The DNA glycosylase NEIL2 plays a vital role in combating SARS-CoV-2 infection

Compromised DNA repair capacity of individuals could play a critical role in the severity of SARS-CoV-2 infection-induced COVID-19. We therefore analyzed the expression of DNA repair genes in publicly available transcriptomic datasets of COVID-19 patients and found that the level of NEIL2, an oxidized base specific mammalian DNA glycosylase, is particularly low in the lungs of COVID-19 patients displaying severe symptoms. Downregulation of pulmonary NEIL2 in CoV-2-permissive animals and postmortem COVID-19 patients validated these results. To investigate the potential roles of NEIL2 in CoV-2 pathogenesis, we infected Neil2-null (Neil2−/−) mice with a mouse-adapted CoV-2 strain and found that Neil2−/− mice suffered more severe viral infection concomitant with increased expression of proinflammatory genes, which resulted in an enhanced mortality rate of 80%, up from 20% for the age matched Neil2+/+ cohorts. We also found that infected animals accumulated a significant amount of damage in their lung DNA. Surprisingly, recombinant NEIL2 delivered into permissive A549-ACE2 cells significantly decreased viral replication. Toward better understanding the mechanistic basis of how NEIL2 plays such a protective role against CoV-2 infection, we determined that NEIL2 specifically binds to the 5’-UTR of SARS-CoV-2 genomic RNA and blocks protein synthesis. Together, our data suggest that NEIL2 plays a previously unidentified role in regulating CoV-2-induced pathogenesis, via inhibiting viral replication and preventing exacerbated proinflammatory responses, and also via its well-established role of repairing host genome damage.

Recently, several studies have indicated that cytokine storm and oxidative stress contribute to the severe outcome of COVID-19 patients (21)(22)(23)(24)(25). Such hyperin ammation and oxidative stress can generate an excessive level of reactive oxygen species (ROS), which consequently cause damage to various cellular macromolecules, including oxidative genome damage that is primarily repaired via the base excision

Results
The low-level expression of NEIL2 in COVID-19 patients correlates with the severity of disease SARS-CoV-2 infection-induced expression of soluble in ammatory mediators increase in ux of in ammatory cells (macrophages, T cells, and, occasionally, neutrophils) to the site of infection, leading to uncontrolled in ammation, pulmonary endothelial leakage, and impairing lung function (7,8,37). CoV-2 infection and host in ammatory responses also generate ROS that are not only signal transducers but are also inducers of host genome damage, thereby triggering a DNA damage response (37). However, in the absence of any report on the mechanistic link between CoV-2 infection and host genome repair, we analyzed RNA-seq data obtained from the bronchoalveolar lavage uids (BALFs) of the individuals suffering from severe and mild COVID-19, along with healthy individuals as controls that are available in public database (GSE 145926). Surprisingly, the level of NEIL2, among other DNA BER/SSBR proteins, was found to be signi cantly lower in severe COVID-19 patients relative to that of the control population ( Fig. 1a-d and Supplementary Fig. 1a, b). Such striking ndings of the transcriptomic pro le in the BALF specimen were validated in three other independent datasets with whole blood transcriptomics (GSE150728, GSE152641 and GSE161777). It was determined that downregulation of NEIL2 took place primarily in the monocyte/macrophage lineages ( Fig. 1e-g and Supplementary Fig. 1c). Importantly, downregulation of NEIL2 correlated well with disease severity, including the patients that failed to recover from COVID-19 ( Fig. 1a-d and 1g). Comparative expression pro ling of uninfected vs. SARS-CoV-2 infected lung epithelial cells (Calu3, GSE147507) also displayed a signi cant decrease in NEIL2 transcript levels post infection (Fig. 1h). Consistent with decreased levels of transcripts, a signi cant reduction in NEIL2 protein was also observed in the SARS-CoV-2-infected lungs, particularly in alveolar epithelial cells, compared to healthy controls (Fig. 1i,j) as analyzed by Immunohistochemical (IHC) analysis of para nembedded lung specimens of COVID-19 patients.
