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ZBTB7A promotes virus-host homeostasis during human coronavirus 229E infection

The cellular fate after infection with human coronaviruses (HCoVs) is typically death. Previous data suggest, however, that the transcriptional state of an individual cell may sometimes allow additional outcomes of infection. Here, to probe the range of interactions a permissive cell type can have with a HCoV, we perform a CRISPR activation screen with HCoV-229E. The screen identified the transcription factor ZBTB7A, which strongly promotes cell survival after infection. Rather than suppressing viral infection, ZBTB7A upregulation allows the virus to induce a persistent infection and homeostatic state with the cell. We also find that control of oxidative stress is a primary driver of cellular survival during HCoV-229E infection. These data illustrate that, in addition to the nature of the infecting virus and the type of cell that it encounters, the cellular gene expression profile prior to infection can affect the eventual fate.


In brief
The fates of infected cells can affect viral pathogenesis. Zhu et al. show that the upregulation of ZBTB7A can convert a cytolytic human coronavirus infection to a persistent, homeostatic one. Continued work in this area may ultimately explain the broad range of clinical presentations associated with respiratory viral infections.

INTRODUCTION
Coronaviruses (CoVs) are a group of enveloped viruses with positive-stranded RNA genomes . They are divided into four genera, a-, b-, g-, and d-CoV, which belong to order Nidovirales, family Coronaviridae, and subfamily Coronavirinae (The International Committee on Taxonomy of Viruses). Currently seven CoVs are known to infect humans. Four of the human CoVs, HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1, are circulating seasonal pathogens and cause mild to moderate respiratory diseases. The three remaining HCoVs, severe acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome (MERS)-CoV, and SARS-CoV-2, are highly pathogenic and pose a severe threat to public health (Fehr and Perlman, 2015;Zhou et al., 2020).
It has been reported that HCoV infections induce cell death, resulting in tissue damage (Lee et al., 2020;Leist et al., 2020;Mangalmurti and Hunter, 2020). These phenomena have been described in some detail for the more severe respiratory HCoVs that infect the respiratory tract in humans: SARS-CoV-2 infection triggers cell death in airway epithelial cells (Li et al., 2020;Zhu et al., 2020), and SARS-CoV infection results in denudation of lung epithelial cells (Nicholls et al., 2003). While the magnitude of tissue damage with the seasonal HCoVs is less, the same general cytopathic effects have been observed both during in vitro and in vivo infections (Jacomy et al., 2006;Me-sel-Lemoine et al., 2012b;Milewska et al., 2014). There are, however, clinical manifestations of disease such as long-term asymptomatic shedders of virus (Agarwal et al., 2020;Avanzato et al., 2020) that suggest that not all infections induce rapid cell death, inflammation, and major tissue damage. In addition, at least with a murine coronavirus, some cells can resist virusinduced killing and intrinsically clear viral infection (Wheeler et al., 2017). These data together support the idea that the range of interactions between CoVs and the host cell may be broader than the exclusive killing of the infected cells. However, potential controllers of non-lytic death cell fates remain mostly undefined.
Genome-wide screening based on clustered regularly interspaced short palindromic repeats (CRISPR) is a powerful tool for the identification of host factors that affect viral infection (Shalem et al., 2015). Recently, multiple groups have performed both CRISPR gain-of-function and loss-of-function screens to identify essential genes and restriction factors for HCoV infections, with an emphasis on the host factors required for productive SARS-CoV-2 infection (Baggen et al., 2021;Biering et al., 2022;Daniloski et al., 2021;Danziger et al., 2022;Grodzki et al., 2022;Hoffmann et al., 2021;Israeli et al., 2022;Kratzel et al., 2021;Mac Kain et al., 2022;Rebendenne et al., 2021Rebendenne et al., , 2022Schneider et al., 2021;Sherman et al., 2022;Trimarco et al., 2021;Wang et al., 2021aWang et al., , 2021bWei et al., 2021;Zhu et al., 2021Zhu et al., , 2022. In order to define host factors that could alter infected cell fate, we decided to perform a CRISPR activation (CRISPRa) screen that would allow us to define host factors that by themselves are sufficient to alter CoV infection outcomes. CRISPRa is an approach based on a catalytically inactive Cas9 protein (dCas9) that recruits transcriptional activators to gene promoter regions by virtue of single guide RNA (sgRNA) targeting, leading transcriptional upregulation (Konermann et al., 2015). We hypothesized that, in addition to increasing our basic understanding of the range of interactions these viruses can have with their host cells, the identification of host factors regulating CoV-induced cell death might help predict disease susceptibilities or serve as potential therapeutic targets.
