key: cord-0318107-6tgi6dnp authors: Victor, Joshua; Deutsch, Jamie; Whitaker, Annalis; Lamkin, Erica N.; March, Anthony; Zhou, Pei; Botten, Jason W.; Chatterjee, Nimrat title: SARS-CoV-2 triggers DNA damage response in Vero E6 cells date: 2021-09-09 journal: bioRxiv DOI: 10.1101/2021.09.08.459535 sha: 35a4e1ac7e53de26f059b384655b9fe28dfde303 doc_id: 318107 cord_uid: 6tgi6dnp The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus responsible for the current COVID-19 pandemic and has now infected more than 200 million people with more than 4 million deaths globally. Recent data suggest that symptoms and general malaise may continue long after the infection has ended in recovered patients, suggesting that SARS-CoV-2 infection has profound consequences in the host cells. Here we report that SARS-CoV-2 infection can trigger a DNA damage response (DDR) in African green monkey kidney cells (Vero E6). We observed a transcriptional upregulation of the Ataxia telangiectasia and Rad3 related protein (ATR) in infected cells. In addition, we observed enhanced phosphorylation of CHK1, a downstream effector of the ATR DNA damage response, as well as H2AX. Strikingly, SARS-CoV-2 infection lowered the expression of TRF2 shelterin-protein complex, and reduced telomere lengths in infected Vero E6 cells. Thus, our observations suggest SARS-CoV-2 may have pathological consequences to host cells beyond evoking an immunopathogenic immune response. pandemic. SARS-CoV-2 is a zoonotic positive-sense RNA virus from the Coronaviridae family of viruses. Since the inception of the pandemic more than 4 million deaths have occurred and some recovered patients have continued to report debilitating, and sometimes new symptoms long after the infection has ended. This condition with lingering symptoms is often referred to as "Long COVID". Although mechanisms are unclear, hypotheses include viral-induced tissue and organ injury, in which SARS-CoV-2 infection alters host cell physiology, and cellular functions might be permanently altered. Hence there is an urgent need to understand the pathobiology of SARS-CoV-2, that might shed light on the causes of long-term symptoms [1] [2] [3] [4] [5] . RNA viruses are the etiologic agents of many prevalent and lethal human diseases 6 . It is well documented that despite the completion of their life cycle within the host cell cytoplasm, RNA viruses can induce significant DNA damage and activate the DNA damage response (DDR) pathway. Both events enable viral replication and modulation of host cell functions. Notably, the positive-sense RNA viruses from the family Coronaviridae to which the SARS-CoV-2 belongs, and the negative-strand Influenza A virus from the Orthomyxoviridae family induces the DDR pathway in host cells [7] [8] [9] . More recently, global phosphorylation mapping 10 and ATR DDR inhibitor studies 5 indicate that SARS-CoV-2 may also engage the DDR pathway to propagate in host cells. A classical DNA damage response is mediated by one of the signaling pathways-ATM, ATR, and DNA-PK kinases 6 . Double strand breaks usually engage the ATM and the DNA-PK pathways, while single-stranded DNA activates the ATR kinase pathway 11 . These pathways activate specific downstream effectors such as CHK1 by ATR and CHK2 by ATM to trigger a cellular response that allows cells to arrest cell cycle progression to repair damaged DNA. The DDR pathway is a critical component of an intracellular defense mechanism that is activated upon detection of lesions on the DNA to facilitate DNA repair by any of the following: base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), non-homologous end-joining (NHEJ) or homologous recombination (HR) to fix the damaged DNA 12 . In the event that DNA repair fails, either programmed cell death is induced, or an alternative pathway of DNA damage tolerance or translesion synthesis (TLS) is triggered 13 to allow cell survival despite the presence of DNA damage. Additionally, it is known that ATR and ATMdependent DNA damage responses associate with telomere dysfunction 14, 15 . Telomeres are specialized DNA-protein structures that play a key role in maintaining genome stability by protecting the ends of chromosomes 16 . The shelterin protein complex, comprised of six proteins-TRF1, TRF2, POT1, TPP1, TIN2 and Rap1specifically associate with telomeric sequences to prevent the chromosomal ends from being recognized as double strand breaks 17 . Depletion or loss of components of the shelterin complex results in telomere shortening 18 . Literature survey suggests that Epstein-Bar Virus (EBV) infections can also destabilize telomeres by downregulating TRF2 expression 19 . Whether SARS-CoV-2 might trigger telomere dysfunction is not known. Herein, we investigated the ability of SARS-CoV-2 to impact the DNA