We next investigated the expression of NEIL2 at mRNA and protein levels in SARS-CoV-2-permissive golden Syrian hamsters (38). Lung specimens were harvested at 5 days post infection (dpi) with SARS-CoV-2 (1 × 10 6 TCID 50 ), when weight loss in the animals reached its peak, for the subsequent assessment of the expression of DNA glycosylases. We found that the expression of NEIL2 protein ( Supplementary Fig. 2a,b) was signi cantly low in infected lungs, compared to uninfected controls. Decreased mRNA levels of NEIL2, but not of OGG1, another oxidized DNA base repair enzyme as control, within the infected lung was further assessed by using real-time quantitative PCR (RT-qPCR) ( Supplementary Fig. 2c). To further con rm these observations, immunoblots were performed for assessing the expression of several BER proteins in the nuclear extracts of uninfected and SARS-CoV-2 infected lungs of hamster at 10 dpi. Again, NEIL2 remains to be the only protein whose expression was signi cantly downregulated upon SARS-CoV-2 infection; in contrast to the expression of other BER proteins, such as OGG1, NEIL1 and AP-endonuclase1 (APE1), which were largely unchanged ( Supplementary Fig. 2d). We reported earlier that the loss of NEIL2 leads to signi cant accumulation of oxidative DNA damage in the animal model and cultured cells (33)(34)(35), we thus analyzed DNA damage accumulation in the lungs of SARS-CoV-2-infected vs. uninfected hamsters using Long Amplicon-based qPCR (LA-qPCR) (39). Indeed, SARS-CoV-2-infected animals showed a signi cant increase in DNA damage accumulation ( Supplementary Fig. 2e), consistent with the outcome of a decreased level of NEIL2 in those animals (33). Together, these data suggest a close link between decreased NEIL2 expression and the severe outcome of SARS-CoV-2 infection.
Correlation of NEIL2 levels and prognosis of COVID-19 based on patient's sex or age Next, we investigated the prognostic potential of NEIL2 expression for COVID-19 severity, such as the need for ICU admission and the use of mechanical ventilator (MV), in COVID-19 and non-COVID-19 patient populations. Patients requiring ICU or MV had considerably lower levels of NEIL2 than non-ICU or non-MV patients (Fig. 2a,b); however, no such relationship was found for other DNA glycosylases, such as OGG1 and NEIL3 ( Supplementary Fig. 3a,b). Moreover, among hospitalized COVID-19 patients, females aged 40 years or less had signi cantly higher NEIL2 levels ( Fig. 2c) which coincided with their shorter duration of hospitalization compared to males in the same age group (Fig. 2d). These ndings support the notion that the sex disparity in COVID-19-related severity/deaths puts males at a greater risk, and that this risk markedly increases with age in both sexes (40). In the case of OGG1/NEIL3, no such age/sexspeci c trends were observed ( Supplementary Fig. 3c,d). Furthermore, a study involving 100 hospitalized COVID-19 patients showed a signi cant correlation between higher NEIL2 levels and a shorter duration of hospitalization (Fig. 2e), unlike OGG1 and NEIL3 levels, which showed no correlation to hospital stay ( Supplementary Fig. 3e,f). Collectively, these observations again support a strong link between NEIL2 de ciency and COVID-19 severity.
Increased morbidity and mortality of Neil2 -/mice upon SARS-CoV-2/MA10 infection The results presented above led us to hypothesize that low levels of NEIL2 play a critical role in an exacerbated outcome of SARS-CoV-2 infection. To test the biological signi cance of NEIL2 in COVID-19 pathogenesis, we utilized the Neil2 -/mouse model developed in our lab (33). Neil2 -/and Neil2 +/+ mice were infected with the mouse-adapted SARS-CoV-2-MA10 (CoV-2/MA10) that could productively infect mice resulting in weight loss and mortality in an age-dependent manner (41), providing an opportunity to explore the impact of NEIL2 on viral infection. Six to seven months old Neil2 +/+ and Neil2 -/mice were infected intranasally (i.n) with 1x10 5 TICD 50 of CoV-2/MA10 strain and were monitored daily for the onset of morbidity (i.e., weight changes and other signs of illness) and mortality (if any). Those animals reaching to the end-stage of clinical disease (>20% weight loss) were euthanized to assess the yields of infectious progeny virus within the lungs. We noted that infected Neil2 -/mice were losing weight more rapidly than the Neil2 +/+ cohort (Fig. 3a) concomitant with the onset of other signs of illness (Fig. 3b).