Using the normally highly cytopathic human seasonal HCoV-229E as a model contemporary CoV, we found that CRISPR activation screening almost exclusively enriched for the host transcription factor ZBTB7A. While upregulation of this factor dramatically limited HCoV-229E-induced cell death, further experiments revealed that ZBTB7A did not prevent infection but rather allowed the virus to induce homeostasis within the host cells in a quasi-persistent infection. To understand the mechanism by which ZBTB7A was allowing this phenotype, we performed RNA sequencing (RNA-seq) both before and during viral infection. While many cellular pathways were dysregulated by the upregulation of this transcription factor, we identified a number of oxidative stress-response genes induced by ZBTB7A. Further experimentation confirmed that control of oxidative damage during infection is a major contributor to cellular resistance of virus-induced killing. These data highlight the need for continued work understanding the true range of infection outcomes during authentic infection as well as how altering cellular fate may affect pathogenesis.

RESULTS
A CRISPR activation screen identifies host factors that prevent HCoV-229E-induced cell killing We were primarily interested in identifying the host factors that were sufficient for the avoidance of cellular death after HCoV infection. We therefore chose to perform a CRISPR activation screen with the expectation that we would enrich for general viral restriction factors as well as modulators of cell fate. For the virus, we chose the seasonal HCoV-229E as we have previously observed strong cytopathic effect in the human hepatoma Huh7 cell line (Trimarco et al., 2021); this allows us to apply a strong selection to the phenotypically altered population of cells. To perform the screen, catalytically inactive Cas9 (dCas9) was stably expressed in our Huh7 cell line and the Calabrese library (Sanson et al., 2018) of sgRNAs were introduced. After allowing sufficient time for gene upregulation, we infected the cells with HCoV-229E ( Figure 1A).
Early after infection, we observed dramatic cellular killing and failed to enrich for resistant cell populations of any appreciable size; we therefore allowed the few remaining cells to co-incubate with the virus for approximately seven additional weeks. Toward the end of this time, resistant populations that were actively proliferating and expanding could be observed. These cells were then collected, the gDNA was extracted, and the sgRNAs present were identified by Illumina sequencing. Despite high diversity of sgRNAs present prior to infection, only a handful of sgRNAs were detected after viral selection, suggesting that the upregulation of most cellular genes is irrelevant for avoidance of virally induced cell killing. One sgRNA predicted to upregulate zinc finger and BTB domain containing 7A (ZBTB7A), however, was strongly enriched in both replicates and represented over 99% of the sgRNA reads in one of the samples ( Figure 1B). After using the MAGeCK bioinformatic pipeline  for statistical analysis, we selected the seven genes with the lowest p values for subsequent testing: ZBTB7A, GEMIN7, SAA2-SAA4, CEBPB, POC5, USP22, and ZBTB7B ( Figure 1C; Table S1).
In order to avoid any potential CRISPR off-target effects during validation studies, we cloned the open reading frames (ORFs) for these seven genes and stably introduced them into Huh7 cells. Quantitative reverse transcription PCR (qRT-PCR) assays showed that the mRNA levels of all candidate genes were increased compared with the control Huh7 cells expressing mCherry ( Figure 1D). These cell lines were then infected with HCoV-229E and cellular viability was observed and quantified via a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay or crystal violet staining. The overexpression of three genes, ZBTB7A, ZBTB7B, and GEMIN7, improved cell viability during infection; however, the effect was by far the most dramatic after ZBTB7A upregulation ( Figures 1E, 1F, and S1).
