stained with DAPI. Scale bar, 10 μm. d, Quantification of DDR activation shown in c. Each dot is a nucleus. e, Images of comet assays of infected or mock-infected Huh7 cells. Scale bar, 100 μm. f, Quantification of comet tail moment shown in e. Horizontal bars represent the median values ± 95% confidence interval (CI) of three independent infections. Source numerical data and unprocessed blots are available in source data.

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To confirm and extend at single-cell resolution the impact of SARS-CoV-2 infection on DDR, we performed quantitative immunofluorescence analyses of the conditions aforementioned. We observed increased numbers of pKAP1S824, pRPAS4/8 and γH2AX foci per cell in infected Huh7 compared with mock-infected cells (Fig. 1c,d). In addition, SARS-CoV-2 infection of human nasal epithelial primary cells (HNEpCs) confirmed DDR activation, as detected by pRPAS4/8 and γH2AX foci (Extended Data Fig. 1c,d).

To directly monitor the impact of the virus on physical DNA integrity, we performed comet assays in Huh7 and Calu-3 cells. We observed DNA fragmentation induction in both SARS-CoV-2-infected cell lines compared with control conditions, as measured by tail moment (Fig. 1e,f and Extended Data Fig. 1e,f).

Damaged DNA released in the cytoplasm can be sensed by the cGAS–STING pathway triggering an inflammatory response31. We therefore investigated cGAS–STING and other inflammatory pathways in cells upon SARS-CoV-2 infection and observed a higher number of micronuclei, which also stained positive for cGAS (Extended Data Fig. 1g,h) in Calu-3 cells, suggestive of the release of damaged nuclear DNA in the cytosol. In infected Huh7 cells, which do not express cGAS and STING32, P38 and STAT1, factors involved in the pro-inflammatory response7, were activated (Extended Data Fig. 1i).

To test the consequences of the activation of these pro-inflammatory pathways, we monitored by quantitative reverse transcription polymerase chain reaction (RT–qPCR) the transcriptional induction of IL6, IL8, CXCL9, CXCL10 and TNFα genes in Huh7 and Calu-3 upon SARS-CoV-2 infection. We detected their significant upregulation in both cell types, although generally stronger in Calu-3 (Extended Data Fig. 1j). Since increased expression of pro-inflammatory genes is consistent with the induction of cellular senescence by SARS-CoV-2, as recently reported33,34, we tested and confirmed the establishment of cellular senescence following SARS-CoV-2 infection in our settings, as demonstrated by increased senescence-associated β-galactosidase (SA-β-gal) activity (Extended Data Fig. 1l,m), augmented P21- and reduced KI67-positive cells (Extended Data Fig. 1n,o), and no significant induction of apoptosis (Extended Data Fig. 1k).

In sum, our results obtained by different techniques and in three independent cell types indicate that SARS-CoV-2 infection causes DNA damage and an altered DDR; this is associated with the induction of pro-inflammatory pathways and cytokines and cellular senescence.

SARS-CoV-2 causes dNTP shortage by decreasing CHK1 levels

While studying activation of individual DDR proteins, we noticed that total CHK1 protein levels progressively decreased in infected Huh7 and Calu-3 cells (Figs. 1a and 2a–d and Extended Data Fig. 2c,d), mainly post-transcriptionally (Extended Data Fig. 2e,f). CHK1 loss is reportedly sufficient to cause DNA replication stress and DNA damage accumulation35. CHK1 controls the expression of the ribonucleoside-diphosphate reductase subunit M2 (RRM2), the small subunit of the RNR enzyme that converts ribonucleoside triphosphates (rNTPs) into dNTPs, necessary for DNA synthesis23,36. By testing RRM2 messenger RNA and protein levels by RT–qPCR, immunoblotting and immunofluorescence, we consistently observed their progressive and significant decrease following SARS-CoV-2 infection (Fig. 2a–d and Extended Data Fig. 2a–f).

Fig. 2: SARS-CoV-2 reduces CHK1 and RRM2 levels leading to dNTP shortage.figure 2

a, Immunoblotting of whole cell lysates of Huh7 infected, or not, with SARS-CoV-2 and analysed at different timepoints post-infection. b, Quantification of protein levels shown in a; values are shown as relative to mock-infected samples. c, Immunofluorescence (IF) images of infected (V+) or mock-infected (V−) Huh7 cells fixed 48 h post-infection; nuclei were stained with DAPI. Scale bar, 10 μm. d, Quantification of CHK1- or RRM2-positive cells shown in c; n = 3 independent experiments. e, dNTP concentration was measured in V− or V+ Huh7 and Calu-3; values are shown as relative to V−. f, Histograms show the percentage of cells in each phase of the cell cycle in V− or V+ Huh7 fixed 48 h post-infection. g, Fraction of V− or V+ Huh7 cells that did not incorporate BrdU (BrdU−) measured by flow cytometry 48 h post-infection. Source numerical data and unprocessed blots are available in source data.