More importantly, 80% of Neil2 -/mice succumbed to infection within 4-6 days compared to 20% mortality in Neil2 +/+ mice (Fig. 3c). Additionally, yields of infectious virus within the lung of Neil2 -/mice were signi cantly higher (~ 20-fold) than that of Neil2 +/+ mice at 2 dpi (Fig. 3d). Furthermore, a signi cant decrease in NEIL2, but not OGG1 or NEIL1, transcript was observed in the lungs of CoV-2/MA10 infected Neil2 +/+ mice ( Supplementary Fig. 4a-c), which is consistent with our nding in SARS-CoV-2 infected permissive hamster model. Such a decrease in expression of NEIL2 at RNA level was subsequently con rmed by the immunoblotting which showed that the levels of NEIL2 protein in infected Neil2 +/+ mice were signi cantly decreased compared to uninfected controls ( Supplementary Fig. 4d). As reduced NEIL2 expressions usually resulted in increased phosphorylated γH2AX (pSer139), a sensitive marker of double strand breaks (39), we investigated whether such a reverse correlation of the expression of NEIL2 and γH2AX could occur in SARS-CoV-2 infected mice. We indeed observed a signi cant increase in γH2AX ( Supplementary Fig. 4d), thereby implying increased DNA double strand break accumulation in the lungs of CoV-2/MA10 infected mice. We validated the increased amount of DNA strand-break accumulation in CoV2/MA10 infected mouse lung using LA-qPCR analysis (Fig. 3e). Of note, we could not measure DNA damage in Neil2 -/mice because those (80%) died within 5 days. Collectively, these results suggest that excessive genome damage due to decreased NEIL2 level contributes to exacerbation of SARS-CoV-2 infection in COVID-19.
CoV2/MA10-infection induced in ammatory responses in Neil2 -/mice Given that NEIL2 is signi cantly downregulated in SARS-CoV-2 infected patients, and in both hamster and mouse models, we conducted a multiplex RT-qPCR analysis of 84 in ammation-associated genes in the lungs of CoV-2/MA10 infected Neil2 -/vs. Neil2 +/+ mice. As shown in Fig. 4a, infected Neil2 -/lungs had elevated (more than 2-fold) expression of ~47 genes and decreased expression of ~8 genes, compared to infected Neil2 +/+ lungs. Interestingly, a signi cant decrease was observed in the anti-viral cytokine IFNγ (p=0.0345). Fig. 4b shows differential expression of some of the critical cytokines in Neil2 -/vs. Neil2 +/+ mice that were found to be highly expressed in severe COVID-19 patients. The multiplex assay was further validated for a subset of signi cantly altered genes using RT-qPCR (Fig. 4c). Collectively, these data imply that NEIL2 de ciency or downregulation following viral infection plays a signi cant role in the host immune response and organ damage that are commonly linked with severe COVID-19.
Given that NEIL2 is an anti-in ammatory protein and in view of emerging success of protein therapy, we tested if exogenously added recombinant NEIL2 (rNEIL2) can limit CoV2-infection induced in ammatory responses. A549 cells expressing angiotensin converting enzyme-2 (ACE-2), the receptor for the SARS-CoV-2 viral entry (42), were transduced with rNEIL2, rNEIL1 or mock as control, and then infected with SARS-CoV-2 at the MOI of 1 for 24 h. TNFα, IL6 and IL1β mRNA levels were all signi cantly reduced in rNEIL2 vs. mock transduced cells (Fig. 4d). In contrast, rNEIL1 transduced cells showed no signi cant change in mRNA levels compared to control cells. Surprisingly, we discovered that rNEIL2 transduced cells had fewer viral progeny (Fig. 4e) and lower viral E-gene expression ( Supplementary Fig. 5), as compared to mock or rNEIL1 transduced cells. Similarly, overexpressing NEIL2 in human gastric adenocarcinoma, AGS cells infected with the human coronavirus 229E strain signi cantly decreased IL6 transcript levels (Fig. 4f, left panel), and displayed lower levels of viral E-gene transcript (Fig. 4f, right panel), in comparison to infected control vector expressing cells. All these ndings clearly suggest that NEIL2 plays a protective antiviral role against SARS-CoV-2 using multiple mechanisms.