damage response and telomere stability in Vero E6 cells. The lysate was mixed thoroughly, followed by addition of one volume of 70% ethanol (Fisher Scientific) and mixed again by pipetting. The lysate was then transferred to the RNeasy spin column provided in the RNeasy kit (Qiagen). The manufacturers protocol was then followed for the rest of the isolation process. Isolated RNA was quantified using the Nanodrop-2000 (ThermoFisher), and then diluted using RNase/DNase free water (VWR Life Sciences) until all the concentration of RNA in each sample was 10ng/µL. The iTaq Universal SYBR Green One-Step Kit (Bio-Rad) was used to run the RT-qPCR reactions, using the manufacturer recommended cycling conditions for the StepOnePlus thermal cycler. The telomere lengths were measured by using the Relative Telomere Length Quantification qPCR kit (ScienCell) which utilizes primers that recognize telomeric repeats (primer Tel) and the resulting gene expression is normalized to the gene expression of a single copy reference (SCR) control that recognizes a 100 bp region on human chromosome 17. 1µL of DNA at a concentration of 1ng/µL was mixed with either 2µL of primer Tel or primer SCR and 10µL PowerTrack SYBR Green Master Mix (ThermoFisher) with the volume of the reaction being brought up to 20µL with ddH2O. The cycling conditions for this reaction were 2 minutes at 95 o C followed by 40 cycles of 15 seconds at 95 o C and 1 minute at 60 o C, with the reaction taking place on the StepOnePlus thermal cycler (ThermoFisher), and the average telomere length was calculated using the manufacturer's instructions. The StepOnePlus software was used to analyze that data and GraphPad Prism 9.0.1 was used for statistical analysis and to the plot the results. All other primers used for RT-qPCR were obtained through ThermoFisher and are listed below. Membranes were imaged with the Li-COR Odyssey CLx, and images were analyzed with Image Studio software. Bands indicating our proteins of interest were normalized to Actin, and SARS-CoV-2 treated results were further normalized to the mock controls. To test the hypothesis that SARS-CoV-2 infection triggers a DDR, we infected Vero E6 cells at an MOI of 0.01 and tested expression of DDR genes after 48 hours (Fig. 1A) . Fig. 1D and 2A) . Within this context, we did not see an enhanced phosphorylation of the ATR protein or the total ATR protein levels in infected cells ( Fig. 2A and 2B) , suggesting that the overall increase in ATR levels corresponding to the increased mRNA levels may have occurred prior to our test time of 48 hours. In fact, we observe an overall reduction in both the total ATR and CHK1 protein levels at 48 hours (Fig. 2B) . Ascertaining the increased expression of ATR and CHK1 in a time course experiment post SARS-CoV-2 infection will be of interest in future studies. Interestingly, we also observed an increase in H2AX phosphorylation protein, despite a lack of an increase in ATM transcript expression. We conclude that SARS-CoV-2 infection activates the host cell ATR DDR pathway, which could provide an unknown proliferation potential to its infectious cycle 5 . ATR activation due to retroviral infections such as Human immunodeficiency Virus 1 To quantify possible effects of an activated ATR DNA damage response in infected cells, we measured telomere lengths both in mock and SARS-CoV-2 infected cells. We used the commercially available qPCR-based Relative Telomere Length Quantification kit and compared the relative amplification of the telomere end to the internal control. Strikingly, we observed that SARS-CoV-2 infection shortens the relative length of telomeres compared to mock controls within 48 hours (Fig. 3A) . In addition, we examined the relative expression of one of the key telomere proteins, TRF2, and found its expression to be significantly suppressed in SARS-CoV-2 infected Vero E6 cells ( Fig. 3B and C) . Whether other cell types, such as rodent or human cells would exhibit a similar telomere phenotype post-SARS-CoV-2 infection needs to be ascertained. TRF2 is one of the most important shelterin complex proteins that ensure telomere end SARS-CoV-2 has made a lasting impact across the globe infecting millions of people, and spurred concern due to long-term health consequences. With the advent of the continuously evolving strains, studying the pathobiological consequences of this infection in recovered patients is vital. This study suggests that in Vero E6 cells, SARS-CoV-2 infection triggers an ATR DNA damage response and affects telomere function. Both ATR activation and telomere instability are associated with genome instability. Further studies are required to expand these findings and ascertain the clinical implications of these results. 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