A new study led by researchers from the University of Vermont, Sidney Kimmel Medical College and the University of Vermont Medical Center that indicates more possible long-term medical conditions arising in Long COVID including various cancers.
The study that also involved medical scientists from Duke University, New York University and Icahn School of Medicine at Mount Sinai found that the SARS-CoV-2 coronavirus is capable of hijacking the host DNA, causing mutagenesis, telomere dysregulation and it also impairs DNA mismatch repair, giving rise to the state of hypermutability.
The study findings were published on a preprint server: Research Square and are currently being peer reviewed. https://www.researchsquare.com/article/rs-1556634/v1
SARS-CoV-2 has infected over 501 million people and caused more than 6 million deaths worldwide(https://coronavirus.jhu.edu/map.html). Apart from the varying severity of clinical symptoms duringactive SARS-CoV-2 infection, about 40% of recovered patients are susceptible to developing long-COVID,where general malaise and debilitating symptoms persist1. Recent reports also indicate thatcardiovascular health and brain structure are negatively impacted in patients irrespective of the symptomseverity during active SARS-CoV-2 infection2, 3. Furthermore, studies indicate senescence-associatedphenotypes and enrichment of aging signatures in infected cells and tissues, suggesting large-scaleuncharacterized cellular damage from SARS-CoV-24, 5.
Therefore, a molecular understanding of the SARS-CoV-2-dependent host-cell pathophysiology will aid in addressing and managing the COVID-19 diseasecourse. Previously, we reported that SARS-CoV-2 infection triggers an ATR (ataxia telangiectasia andRad3-related protein) DNA damage response (DDR) in Vero-E6 cells6. Typically, activated DDR serves as amolecular link to engage different DNA repair pathways or evoke translesion synthesis (TLS) to bypassirreparable damage7.
An engaged TLS pathway not only propels DNA mutagenesis but also regulatesmetabolic processes8–10. Dysregulation of these pathways causes genome instability, which can bephenotypically quantied as differential expression of molecules of these pathways and different geneticalterations at the DNA7.To test whether SARS-CoV-2 triggers genome instability, we rst quantied relative transcript levels of theDDR, DNA repair, and TLS genes at 48 hours post-infection in A549-ACE2 + cells. We found upregulationof the DDR genes6, 11 (ATM, ATR, including CHK1), in addition to the increased expression of speciedDNA repair genes from double-strand break repair (DSBR: BRCA1, MRE11A, PARP1, and RAD51),nucleotide excision repair (NER: XPA), and the major mutagenic TLS genes (POLh, POLk, POLi, REV1, and REV7) (Fig. 1A). Similarly, ATR expression was detected in the lung tissue of the Golden Syrian hamster at30 days post-SARS-CoV-2 infection (Supplementary Fig. 1A).
At the protein level, each factor exhibited aunique pattern of upregulation with the peak expression levels between 4 to 8 hours post-infection(Fig. 1B). Such a unique expression pattern was not observed in inuenza A virus-infected A549-ACE2 + cells (Supplementary Fig. 1C-D), where a different set of DDR genes (DDB2, DDB1, DDIT4, SMC5, etc.)were upregulated. Further, immunohistochemical analysis of the human autopsy COVID-19 lung tissuesshowed an increased expression of gH2AX compared to their PMI (post-mortem interval)-matchedcontrols (Fig. 1C and Supplementary Fig. 2), just as was observed in lung tissue of Golden Syrianhamster up to 30 days post-SARS-CoV-2 infection (Fig. 1D-E).
Interestingly, 53BP1, an importanttransducer of DNA damage and genome instability12, was highly expressed in the terminal bronchioles,but the overall expression in the surrounding lung tissue was less pronounced (Fig. 1C andSupplementary Fig. 2). Within a limited group of patients investigated at least three months followingacute COVID, longitudinal expression of 53BP1 at three intervals six months apart following the rst visitshowed a signicant decrease in expression in three of the ve patients (Fig. 1F).
These results suggestSARS-CoV-2 infection modulates the expression of genome instability markers in cells, autopsy lungtissues, Golden Syrian hamster lung tissue, and sera from post-COVID patients.Second, we assessed telomere dysfunction, an important genome instability marker13, by quantifyingtelomere length and expression of key telomere maintenance proteins.
We found signicant telomereinstability—marked by a reduction and lengthening of telomeres—in autopsy patient lung tissues, infectedA549-ACE2 + cells, and lung tissue of Golden Syrian hamster for 30 days post-SARS-CoV-2 infection(Supplementary Fig. 3A, 3B, and 4). Further, expression of the two shelterin proteins, TRF2 and POT1,which encapsulate telomeres into protective units, was signicantly repressed in autopsy lung tissuesand infected cells, in contrast to the elevated hTERT expression in infected A549-ACE2 + cells and thelung tissue of Golden Syrian hamster 30 days post-SARS-CoV-2 infection (Supplementary Fig. 1A, 2, and3C-3D).
Because different cell lines exhibited distinct telomere lengths, SARS-CoV-2 may be impacting thetelomere biology uniquely in different tissues.Since SARS-CoV-2 increases the expression of mutagenic TLS polymerases, we next tested a two-foldhypothesis: a) whether SARS-CoV-2-dependent increased TLS expression inadvertently causes host cellgenetic alterations, and b) whether inhibiting the TLS pathway diminishes the deleterious consequencesof SARS-CoV-2 infection. Figure 1G and Supplementary Fig. 5 show a 120% increase in mutationfrequency at the HPRT (hypoxanthine phosphoribosyltransferase) gene, suggesting a general increase inthe mutational burden in infected cells. Likewise, other mutability events, such as microsatellite instability(MSI), where insertions or deletions occur at a high frequency at repetitive DNA14, were high not only inA549-ACE2 + infected cells but also in most of the autopsy lung tissues compared to the PMI-matchedcontrols (Fig. 1H and Supplementary Fig. 6).