NEIL2 interacts with 5'-UTR of SARS-CoV-2 RNA
Like other b-CoVs, SARS-CoV-2 possesses a long RNA genome anked by 5'-and 3'-untranslated regions (UTRs), containing regulatory cis-acting elements and very stable secondary RNA structure, essential for translation and RNA synthesis (43)(44)(45). Several host proteins interact with the 5'-and 3'-UTRs of viral RNA either to facilitate or hinder viral protein and RNA synthesis (43,(45)(46)(47)(48). The suppression of viral progeny and E-gene expression in the presence of rNEIL2 prompted us to test whether NEIL2 is directly involved in the regulation of the viral life cycle via its interaction with CoV-2 RNA. We cloned the 5'-UTR of SARS-CoV-2 mRNA upstream of Green Fluorescence protein (eGFP) in the pcDNA3.1 vector (CoV2-5'-UTR-eGFP, Fig  5a) and transfected into human lung epithelial BEAS-2B cells, stably expressing FLAG-tagged NEIL2 (NEIL2-FLAG). Sixteen hours post transfection, the cell lysates were subjected to RNA chromatin immunoprecipitation (RNA-ChIP) using anti-FLAG antibody, followed by RT-qPCR analysis. Indeed, we detected strong association of NEIL2 with full length SARS-CoV-2 5'-UTR, but not with the 5'-UTR of several host genes; GAPDH, HPRT and DNA polymerase β (POLB) as controls in the RNA-ChIP analysis ( Fig. 5b). Control reactions without reverse transcriptase ruled out the possibility of DNA contamination in the samples ( Supplementary Fig. 6a).

NEIL2 suppresses SARS-CoV-2-5'-UTR-mediated protein expression
To examine the regulatory function of NEIL2 in viral protein expression, we transfected SARS-CoV-2-5'-UTR-eGFP plasmid (Fig. 4a) or UTR-Less-eGFP plasmid into NEIL2-FLAG overexpressing or control BEAS-2B cells; and GFP uorescence was analyzed as a measure of expression of the protein, 12-16 h post transfection. Both SARS-CoV-2-5'-UTR-eGFP and UTR-less-eGFP plasmid transfected cells showed comparable GFP DNA (as a measure of transfection e ciency) in NEIL2 overexpressing vs. control cells as analyzed by qPCR ( Supplementary Fig. 7a). Intriguingly, we observed that the GFP expression was signi cantly decreased at the protein level (Fig. 6a), but not at the transcript level ( Supplementary Fig. 7b) in NEIL2 overexpressing cells compared to control cells, when transfected with the SARS-CoV-2-5'-UTR-eGFP construct. However, no signi cant change in GFP expression was observed between control or NEIL2 overexpressing cells transfected with UTR-Less-eGFP construct (Fig. 6b). Furthermore, siRNA mediated NEIL2 depletion ( Supplementary Fig. 7c) in HEK-293 cells resulted in signi cantly higher expression of GFP in cells transfected with SARS-CoV-2-5'-UTR-eGFP construct compared to control siRNA-treated (siControl) cells (Fig. 6c), while the UTR-Less-eGFP plasmid transfected into NEIL2-de cient (siNeil2) cells showed only a modest decrease in GFP expression (Fig. 6d). Collectively, these data suggest that NEIL2 binds to 5'-UTR of CoV-2 RNA and blocks the translational machinery and thus, decreased GFP expression. However, in the absence of NEIL2, the 5'-UTR was readily accessible to the host protein synthesis machinery resulting in increased GFP expression. All these data strongly suggest a role of NEIL2 in inhibition of SARS-CoV-2 protein synthesis.

Discussion
Dysregulation and often exacerbation of immune responses caused by viral infections could result in severe tissue damage, eventually leading to multiorgan failure and death. The excessive reactive oxygen species generated as a result cause damage to genomic DNA and activate DNA damage response (DDR) pathways (52,53). We thus postulated that individuals with compromised DNA repair capacity would be more prone to severe CoV-2 infection. However, to date, there is no report describing the linkage between SARS-CoV-2 infection and host genome damage-induced signaling, or the role of DNA repair proteins therein. Here we report that the level of the DNA glycosylase, NEIL2, is signi cantly low at both transcript and protein levels in severe COVID-19 patients. We further investigated the role of NEIL2 in SARS-CoV-2 infection and pathogenicity using permissive animal models. Using the mouse adaptive strain of SARS-CoV-2, CoV-2/MA10, which captures various aspects of severe COVID-19 disease such as elevated cytokines, the loss of pulmonary function linked to ARDS, and the spectrum of morbidity and mortality of COVID-19 disease in an age dependent manner (41), we found that CoV-2/MA10 infected Neil2 −/− mice had higher viral load, signi cant weight loss and mortality within 6-7 days. CoV-2/MA10 infection also signi cantly decreased NEIL2 levels with a concomitant increase in DNA damage in the lungs of wild type mice compared to uninfected animals. SARS-CoV-2 infected golden Syrian hamsters also showed decreased expression of NEIL2 and higher DNA damage. These observations are in accordance with earlier studies that Neil2 −/− mice accumulate signi cant amount of DNA damage (33).