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Then, we measured individual dNTP concentrations in SARS-CoV-2 infected Huh7 and Calu-3 cells and observed reduced levels of cellular dNTPs compared with mock-infected conditions (Fig. 2e).

dNTP shortage can impair DNA synthesis, ultimately hampering S-phase progression23,27. To monitor cell-cycle progression, we measured DNA content in infected or mock-infected cells by propidium iodide (PI) staining followed by flow cytometry analysis. We observed a significant accumulation of infected cells in S-phase compared with control samples (Fig. 2f and Extended Data Figs. 3d and 8c). This was confirmed by strongly reduced levels of CDT1, a G1-phase marker37,38,39 (Extended Data Fig. 2g). By pulse labelling with 5-bromo-2′-deoxyuridine (BrdU) for 1 h before flow cytometry, we observed an increased percentage of BrdU-negative cells in S-phase in infected samples (Fig. 2g and Extended Data Figs. 3d and 8c). Altogether, these results indicate reduced dNTP levels and impaired S-phase progression following infection.

To determine the causal role of reduced dNTP levels, we tested the impact of dN supplementation to culture medium of infected cells. We observed that dN supplementation was sufficient to reduce DDR activation as shown by immunofluorescence and immunoblots of γH2AX, pRPAS4/8 and pKAP1S824 (Fig. 3a–d), DNA damage accumulation detected by comet assays (Fig. 3e,f) and transcription of several pro-inflammatory cytokines (Fig. 3g).

Fig. 3: dN supplementation is sufficient to reduce DNA damage and inflammation.figure 3

a, Immunofluorescence (IF) images of V− or V+ Huh7 cells, treated or not with dNs, fixed 48 h post-infection; nuclei were stained with DAPI. Scale bar, 10 μm. b, Quantification of DDR activation shown in a. Each dot is a nucleus. c, Immunoblots of Huh7 cells treated as in a. d, Quantification of protein levels shown in c. Values are normalized to untreated V+ cells. e, Images of comet assays of Huh7; conditions are as in a. Scale bar, 100 μm. f, Quantification of comet tail moment shown in e; horizontal bars represent the median values ± 95% CI of three independent infections. g, RT–qPCR of pro-inflammatory cytokine expression in V− or V+ Calu-3 cells, treated or not with dNs. Values are shown as relative to RPLP0 mRNA. Source numerical data and unprocessed blots are available in source data.

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Overall, these results are consistent with a model in which SARS-CoV-2 gene products cause CHK1 loss, which reduces RRM2 levels and consequently the pool of available dNTPs, causing impaired DNA replication and S-phase progression, DNA damage accumulation and ultimately fuelling an inflammatory response. Supplementation of dNs is sufficient to tame these events.

CHK1 loss is sufficient to cause DNA damage and inflammation

To determine whether CHK1 loss is sufficient to recapitulate the events here described following SARS-CoV-2 infection, we studied the impact of CHK1 depletion by RNA interference. Consistent with previous reports23, we observed by flow cytometry that cells knocked down for CHK1 accumulate in S-phase (Extended Data Figs. 3a–d and 8c) and pulse labelling with BrdU for 1 h before flow cytometry analysis revealed a higher fraction of BrdU-negative S-phase cells compared with control samples (Extended Data Figs. 3d and 8c).

We also observed that CHK1 depletion was sufficient to reduce RRM2 levels and cause DNA damage, as shown by increased pRPAS4/8 and γH2AX signals (Extended Data Fig. 3e,f). In addition, CHK1 knockdown led to the activation of P38 and STAT1 (Extended Data Fig. 3e,f) and formation of γH2AX foci and micronuclei, often positive for cGAS (Extended Data Fig. 3g–j), indicating that CHK1 loss in infected cells probably contributes to the activation of pro-inflammatory pathways. Indeed, cells depleted for CHK1 displayed increased expression of most of the cytokine and chemokine genes tested (Extended Data Fig. 3k,l) and increased secretion of IL6, CXCL9 and CXCL10 in Calu-3 cells as monitored by immunoassays (Extended Data Fig. 3m).

In sum, CHK1 loss is sufficient to recapitulate several of the events observed in SARS-CoV-2 infected cells, namely, RRM2 reduction, S-phase progression impairment, DNA damage and secretion of inflammatory cytokines.