ZBTB7A overexpression allows a quasi-persistent infection of Huh7 cells As we were unable to find previous reports of a role for ZBTB7A during viral infection, we decided to further characterize how this protein was protecting cells from virus-induced killing. First, we expanded upon our qRT-PCR data and confirmed ZBTB7A overexpression at the protein level ( Figure 2A). We next wanted to define whether the inhibition of cell killing was by restricting initial infection or preventing infected cell death. Immunofluorescence detection of the viral N protein after infection revealed that ZBTB7A upregulation only modestly reduced HCoV-229E infection ( Figure 2B). Further, viral RNA levels and infectious viral titer from ZBTB7A-Huh7 cells were similarly only slightly, but still significantly, reduced (Figures 2C and 2D). Similar experiments at different temperatures and over a time course revealed similar trends, with a reduction in infection observable early after infection ( Figures S2 and S3). To rule out cell-type-specific effects of ZBTB7A, human lung fibroblasts (MRC-5) and alveolar basal epithelial (A549) cells were transduced with lentivirus expressing ZBTB7A. HCoV-229E infection was again only modestly restricted, and ZBTB7A preserved most of the MRC-5 cells on the plate ( Figure S4). Infection was similarly restricted in A549 cells, but, as these cells do not normally undergo appreciable lytic death after HCoV-229E infection, we were unable to measure that phenotype ( Figure S5). These results together suggested that the effects of ZBTB7A upregulation are not restricted to Huh7 cells and that the cell-protective effects after HCoV-229E infection are not solely attributable to inhibition of viral infection.
Due to the limited direct antiviral effects of ZBTB7A overexpression, we hypothesized that the protein may instead be preventing infected cell death. We therefore conducted a long-term culture of HCoV-229E-infected cells and monitored cell death via flow cytometry. Indeed, we found that, while there was an initial dip in the percentage of live cells with ZBTB7A overexpression, cellular viability returned almost to pre-infection levels within a week post infection in contrast to the control cells, which were all killed by 4 days post infection (DPI) ( Figures 2E and S6). As these data could be explained by cell tolerance of infection or rapid cell intrinsic clearance of the virus, we next repeated the experiment but performed immunofluorescence microscopy for the viral N protein. Strikingly, we found that viral infection was continuously detected at similar levels from 1 to 15 DPI (Figure 2F). Intracelluar viral RNA and infectious virus in the supernatant of ZBTB7A-Huh7 cells also remained high across the entire time course (within $10-fold), although they were reduced relative to control at early time points ( Figure 2G).
While the virus was clearly maintained in the ZBTB7A overexpression cultures, we wanted to distinguish between viral ''persistence'' in infected cells and consistent low-level spread of virus followed by cellular killing. We therefore designed a co-culture experiment where ZBTB7A-overexpressing cells infected with HCoV-229E were transduced with lentivirus expressing eGFP, a reporter protein that did not affect HCoV-229Einduced cytopathic effect ( Figure S7). These infected fluorescent cells (or mock-infected fluorescent control cells) were then mixed with non-fluorescent, mock-infected ZBTB7A-overexpression cells at a 1:1 ratio and co-cultured for 23 days. If the virus was killing infected cells and spreading to new cells in the culture, we would expect the eGFP-positive cells to be eliminated from the cultures over time ( Figure 2H). The results showed that, while eGFP-positive cell numbers were slightly reduced early after co-culture, 8 days later, the proportion of eGFP-positive cells then mostly stabilized ( Figure 2I). The proportion of eGFP-positive cells in uninfected control similarly displayed no significant change over the time course ( Figure 2J). Finally, to demonstrate that infected cells can actively divide, we performed an 5-ethynyl-2'-deoxyuridine (EdU)-based proliferation assay and could detect label in cells positive for viral protein ( Figure S8). These data together support a model wherein ZBTB7A does not prevent or allow clearance of infection but at least partially allows the infected cells to better tolerate viral replication long term.
Our next question was whether this phenotype could simply be the avoidance of apoptosis, a cell death pathway known to be induced by CoV infection (Chu et al., 2016;Li et al., 2020). We first asked whether HCoV-229E infection activates the effector caspase proteins of the apoptosis pathway and whether ZBTB7A would affect that activation. Viral infection did indeed activate caspase 3/7, and ZBTB7A overexpression modestly but significantly reduced that activity ( Figure 2K). To directly test whether apoptosis suppression could recapitulate the ZBTB7A phenotype, we treated HCoV-229E-infected cells with the caspase inhibitors Ac-DEVD or Z-VAD and infected with HCoV-229E. While caspase 3/7 activity was suppressed by drug treatment (Figures 2L and 2M), we did not detect an effect on either viral infection or cell survival in Huh7 cells (Figures 2N-2P). Thus, while we could not formally rule out a minor role for inhibition of apoptosis in the ZBTB7A-mediated avoidance of viral killing, it did not appear to be the major driver of the phenotype.