In addition to the canonical function of repairing genome damage, the work presented here also elucidated two non-canonical functions of NEIL2 that can explain its protective role against SARS-CoV-2 infection. We recently reported that NEIL2 acts as a repressor of NF-κB, a transcriptional activator of proin ammatory genes (36). We have demonstrated that expression of in ammatory genes is signi cantly higher in Neil2 −/− mice; however, intrapulmonary delivery of rNEIL2 prevented TNFα-induced NF-κB recruitment to the promoters of cytokine genes both in vitro and in vivo (36). Similarly, we found that transduction of rNEIL2 in A549-ACE2 cells signi cantly inhibited SARS-CoV-2 induced TNFα, IL6 and IL1β expression, further con rming anti-in ammatory role of NEIL2. Therefore, NEIL2 is able to mitigate the viral-induced 'cytokine storm' in the host by acting as a repressor of proin ammatory gene expression.
The success of viruses as pathogens depends on their ability to actively reprogram the host cell antiviral defense mechanisms. Activation of antiviral innate immune signaling cascade generally begins with recognition of viral genomes by intracellular pattern recognition receptors (54,55) or by a set of zinc nger proteins (ZFPs), such as zinc-nger antiviral proteins (ZAP and PARP13), monocyte chemoattractant protein 1-induced protein 1 (MCPIP1) and ZCCHC7 that detect viral RNAs and elicit subsequent antiviral responses. In most known cases, these ZFPs recruit both the 5′-and 3′-mRNA decay machinery to degrade the target RNAs (56-60). Suppression of viral replication can occur via translational repression of its own proteins, as has been shown for in uenza A virus NS1 mRNA by zinc nger protein 36 (58). ZAP also has an inhibitory effect on the viral translation by disrupting the interaction between eIF4G and eIF4A (61). Similarly, we provide evidence here that NEIL2, a ZFP (62), also directly interacts with the CoV-2 5'-UTR and blocks protein synthesis. This data is in accordance with previous reports showing that host protein impedes viral protein synthesis via binding to 5'-or 3'-UTRs of viral RNA (58, 61). The translational initiation for SARS-CoV-2 RNA is not completely understood. Some reports suggest that the translation mechanism of SARS-CoV-2 RNA is independent of cap-binding translation factors (eIF4E and eIF4F) (43). Also, the CoV-2-5'-UTR is rich in GC content and is capable of forming internal ribosome entry sites to recruit host ribosomes for translating its RNA (63). We postulate that NEIL2 binding to the 5'-UTR will inhibit ribosome entry or interfere with the assembly of translational machinery and inhibit viral protein synthesis, which warrants further investigation in the future.
The interplay between the virus and many host factors plays critical roles in determining the nal outcomes of viral infection. Here, we show that NEIL2 is an important host factor for providing protection against SARS-CoV-2 infection. Based on the work presented here, we propose that if the levels of NEIL2 in hosts are low, or the virus is able to signi cantly diminish the levels of NEIL2, there is a greater chance for successful viral life cycle. In support of this notion, we found a correlation between NEIL2 levels and differences in age or sex associated with the risk of severe COVID-19. Additionally, lower levels of NEIL2 not only correlated with a longer period of hospitalization but also with higher instances of admission to ICU or the requirement of MVs among patients with severe disease. Moreover, several studies pointed towards the activation of NF-κB-mediated in ammation to explain the importance of age and sex in COVID-19 severity (40, 64-66). The NF-κB pathway induces a pro-in ammatory phenotype, known as in amm-aging, in older patients that is associated with increased levels of oxidative stress, thus driving the sustained levels of in ammation and DNA damage leading to cellular DDR, and further expression of IL-6 (64, 65). It is imperative that NEIL2 coordinates with other host factors to mount a defense against viral infection. Future experiments are required to delineate the coordination between NEIL2 and different host factors. Collectively, here we demonstrate multi-faceted functions of the host DNA repair enzyme NEIL2, where it unconventionally regulates COVID-19 pathogenesis, by decreasing host in ammatory response, inhibiting CoV-2 replication, and repairing host genome damage, thereby mitigating disease severity. Finally, the ability of rNEIL2 to neutralize the effect of SARS-CoV-2 in cultured cells, suggests that it has a strong therapeutic potential as a biologic against COVID-19.