SARS-CoV-2 ORF6 and NSP13 trigger CHK1 protein degradation

To identify the viral gene products responsible for CHK1 downregulation, we individually expressed 24 of the 26 annotated SARS-CoV-2 proteins40 (SARS-CoV-2 reference genome, NC_045512.2) and analysed by immunoblotting their impact on CHK1 levels. Among the gene products tested, ORF6 and NSP13 were the factors with the strongest and most consistent impact on CHK1 protein levels (Fig. 4 and Extended Data Fig. 4a,b). Their sole expression was also sufficient to reduce RRM2 levels and increase γH2AX and RPA phosphorylation (S4/8) (Fig. 4a–d and Extended Data Fig. 4a,b).

Fig. 4: SARS-CoV-2 ORF6 or NSP13 expression is sufficient to cause CHK1 loss.figure 4

a, Immunofluorescence (IF) images of Huh7 cells expressing Strep-tagged SARS-CoV-2 ORF6 or NSP13 fixed 48 h post-transfection and stained for DDR markers; GFP was used as control; staining with anti-Strep-tag was used to label transfected cells; nuclei were stained with DAPI. Scale bar, 10 μm. b, The histograms show the percentage of CHK1- or RRM2-expressing cells among the transfected ones (Strep-Tag+) as determined in a; n = 3 independent experiments (n = 4 for GFP-expressing cells in RRM2 analysis). The dot plots show the number of γH2AX or pRPAS4/8 foci in the samples described in a. c, Representative immunoblots of whole cell lysates from Huh7 cells transfected with plasmids encoding for HA-tagged SARS-CoV-2 ORF6, or Strep-tagged SARS-CoV-2 NSP13, or empty vector (EV) as a control. Where present, dashed lines indicate where the blot was cropped. d, Quantification of protein levels shown in c; values are the mean ± s.e.m. of four independent experiments and shown as relative to the control sample (EV). Source numerical data and unprocessed blots are available in source data.

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ORF6 causes CHK1 degradation through the proteasome pathway

SARS-CoV-2 ORF6 has been shown to associate with the nuclear pore and to interfere with proteins’ nuclear–cytoplasmic trafficking41,42.

We observed that SARS-CoV-2-infected cells displayed cytoplasmic CHK1 localization compared with mock-infected cells in which CHK1 is mainly nuclear (Fig. 2c). It has been reported that accumulation of CHK1 in the cytoplasm leads to its degradation through the UPS43. To investigate the potential involvement of ORF6 in the cytoplasmic accumulation of CHK1, we took advantage of a point mutant form of ORF6 (ORF6M58R) unable to interact with the nuclear pore complex41. Interestingly, CHK1 protein levels did not decrease in Huh7 cells expressing the mutant ORF6, as detected both by immunofluorescence and immunoblots (Fig. 5a–d). Consistent with that, mutant ORF6 expression also had no impact on RRM2 protein levels and DNA damage accumulation as detected by pRPAS4/8 and γH2AX (Fig. 5c,d). This suggests that ORF6, by altering CHK1 nuclear–cytoplasm shuttling, may cause its degradation.

Fig. 5: ORF6 and NSP13 causes CHK1 reduction through the proteasome and autophagy pathways, respectively.figure 5

a, Images of Huh7 expressing GFP (negative control), ORF6 or its mutant form ORF6M58R. b, Quantification of the percentage of transfected cells expressing CHK1 shown in a. c, Immunoblotting of Huh7 treated as in a; EV-transfected cells were used as negative control. d, Quantification of the protein levels shown in c. Values are the mean ± s.e.m. of four independent experiments. e, Confocal images of GFP- or ORF6-expressing Huh7 ± MG132. f, Quantification of nuclear (n) and cytoplasmic (c) CHK1 levels in the cells described in e. g, Immunoblots of the samples described in e; EV-transfected cells were used as negative control. h, Quantification of the protein levels shown in g. i, Ubiquitination assay of CHK1 immunoprecipitated from ORF6- or ORF6M58R-expressing Huh7 ± MG132. j, Quantification of the samples shown in i. Values are shown as relative to immunoprecipitated CHK1 amounts (IP-CHK1); n = 3 independent experiments. k, Immunofluorescence (IF) images of GFP- or NSP13-expressing Huh7 ± BafA1 or CQ. l, CHK1 quantification in cells described in k. Values are the mean ± s.e.m. of four independent experiments, except for GFP and NSP13 not treated (NT) conditions (n = 6). m, IF images of NSP13-expressing Huh7 treated with BafA1 (1 h). Arrow points to CHK1 and P62 co-localization. n, Quantification of the percentage of cells displaying co-localizing CHK1 and P62 signals shown in m. o, IF images of CHK1 levels in Huh7 transfected with the indicated siRNAs before viral NSP13 overexpression. p, Quantification of the percentage of CHK1-expressing cells in the transfected samples represented in o. Values are the mean ± s.e.m. of four independent experiments. Scale bar, 10 μm and DAPI-stained nuclei in a,e,k,m and o. Source numerical data and unprocessed blots are available in source data.