ZBTB7A modulates gene pathways related to control of oxidative stress As a nuclear transcription factor, ZBTB7A has been reported to regulate a number of cellular pathways Pessler and Hernandez, 2003;Pittol et al., 2018). To identify genes differentially regulated by ZBTB7A that may explain the persistent infection phenotype, we compared gene expression between control and ZBTB7A-overexpression cells with and without HCoV-229E infection using RNA-seq ( Figure 3A and Table S2). We detected a slight reduction in viral RNA levels between control and ZBTB7A cells, consistent with our previous findings of a minor effect of ZBTB7A overexpression on viral replication ( Figure 3B). Turning our attention to the host transcriptional profile, we compared mock or virally infected-control and ZBTB7A-overexpression cells and observed hundreds of genes differentially regulated by ZBTB7A (Tables S3 and  S4). Gene Ontology analysis of the differentially regulated genes revealed that diverse metabolic processes were affected (Tables S5 and S6). When considering ZBTB7A-regulated transcriptional changes with an eye to understanding the viral homeostasis phenotype, we found that oxidative-stress-related genes and their associated pathways were upregulated in both mock-infected and infected samples (Figures 3C and 3D). Oxidative stress is frequently linked to inflammation and cell death processes (Imai et al., 2008;Xu et al., 2019), but not necessarily through the classical apoptosis pathway (Fiers et al., 1999;Gudipaty et al., 2018;Tait et al., 2014). As some of the oxidativestress-response-related genes upregulated by ZBTB7A, such as AKR1C3 and APOA4, can act as general controllers of the oxidative stress response (Qin et al., 1998;Xiong et al., 2014), we decided to focus on the control of oxidative damage as a potential explanation for our HCoV-229E infection phenotype.
First, we confirmed, via qRT-PCR, upregulation of the expected oxidative-response-related genes predicted by RNAseq ( Figure 3E). Next, we examined the expression of a panel of more canonical antioxidant enzymes and found that some, but not all, were also upregulated when ZBTB7A was overexpressed ( Figure 3F). To verify whether ZBTB7A regulates oxidative-response-related genes by direct promoter binding, we constructed a ZBTB7A-FLAG overexpression Huh7 cell line ( Figure 3G). As the FLAG-tag fusion at the N terminus did not affect the cell survival during the infection phenotype ( Figure 3H), we used this cell line to perform chromatin immunoprecipitation (ChIP) experiments. Previous research has shown that ZBTB7A functions as a widespread promoter-associated protein and can bind to thousands of genomic sites (Han et al., 2019;Pittol et al., 2018). Perhaps unexpectedly, we could find our tagged and overexpressed ZBTB7A enriched at promoters of a number of the upregulated oxidative-response-related genes; however, we did observe some specificity as the promoter for IFNA1 did not show a similar enrichment ( Figure 3I). Thus, while not ruling out indirect effects on gene transcription, these data are consistent with a direct role for ZBTB7A in promoting the transcription of antioxidant genes.

Control of oxidative stress contributes to cellular survival during HCoV-229E infection
We next tested whether the gene expression changes mediated by ZBTB7A overexpression would affect biological control of oxidative damage. Indeed, treatment of control or ZBTB7A-overexpression Huh7 cells with hydrogen peroxide (H 2 O 2 ) revealed an enhanced tolerance to oxidative damage when ZBTB7A was present (Figures 4A and 4B). Next, we tested whether HCoV-229E infection induced appreciable reactive oxygen (L) Caspase 3/7 activity assay after apoptosis-related inhibitor treatment. Huh7 cells were infected with HCoV-229E at 0.5 MOI and treated with 30 mM Z-VAD or 30 mM Ac-DEVD at 30 hpi. Samples were analyzed at 2 DPI. Images representative of n = 4 biological replicates. (M) Fluorescence intensity of caspase 3/7 activity calculated from the experiment shown in (L) via Image J. n = 4 biological replicates. (N) Viral RNA detection after caspase inhibitor treatment. Huh7 cells were infected and treated as described for (L). Cells were collected at 2 DPI for qRT-PCR. n = 4 biological replicates. ND, not detected. (O) Viral titer after caspase inhibitor treatment. Huh7 cells were infected and treated as described in (L). n = 4 biological replicates. (P) Cell viability after caspase inhibitor treatment. Huh7 cells were infected and treated as described in (L). MTT assays were conducted at 3 DPI. N = 5 biological replicates. All panels are representative of two independent experiments. Unless otherwise noted, p values were calculated by unpaired two-tailed Student's t tests. For all panels, *p < 0.05; **p < 0.001; ns, not significant. Scale bars, 100 mm. Data shown as mean ± SD.