Analysis of RNASeq Datasets
Publicly available COVID-19 gene expression databases were downloaded from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus website (GEO) (67-69). If the dataset was not normalized, RMA (Robust Multichip Average) (70,71) was used for microarrays and TPM (Transcripts Per Millions) (72,73) was used for RNASeq data for normalization. We used log2 (TPM+1) to compute the nal log-reduced expression values for RNASeq data. Accession numbers for these crowd sourced datasets are provided in the gures and manuscript. Single Cell RNASeq data from GSE145926 was downloaded from GEO in the HDF5 Feature Barcode Matrix Format. The ltered barcode data matrix was processed using Seurat v3 R package (74). Pseudo bulk analysis of GSE145926 data was performed by adding counts from the different cell subtypes and normalized using log2 (CPM+1). All of the above datasets were processed using the Hegemon data analysis framework (75)(76)(77).

Immunohistochemistry (IHC)
COVID-19 samples were inactivated by storing in 10% formalin for 2 days and then transferred to zincformalin solution for another 3 days. The deactivated tissues were transferred to 70% ethanol and cassettes were prepared for tissue sectioning. The slides containing hamster and human lung tissue sections were de-para nized in xylene (Sigma-Aldrich, catalog no. 534056) and rehydrated in graded alcohols to water. For NEIL2 antigen retrieval, slides were immersed in Tris-EDTA buffer (pH 9.0) and boiled for 10 minutes at 100°C inside a pressure cooker. Endogenous peroxidase activity was blocked by incubation with 3% H 2 O 2 for 10 minutes. To block non-speci c protein binding 2.5% goat serum (Vector Laboratories, catalog no. MP-7401) was added. Tissues were then incubated with rabbit anti-NEIL2 polyclonal antibody (in house, 62) for 1.5 h at room temperature in a humidi ed chamber and then rinsed with TBS or PBS 3x, 5 minutes each. Sections were incubated with horse anti-rabbit IgG (Vector Laboratories, catalog no. MP-7401) secondary antibodies for 30 minutes at room temperature and then washed with TBS or PBS 3x, 5 minutes each; incubated with DAB (3,3'-diaminobenzidine tetrahydrochloride) (Thermo Scienti c, catalog no. 34002), counterstained with hematoxylin (Sigma-Aldrich, catalog no. MHS1) for 30 seconds, dehydrated in graded alcohols, cleared in xylene, and cover slipped. Epithelial and stromal components of the lung tissue were identi ed by staining duplicate slides in parallel with hematoxylin and eosin (Sigma-Aldrich, catalog no. E4009) and visualizing by Leica DM1000 LED (Leica Microsystems, Germany).

IHC Quanti cation
IHC images were randomly sampled at different 300x300 pixel regions of interest (ROI). The ROIs were analyzed using IHC Pro ler (78). IHC Pro ler uses a spectral deconvolution method of DAB/hematoxylin color spectra by using optimized optical density vectors of the color deconvolution plugin for proper separation of the DAB color spectra. The histogram of the DAB intensity was divided into 4 zones: high positive (0 to 60), positive (61 to 120), low positive (121 to 180) and negative (181 to 235). High positive, positive, and low positive percentages were combined to compute the nal percentage positive for each ROI. The range of values for the percent positive is compared among different experimental groups.
Lung tissue specimens from the rapid autopsy procedure Lung specimens from COVID-19 positive human subjects were collected using autopsy procedures at the University of California San Diego (the study was IRB Exempt) following guidelines from the CDC and CAP autopsy committee. All donations to this trial were obtained after telephone consent followed by written email con rmation with next of kin/power of attorney per California state law (no in-person visitation could be allowed into the COVID-19 ICU during the pandemic).

Animals
Lung samples were collected from 8 week-old Syrian hamsters post SARS-CoV-2 infection conducted exactly as in a previously published study (38). Brie y, lungs from hamsters challenged with SARS-CoV-2 (1 × 10 6 PFU) were harvested on day 5 (peak weight loss) and NEIL2 protein and mRNA levels were analyzed by IHC and quantitative Polymerase Chain Reaction (qPCR), respectively. Nuclear extract was prepared from the uninfected and infected hamsters lungs at 10 days post infection, and DNA was extracted from the same samples for Long Amplicon qPCR (LA-qPCR). The generation of Neil2 -/mice (C57BL/6J congenic) background was described previously (33). Six-month-old, Neil2 +/+ and Neil2 -/-(16 each) mice were challenged with 1x10 5 TCID 50 mouse adapted strain of SARS-CoV-2 MA10 (CoV2/MA10) and observed daily for body weight change, mortality and clinical score/wellbeing. Clinical wellbeing of mice was scored based on a 1-4 standardized grading system. Score 1 is healthy; score 2 is with ru ed fur and lethargic; score 3 is with additional clinical sign such as hunched posture, orbital tightening, increased respiratory rate, and/or > 15% weight loss; score 4 is showing dyspnea, reluctance to move when stimulated, or ≥ 20% weight loss that needs immediate euthanasia. Six mice from each group were euthanized at 2 day post-infection to assess the lung viral load by TCID 50 .
