In a recent study published in Nature Cell Biology, researchers assessed the association between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and deoxyribonucleic acid (DNA) damage and cellular senescence.
Several cellular mechanisms, such as the autophagy pathway, the DNA damage response, and the ubiquitin-proteasome system (UPS), can be affected by viral infections (DDR). Although the interaction between DDR and certain DNA viruses has been examined, significantly less about ribonucleic acid (RNA) viruses is understood.
It has been proposed that SARS-CoV-2 infection engages components involved in DDR machinery; however, a comprehensive characterization and mechanistic investigation of the effect of SARS-CoV-2 infection on DDR engagement and genome integrity are lacking.
About the study
In the present study, researchers demonstrated that SARS-CoV-2 damages DNA and triggers an altered DNA damage response.
Immunoblotting was used to examine the activation of the DDR pathways in a human cell line called Huh7, which naturally permits SARS-CoV-2 at various time-points following infection with SARS-CoV-2. As a negative control, the team utilized mock-infected cells. On the other hand, as a positive control, cells were subjected to hydroxyurea (HU), which generates DNA replication stress and elicits the ataxia telangiectasia and Rad3-related (ATR)-checkpoint kinase 1 (CHK1) axis, or ionizing radiation (IR), which causes double-strand breaks (DSBs) and stimulates the ataxia-telangiectasia mutated (ATM)-CHK2 pathway.
Quantitative immunofluorescence investigations were conducted to confirm and expand the effect of SARS-CoV-2 infection at the single-cell resolution on DDR. The team examined cyclic-guanosine monophosphate–adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) along with other inflammatory pathways in cells infected with SARS-CoV-2.
Furthermore, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunofluorescence, and immunoblotting were employed to determine the amounts of ribonucleotide reductase M2 (RRM2) messenger RNA and protein. To evaluate if CHK1 depletion is enough to recreate the events after SARS-CoV-2 infection, the researchers investigated the effect of CHK1 depletion by RNA interference.
To recognize the viral gene products that cause CHK1 downregulation, the team expressed 24 out of the 26 identified SARS-CoV-2 proteins and analyzed their effect on CHK1 levels using immunoblotting.
SARS-CoV-2 infection induced the autophosphorylation and consequent stimulation of the master kinases DNA-protein kinase and ATM, but not ATR. CHK2, the immediate downstream target of ATM, was not phosphorylated at its activation site, and neither was CHK1. In addition, P53 was not extensively phosphorylated on S15, a target site for ATM/ATR. Kruppel-associated box)-associated protein 1 (KAP1), a chromatin-bound ATM target, was significantly phosphorylated alongside phosphorylated ƔH2AX and replication protein-A (RPA), indicators of double-strand breaks and single-strand breaks, respectively.
Infected human pulmonary epithelial Calu-3 cells produced comparable outcomes. Infection of human nasal epithelial primary cells (HNEpCs) with SARS-CoV-2 confirmed DDR activation, as indicated by pRPAS4/8 and H2AX foci. As evaluated by tail moment, DNA fragmentation was induced in both SARS-CoV-2-infected cell lines relative to control conditions.
In Calu-3 cells, an increased number of micronuclei stained positively for cGAS suggested the generation of damaged nuclear DNA into the cytosol. In SARS-CoV-2-infected Huh7 cells, which did not express cGAS and STING32, factors responsible for pro-inflammatory response, namely P38 and signal transducer and activator of transcription 1 (STAT1), were activated.
Following the SARS-CoV-2 infection, the team observed a steady and considerable decline in their numbers. Deoxynucleoside triphosphate (dNTP) deficiency can hinder DNA synthesis, hence impeding S-phase progression. Compared to control samples, the researchers detected a considerable concentration of infected cells in the S-phase. Also, an increase in the proportion of BrdU-negative S-phase cells in infected samples was noted. Following infection, these findings indicate decreased dNTP levels and hindered S-phase development.
Flow cytometry demonstrated that cells knocked down for CHK1 aggregated in S-phase. Pulse labeling with BrdU prior to flow cytometry analysis revealed a greater proportion of BrdU-negative S-phase cells in comparison to control samples. The team also noted that the depletion of CHK1 was sufficient to decrease RRM2 levels and result in DNA damage.
Moreover, CHK1 knockdown resulted in the activation of STAT1 and P38 as well as the generation of H2AX foci and micronuclei that were frequently positive for cGAS. This indicated that CHK1 depletion in infected cells likely contributed to the stimulation of pro-inflammatory pathways. As shown by immunoassays, cells depleted of CHK1 demonstrated increased expression of the majority of cytokine and chemokine genes as well as enhanced secretion of interleukin (IL)-6, CXCL9, and CXCL10 in Calu-3 cells.
ORF6 and NSP13 were the investigated gene products having the most robust and consistent effect on CHK1 protein levels. Also, their expression alone was sufficient to decrease RRM2 levels and enhance the phosphorylation of gamma-H2AX and RPA. It has been demonstrated that SARS-CoV-2 ORF6 is associated with the nuclear pore and interferes with the nuclear-cytoplasmic transport of proteins.
The study findings showed that SARS-CoV-2-induced DNA damage activated a cell-intrinsic pro-inflammatory pathway that, in conjunction with the immunological response, generated the intense inflammatory response found in COVID-19 patients.
The team presented a model to enhance the knowledge of SARS-CoV-2-induced cellular senescence by providing a mechanism for the creation of DNA damage and the stimulation of DDR pathways as well as a pro-inflammatory program.
- Jaycox, J. et al. (2023) "SARS-CoV-2 mRNA vaccines decouple anti-viral immunity from humoral autoimmunity", Nature Communications, 14(1). doi: 10.1038/s41467-023-36686-8. https://www.nature.com/articles/s41467-023-36686-8
Posted in: Genomics | Medical Science News | Medical Research News | Disease/Infection News
Tags: Adenosine, Ataxia, Ataxia-Telangiectasia, Autoimmunity, Autophagy, Cell, Cell Biology, Cell Line, Chemokine, Chromatin, Coronavirus, Coronavirus Disease COVID-19, covid-19, CXCL10, Cytokine, Cytometry, DNA, DNA Damage, DNA Replication, DNA Synthesis, Flow Cytometry, Gene, Genes, Genome, immunity, Immunoassays, Interferon, Interleukin, Kinase, Phosphorylation, Polymerase, Polymerase Chain Reaction, Protein, Respiratory, Ribonucleic Acid, RNA, RNA Interference, SARS, SARS-CoV-2, Severe Acute Respiratory, Severe Acute Respiratory Syndrome, Stress, Syndrome, Transcription, Ubiquitin
Bhavana Kunkalikar is a medical writer based in Goa, India. Her academic background is in Pharmaceutical sciences and she holds a Bachelor's degree in Pharmacy. Her educational background allowed her to foster an interest in anatomical and physiological sciences. Her college project work based on ‘The manifestations and causes of sickle cell anemia’ formed the stepping stone to a life-long fascination with human pathophysiology.
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