Cell Reports 41, 111540, October 25, 2022 5 Article ll OPEN ACCESS species (ROS) in our experimental system. Staining for ROS via fluorogenic probe in the presence or absence of ZBTB7A revealed ROS were clearly induced by viral infection and dramati-cally suppressed by ZBTB7A (Figures 4C, 4D, and S9). This induction of ROS is consistent with reports from other CoV infection systems; SARS-CoV-2 causes pulmonary diseases   (Sawalha et al., 2020), and an ROS imbalance has been reported in SARS-CoV-infected mice (Nicholls et al., 2003).
To determine whether increased control of ROS was sufficient to prevent CoV-induced cell death, we utilized the superoxide scavengers tiron and trolox. As expected, tiron or trolox treatment both significantly decreased ROS activity during HCoV-229E infection (Figures 4E and 4F). While viral replication was only slightly affected by tiron or trolox treatment ( Figures 4G  and 4H), MTT assays and live cell imaging revealed that both treatments promoted cellular survival during infection at multiple time points (Figures 4I-4K). Staining for the HCoV-229E N protein revealed that, after tiron and trolox treatment, the live cells were also predominantly positive for viral antigen ( Figure 4L). We repeated experiments in MRC-5 cells and again found good concordance with the Huh7 experiments ( Figure S10). Thus, artificial suppression of ROS phenocopies ZBTB7A overexpression during HCoV-229E infection. Finally, as the previous experiments were done in the context of ZBTB7A overexpression, we tested the effects of endogenous levels of ZBTB7A. Huh7 cells were transfected with siRNA to reduce ZBTB7A transcript and protein levels ( Figures 4M and 4N). While knockdown of endogenous ZBTB7A did not affect HCoV-229E RNA levels or release of infectious particles, it promoted ROS activity and cell death after infection (Figures 4O-4R). These data together suggest that, while ZBTB7A activity is important for many cellular pathways, its ability to facilitate control of ROS signaling drives the uncoupling of HCoV-229E infection and cell death.

DISCUSSION
To fully understand the molecular basis of viral pathogenesis, a complete understanding of the potential outcomes of infection is necessary. In this study, we wanted to identify the host factors that could modulate CoV-induced cell death using a CRISPR activation screen. We identified that high levels of the host gene ZBTB7A could allow the establishment of a quasi-homeostasis between the virus and the infected cell, at least after HCoV-229E infection. Through RNA-seq and subsequent mechanistic analysis, we identified a number of ZBTB7A-regulated host pathways, with control of oxidative stress being particularly important for modulating host cell fate. While previous work with ZBTB7A has been primarily in the field of cancer biology (Jeon et al., 2008;Wang et al., 2013) and no links between ZBTB7A and viruses have previously been reported, it is clear that this host factor can alter the outcomes of infection.
While we have shown that control of ROS contributes to the ability of a cell to tolerate viral replication, how the full range of ZBTB7A-induced changes contributes to the viral/host homeostasis phenotypes remains incompletely answered. For example, ZBTB7A likely has differential effects on the various cell death pathways. We could not recapitulate the HCoV-229E cell survival phenotype with caspase inhibitors, suggesting that the virally/ROS-induced death pathway is at least partially caspase independent. This is consistent with previous reports showing a variety of cell death pathways are induced by HCoV infections (Lee et al., 2020;Mesel-Lemoine et al., 2012a); the relative contributions of different cell death pathways (and associated effects of ZBTB7A) during HCoV infections, however, remain an important area of future study. Additionally, while ROS inhibitor treatment increased cell survival, its effect did not fully recapitulate the ZBTB7A overexpression phenotype. We therefore believe it is likely that a number of alterations to cell physiology (in addition to the pathways that we tested) all contribute to the ability of the cell to harbor a normally lytic virus long term. By far, most of the pathways identified in our Gene Ontology analysis were related to metabolic alterations of the cell. We suspect that, in addition to avoiding cell death pathways and preventing oxidative damage, overall metabolic reprogramming of cells is likely required to balance the requirements of both the host and the virus for various metabolites.