Cell culture and Transient transfection Human bronchial epithelium cell line, BEAS2B (ATCC® CRL-9609™) stably expressing NEIL2-FLAG and human embryonic kidney cells (HEK293; 81) were grown at 37°C and 5 % CO2 in DMEM/F-12 (1:1) containing 10 % fetal bovine serum, 100 units/ml penicillin and 100 units/ml streptomycin. For all experiments, 50-60 % con uent cells were used. We routinely test mycoplasma contaminations in all our cell lines using the PCR-based Venor™ GeM Mycoplasma Detection Kit (Sigma, catalog no. MP0025). Control or stable BEAS-2B cells at approximately 70 % con uency were transiently transfected with vector expressing GFP with (SARS-CoV2-5'-UTR-eGFP construct) or without (UTR-Less-eGFP construct) UTR (100 ng) using Lipofectamine TM 2000, according to the supplier's protocol. To monitor transfection e ciency, a reporter gene construct (0.25 µg) containing β-galactosidase downstream to the SV40 promoter was cotransfected. Cells were allowed to recover for 16 h in media with serum and then GFP orescence was measured using an ECHO orescent microscope. Total RNA and DNA were isolated for subsequent qPCR analysis.
Gene expression with real time-qPCR (RT-qPCR) Total RNA extraction was performed from cells using TRIzol™ Reagent (Invitrogen™, catalog no. 15596026). Genomic DNA was removed and up to 2 μg RNA was used to synthesize cDNA with a PrimeScriptTM RT Kit with gDNA Eraser (TaKaRa, catalog no. RR047A). qPCR was carried out using TB Green™ Premix Ex Taq™ II (Tli RNase H Plus; TaKaRa, catalog no. RR820A) in Applied Biosystems™ 7500 Real-Time PCR Systems with thermal cycling conditions of 94°C for 5 min, (94°C for 10 s, and 60°C for 1 min) for 40 cycles, and 60°C for 5 min. The target mRNA levels were normalized to that of Gapdh. In each case, DNase-treated RNA samples without reverse transcriptase were used to rule out genomic DNA contamination.

RNA Chromatin immunoprecipitation and quantitative PCR (RNA-ChIP)
RNA-ChIP assays were performed as described earlier (39). Brie y, cells were cross-linked in 1% formaldehyde for 10 min at room temperature. Then 125 mM Glycine was added to stop crosslinking and samples were incubated for 5 min at room temperature. Samples then were centrifuged at 1000 xg at 4°C for 5 min to pellet the cells. The cells were incubated in buffer A (5 mM HEPES, 85 mM KCl, 0.5% NP-40 and 1X Protease inhibitor cocktail (Roche, catalog no. 4693132001)) for 10 min at 4 °C, then washed once with buffer B (buffer A minus NP-40) at 2,500 xg for 5 min to pellet the nucleus. The nuclear pellet was re-suspended in sonication buffer containing 50 mM Tris-HCl pH 8.0, 10 mM EDTA and 1% SDS with 1X Protease inhibitor cocktail and sonicated to an average DNA size of ~300 bp using a sonicator (Qsonica Sonicators). The supernatants were diluted with 15 mM Tris-HCl pH 8.0, 1.0 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.01% SDS and protease inhibitors, and incubated with ChIP grade anti-FLAG (Millipore, catalog no. F1804) or normal IgG (Santa Cruz, sc-2025) antibodies overnight at 4°C. Immunocomplexes (ICs) were captured by Protein A/G PLUS agarose beads (Santa Cruz, catalog no. sc-2003) that were then washed sequentially in buffer I (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton-X-100 and 0.1 % SDS); buffer II (same as buffer I, except containing 500 mM NaCl); buffer III (1% NP-40, 1% sodium deoxycholate, 10 mM Tris-HCl pH 8.0, 1 mM EDTA); and nally with 1X Tris-EDTA (pH 8.0) buffer at 4°C for 5 min each. 50 U ml −1 of RNase inhibitor (Roche, catalog no. 03335402001) was added to buffers A and B, sonication and IP buffers, and 40 U ml −1 to each wash buffer. The ICs were extracted from the beads with elution buffer (1% SDS and 100 mM NaHCO 3 ) and de-crosslinked for 2 h at 65°C. RNA isolation was carried out in acidic phenol-chloroform followed by ethanol precipitation with GlycoBlue (Life Technologies, catalog no. AM9516) as a carrier. Genomic DNA was removed and reverse transcription was performed using a PrimeScript RT Kit with gDNA Eraser (TaKaRa, catlog no. RR047A). RNA-ChIP samples were analyzed by qPCR using speci c primers. qPCR data are represented as percentage input after normalization to IgG.