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We therefore probed the engagement of UPS in ORF6-dependent CHK1 loss. Treatment of ORF6-expressing Huh7 cells with MG132—a proteasome inhibitor—recovered CHK1 protein levels, detected mostly in the cytoplasm (Fig. 5e–h), consistent with impaired protein trafficking of ORF6-expressing cells.

To demonstrate that SARS-CoV-2 ORF6 expression causes CHK1 poly-ubiquitination, a prerequisite of proteosome-dependent degradation, we immunoprecipitated endogenous CHK1 in Huh7 cells expressing either ORF6 or ORF6M58R, treated or not with MG132, and probed for ubiquitinated CHK1. Proteasomal inhibition led to a higher accumulation of ubiquitinated CHK1 in ORF6-expressing cells compared with samples that overexpressed ORF6M58R (Fig. 5i,j).

These results indicate that SARS-CoV-2 ORF6 prevents CHK1 nuclear import, causing its accumulation in the cytoplasm and its consequent degradation through the UPS.

NSP13 causes CHK1 degradation through autophagy

It has been shown that NSP13 can promote protein degradation in an autophagy-dependent manner44. Therefore, to test whether CHK1 loss in NSP13-expressing cells was dependent on the autophagic route, we transiently expressed the viral NSP13 gene in Huh7 cells in the presence of either Bafilomycin A1 (BafA1) or chloroquine (CQ), two specific inhibitors of autophagy45; efficacy was confirmed by the observed accumulation of P62 cytoplasmic aggregates45 (Extended Data Fig. 5a,b). NSP13-mediated reduction of CHK1 protein levels was abolished by both treatments (Fig. 5k,l). A short BafA1 exposure highlighted a clear co-localization of CHK1 with P62 cytoplasmic aggregates, suggesting its accumulation in autophagosomes (Fig. 5m,n). To confirm and extend these results, we individually knocked down Beclin 1 (BECN1) and LC3B—two key regulators of autophagy46—and observed a significant restoration of CHK1 protein levels in NSP13-expressing cells (Fig. 5o,p and Extended Data Fig. 5c,d).

These results indicate that SARS-CoV-2 NSP13 causes the accumulation of CHK1 in the cytoplasm, where it co-localizes with P62, in this way promoting its degradation through autophagy.

N-protein impairs 53BP1 recruitment at DSB and hinders NHEJ

We noticed that γH2AX foci accumulation was not accompanied by co-localizing 53BP1 foci in SARS-CoV-2-infected Huh7, Calu-3 and HNEpC (Fig. 6a–d and Extended Data Fig. 6a,b), despite unaltered 53BP1 protein levels (Extended Data Fig. 6c).

Fig. 6: SARS-CoV-2 N suppresses 53BP1 activation and inhibits repair by NHEJ.figure 6

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.

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SARS-CoV-2 N-protein is an RNA-binding protein capable to undergo RNA-dependent LLPS21,22,47,48,49. We previously reported that also 53BP1 phase-separates in an RNA-dependent manner17. To test the potential impact of N-protein on 53BP1 foci formation, we expressed the viral N gene40 in Huh7 cells and exposed them to IR. We observed that irradiated cells expressing N showed increased numbers of γH2AX foci per cell, but fewer 53BP1 foci compared with control cells (Fig. 6e,f). To reduce the possibility of an indirect effect mediated by altered gene expression, we micro-injected purified recombinant N-protein into the nuclei of irradiated cells stably expressing 53BP1-GFP50 and immediately studied the kinetics of 53BP1 foci by live imaging. We observed 53BP1 foci number decreasing with a faster (~8.5-fold) kinetic in cells injected with the N-protein compared with control cells (Fig. 6g, Extended Data Fig. 6d and Supplementary Video 1).

Next, we sought to elucidate the molecular mechanisms underlying N-protein impact on 53BP1 functions. In co-immunoprecipitation experiments, 53BP1 did not interact with N-protein (Extended Data Fig. 6e). We previously reported that dilncRNAs generated at DSB drive LLPS of 53BP1 (refs. 16,17). Intriguingly, both viral and cellular RNAs have been reported to associate with N-protein and promote its phase separation48, as we confirmed (Extended Data Fig. 6f,g).

Since N-protein, although mainly cytoplasmic, also localizes in the nucleus51,52,53 (Extended Data Fig. 6j,k), we tested whether N associates with cellular dilncRNAs by performing RNA immunoprecipitation (RIP) against the N-protein in NIH2/4 cells, which we previously characterized for the expression of dilncRNAs upon DSB induction by I-SceI endonuclease

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