Along those lines, we focused some efforts on understanding which ZBTB7A pathways may mediate the early suppression of infection. We found the alcohol dehydrogenase 1 (ADH1) gene family, including ADH1A, ADH1B, and ADH1C, were induced by ZBTB7A after viral infection (Table S2). It has been reported that expression of these genes is sufficient to complete the retinoic acid biosynthesis pathway in Huh7 cells and, interestingly, retinoic acid signaling has been reported to have antiviral activity (Cho et al., 2016;Liang et al., 2020;Trottier et al., 2008). Whether ZBTB7A does indeed mediate activation of retinoic acid metabolism and thereby contribute to the restriction of viral infection is an area that requires future study.
Perhaps the most important remaining question raised by this study is whether differences in physiological levels of ZBTB7A across different tissues or cell types truly modulate cell fate during a natural infection. ZBTB7A is expressed in virtually all tissues, with particularly high expression in the brain, lung, gastrointestinal (GI) tract, and lymphoid tissues (Thul et al., 2017;Uhlen et al., 2015), and therefore could be affecting cell fate during respiratory HCoV-229E infection. Whether or not there is any specific induction of the protein in cells after viral infection or innate immune activation remains an open question. It also remains unclear whether ZBTB7A levels mediate a dose-dependent effect on cellular survival, or if a threshold of expression is required for a binary survival versus the normal death fate. We suspect that the general activities of the protein normally establish a cellular environment that contributes to the amount of time that a cell can tolerate infection prior to death. However, it remains possible that cells that rapidly die or tolerate the virus for a disproportionately long time may do so in part because of fluctuations in the expression levels of ZBTB7A. The answers to these questions, as well as whether or not cellular fates after infection with other HCoVs are affected by ZBTB7A, will require additional investigation.
In summary, we have identified a previously unappreciated cellular fate after HCoV-229E infection, the establishment of a (R) Proportion of live cells after ZBTB7A knockdown. Huh7 cells treated as described in (O). Dead cells were stained at the indicated time points and analyzed by flow cytometry. n = 4 biological replicates. p values were calculated by two-way ANOVA. Unless otherwise indicated, all panels are representative of two independent experiments. For all panels, p values were calculated by unpaired two-tailed Student's t tests. *p < 0.05; **p < 0.001; ns, not significant. Scale bar, 100 mm. Data shown as mean ± SD. Article ll OPEN ACCESS quasi-homeostatic state with the host cell. This fate alteration is mediated by ZBTB7A, a host transcription factor, which broadly alters the host cell physiology but likely prevents cell death in no small part due to control of oxidative damage during infection. Future work understanding this, and similar processes, may ultimately allow for a mechanistic understanding of certain clinical manifestations of respiratory virus disease, such as asymptomatic long-term viral shedders. Such insights will undoubtedly not only increase our understanding of viral pathogenesis but may also identify next-generation intervention strategies.

Limitations of the study
In the current study, we reveal a role for ZBTB7A in virus-host interactions. Although the control of ROS regulated by ZBTB7A was vital for cell survival during infection, the extent that other effects mediated by ZBTB7A expression (e.g., metabolic reprogramming) are required to establish a virus/host balance is still unclear. Additionally, our study was mainly focused on HCoV-229E infection and was conducted in immortalized cell culture systems. Further study will be required to understand whether other coronaviruses are similarly affected and whether or not the phenotypes extend in vivo.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Nicholas S. Heaton (nicholas.heaton@duke.edu).

Materials availability
All plasmids and cell lines generated in this study are available from the lead contact upon request.
Data and code availability CRISPR sequencing data and RNA sequencing data have been deposited at NCBI GEO and are publicly available. Accession numbers are listed in the key resources table. This paper does not report original code and any additional information required to reanalyze the data reported in this work paper is available from the lead contact upon request.
Human coronavirus 229E (HCoV-229E), strain VR-740, was obtained from ATCC. Huh7 cells cultured in 15 cm dish were inoculated with 0.05 MOI HCoV-229E at 37 C. At 1-h post infection (hpi) the supernatant was replaced with 20 mL complete DMEM. The media supernatant was collected 72 hpi and stored in aliquots at À80 C. Plaque assay was conducted on Huh7 cells to calculate virus titer, as described previously (Trimarco et al., 2021).