The novel Corona virus nsp12 (GenBank: MN908947) gene, cloned into a modi ed pET 24b vector, with the C-terminus possessing a 10 × His-tag, was a gift from Dr. Whitney Yin. The plasmid was transformed into E. coli BL21 (DE3), and the transformed cells were cultured at 37 °C in LB media containing 100 mg/L ampicillin. After the OD 600 reached 0.8, the culture was cooled to 16 °C and supplemented with 0.5 mM IPTG. After overnight induction, the cells were harvested through centrifugation, and the pellets were re-suspended in lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 4 mM MgCl2, 10% glycerol). The rest of the procedure is same as above with following modi cations: the His-tagged protein was eluted with an imidazole gradient (80-250 mM imidazole in buffer containing 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 4 mM MgCl 2 , 10 % glycerol). Similarly, nsp7 and nsp8 genes, individually cloned in pET22b and pET30a+ vectors, respectively, were expressed in E. coli as described in case of NEIL proteins. After elution, the peak protein fractions of these proteins were dialyzed against Buffer D (20 mM Tris-HCl, pH 8.0, 250 mM NaCl, 1 mM DTT, 25% glycerol) and stored at -20°C in aliquots.
For the RdRp assay, the 5'-monophosphorylated RNA templates (the portion of the template which is complementary to the 4-nt primer is underlined, Supplementary Table)  Sequences of the oligonucleotide (oligo) probes used for RNA-EMSAs are listed in Supplementary Fig 3D. RNA-EMSA was performed as described before (83), with some modi cations. Brie y, [α-32 P]ATP labelled RNA oligo probes were incubated with 150-300 ng of puri ed protein in a binding buffer containing 10 mM Tris-Cl buffer (pH 7.6), 15 mM KCl, 5 mM MgCl 2 , 0.1 mM DTT, 10 units of RNase inhibitor, 1 mg BSA, and 0.2 mg/ml yeast tRNA in a 10 μl reaction volume. After a 15-min incubation on ice, 100 mg/ml of heparin was added and incubated for 10 min. RNA-protein complexes were resolved on a 5 % nondenaturing polyacrylamide gel at 120 V using 0.5x Tris-borate-EDTA as the running buffer at 4°C. Gels were xed in an Acetone: Methanol: H 2 O (10:50:40) solution for 10 min, exposed to a Phosphor screen for 12-16 h and scanned using Typhoon FLA 7000 phosphorimager.
Long Amplicon qPCR (LA-qPCR) assay Lung tissues from freshly euthanized uninfected and SARS-CoV-2 infected hamsters and mice were used for DNA damage analysis. Genomic DNA was extracted using the Genomic tip 20/G kit (Qiagen) per the manufacturer's protocol, to ensure minimal DNA oxidation during the isolation steps. The DNA was quantitated by Pico Green (Molecular Probes) in a black-bottomed 96-well plate and gene-speci c LA qPCR assays were performed as described earlier (39) using Long Amp Taq DNA Polymerase (New England BioLabs). The LA-qPCR reaction was set for all genes from the same stock of diluted genomic DNA sample, to avoid variations in PCR ampli cation during sample preparation. Preliminary optimization of the assays was performed to ensure the linearity of PCR ampli cation with respect to the number of cycles and DNA concentration (10-15 ng). The nal PCR reaction conditions were optimized at 94°C for 30 s; (94°C for 30 s, 55-60°C for 30 s depending on the oligo annealing temperature, 65°C for 10 min) for 25 cycles; 65°C for 10 min. Since ampli cation of a small region is independent of DNA damage, a small DNA fragment (~200-500 bp) from the corresponding gene(s) was also ampli ed for normalization of ampli cation of the large fragment. The ampli ed products were then visualized on gels and quantitated with ImageJ software (NIH). The extent of damage was calculated in terms of relative band intensity with the uninfected control mice/hamster sample considered as 100.