Furthermore, we observed a signicant reduction inexpression of the mismatch repair (MMR) proteins, MSH2, MLH1 and MSH6 (Fig. 1I and SupplementaryFig. 7A) in A549-ACE2 + cells infected with SARS-CoV-2. To determine MMR status in patients post-COVID,we tested the longitudinal expression of MSH2 protein in patient sera and found it to be signicantly reduced in two of the ve tested patients (Fig. 1J). Elevated MSI and decient MMR (dMMR) are ahallmark of certain cancers14; whether long-COVID patients with the said changes would be at risk forcancer needs further longitudinal analysis.
To determine whether TLS inhibition might suppress the noted mutagenic events, we tested whether TLSinhibitor, JH-RE-06, that specically targets the REV7 interface of REV1 TLS polymerase15, suppressesgenetic alterations in host cell DNA. Figure 2A and B show that JH-RE-06 treatment suppresses both theSARS-CoV-2-dependent HPRT mutagenesis and MSI in infected A549-ACE2 + cells, suggesting thatincreased expression of TLS polymerases indeed contributes to the elevation of mutagenic events andthat therapeutic inhibition of TLS can suppress SARS-CoV-2-dependent deleterious consequences.Encouraged by this result, we tested whether other genome instability markers were also repressed by theJH-RE-06 treatment in SARS-CoV-2 infected cells.
Figure 2C shows that JH-RE-06 treatment of the A549-ACE2 + cells suppressed transcript expression of all the DDR, TLS, and DNA repair genes. Likewise, theenhanced expression of gH2AX in SARS-CoV-2 infected A549-ACE2 + cells at 48 hours was suppressed byup to 40-fold post-JH-RE-06 treatment (Fig. 2D-2E and Supplementary Fig. 7B). Interestingly, JH-RE-06treatment did not rescue telomere instability in SARS-CoV-2 infected A549-ACE2 + cells (Fig. 2C andSupplementary Fig. 7C), suggesting that SARS-CoV-2 may impact telomere instability by an independentpathway.
Most unexpectedly, we observed that the compound JH-RE-06 was also able to directly suppress theproliferation of SARS-CoV-2 in three independent cell lines—Vero, A549-ACE2+, and Calu-3 cells as notedby the relative N content in cells (Fig. 2F, 2G and Supplementary Fig. 8). This surprising result of JH-RE-06-dependent suppression of SARS-CoV-2 proliferation was also observed in the STAT1KO cell line,suggesting independence from the immune pathway (Fig. 2H) and a possible role of REV1 in SARS-CoV-2propagation. Because siREV1 knockdown in A549-ACE2 + cells also suppressed SARS-CoV-2 propagation(Fig. 2I), we conclude that REV1 has a specic role in virus propagation in cells.
Because REV1 inhibitionwas recently shown to trigger autophagy10, we tested whether JH-RE-06 treatment induces autophagy tolimit SARS-CoV-2. On its own, SARS-CoV-2 infection steadily increases LC3 expression over time, withoutan increase in p62 (Fig. 2J and Supplementary Fig. 9A). However, JH-RE-06 treatment signicantlyincreases the expression of p62 and LC3 in SARS-CoV-2 infected cells (Fig. 2K and SupplementaryFig. 9B), indicating that JH-RE-06 treatment upregulates p62 expression that might promote lysosomaldegradation of SARS-CoV-2, limiting its propagation in cells.
Further studies are needed to delineate theexact mechanism by which JH-RE-06-dependent autophagy suppresses SARS-CoV-2 proliferation16.We next reexamined existing RNA sequence data because REV1’s engagement with viruses, particularlySARS-CoV-2, was unknown. We unexpectedly observed a gene enrichment for viral myocarditis in theREV1KO mouse embryonic broblasts17 (KEGG pathway mmu05416 from(https://www.genome.jp/kegg/pathway/hsa/hsa05416.html), which prompted us to test whether JH-RE-06 treatment might suppress one of the key factors, CASP9, involved in SARS-CoV-2-dependent increasein myocarditis18.
Figure 2E and Supplementary Fig. 9C show that treatment of cells with the JH-RE-06 inhibitor suppresses CASP9 expression, suggesting mechanisms of genome instability might associatewith myocarditis with therapeutic implications during long-COVID.While genome instability is considered a hallmark of some cancers and can be associated with otherhuman diseases, large-scale and long-term human studies are required to establish whether SARS-CoV-2infection will be a risk factor for developing these diseases.
For instance, RNA-viruses such as HTLV-1and HCV that are known to promote oncogenesis typically manifest over several years and rely on hostgenetic variability and environmental factors to develop cancer19. This study has some limitations: 1) thesample size for the clinical post-COVID specimens is low, 2) the follow-up period for the post-COVIDpatients is short when considering the time frame for carcinogenesis, and 3) the mechanisms of dMMRare unknown at the molecular.
Additionally, within the hamster animal model, at 60 days post-infection,when Nucleocapsid (N) expression dissipates, some genome instability markers, gH2AX, ATR, TERT, andtelomere length alterations, return to baseline levels (Supplementary Fig. 1A and 7D). However, theircharacterization in long-COVID patients remains. Collectively, we report that SARS-CoV-2 infection triggersgenome instability quantied as modulated expression of various biomarkers (DDR, DNA repair, andTLS), telomere instability, and enhanced host cell mutagenesis in cultured cells, hamster model, and post-COVID patients. Treatment of cells with a TLS inhibitor, JH-RE-06, reverses these phenotypes, suggestinga strong therapeutic potential for COVID-19