BRD2 inhibition blocks SARS-CoV-2 infection by reducing transcription of the host cell receptor ACE2

SARS-CoV-2 infection of human cells is initiated by the binding of the viral Spike protein to its cell-surface receptor ACE2. We conducted a targeted CRISPRi screen to uncover druggable pathways controlling Spike protein binding to human cells. We found that the protein BRD2 is required for ACE2 transcription in human lung epithelial cells and cardiomyocytes, and BRD2 inhibitors currently evaluated in clinical trials potently block endogenous ACE2 expression and SARS-CoV-2 infection of human cells, including those of human nasal epithelia. Moreover, pharmacological BRD2 inhibition with the drug ABBV-744 inhibited SARS-CoV-2 replication in Syrian hamsters. We also found that BRD2 controls transcription of several other genes induced upon SARS-CoV-2 infection, including the interferon response, which in turn regulates the antiviral response. Together, our results pinpoint BRD2 as a potent and essential regulator of the host response to SARS-CoV-2 infection and highlight the potential of BRD2 as a novel therapeutic target for COVID-19.


Introduction
The ongoing COVID-19 pandemic is a public health emergency. As of September 2021, SARS-CoV-2, the novel coronavirus causing this disease, has infected over 200 million people worldwide, causing at least four and a half million deaths (https://covid19.who.int). New infections are still rapidly increasing despite current vaccination programs. The emergence of novel viral variants with the potential to partially overcome vaccine-elicited immunity highlights the need to elucidate the molecular mechanisms that underlie SARS-CoV-2 interactions with host cells to enable the development of therapeutics to treat and prevent COVID-19, complementing ongoing vaccination efforts.
SARS-CoV-2 entry into human cells is initiated by the interaction of the viral Spike protein with its receptor on the cell surface, Angiotensin-converting enzyme 2 (ACE2). To uncover new therapeutic targets targeting this step of SARS-CoV-2 infection, we conducted a focused CRISPR interference (CRISPRi)-based screen for modifiers of Spike binding to human cells. We expected that ACE2 and factors regulating ACE2 expression would be major hit genes in this screen. A second motivation for identifying regulators of ACE2 was the fact that ACE2 affects inflammatory responses and is itself regulated in the context of inflammation [1][2][3] . Inflammatory signaling, in particular the type I interferon response, is known to be misregulated in the most severely affected COVID-19 patients [4][5][6][7] . Therefore, regulators of ACE2 expression would likely be relevant for COVID-19 in human patients, as suggested by clinical data 8 .
Previous CRISPR screens have been performed in cell-based models of SARS-CoV-2 infection that overexpressed an ACE2 transgene 9,10 , represented cell types not primarily targeted by SARS-CoV-2 11 , or were non-human cells 12 . While these studies elucidated major features of SARS-CoV-2 biology, we reasoned that the cell lines used would not have enabled the discovery of regulators of ACE2 expression in relevant human cell types.
Here, we selected a lung epithelial cancer cell line, Calu-3, which endogenously expresses ACE2, to perform a targeted CRISPRi screen to find novel regulators of Spike protein binding. We found that the strongest hit genes are potent regulators of ACE2 levels. Knockdown of these genes reduced or increased ACE2 levels transcriptionally, and prevented or enhanced, respectively, SARS-CoV-2 infection in cell culture.
We identified the transcriptional regulator Bromodomain-containing protein 2 (Brd2) as a major node for host-SARS-CoV-2 interaction. Brd2 is part of the Bromodomain and Extra-terminal domain (BET) family of proteins that includes Brd3, Brd4 and BrdT. BETs are being explored as targets for a number of cancers 13 . These proteins are known to be master transcriptional regulators and serve to bridge chromatin marks (mostly acetyl-lysines) to the transcriptional machinery 14 . We found binding (ACE2 and BRD2), and three genes knockdown of which increased Spike-RBD-binding (CDC7, COMP and TRRAP).

Hit genes modulate ACE2 levels and affect infection with SARS-CoV-2
Since Spike-RBD binding is dependent on ACE2 expression as shown above, we hypothesized that other hit genes might act by modulating ACE2 levels. Western Blots for ACE2 levels in Calu-3 cell lines expressing sgRNAs against validated target genes (hereafter referred to as knockdown lines) indeed revealed marked changes in ACE2 protein levels. For hits associated with lower levels of Spike-RBD binding in the primary screen, we observed lower levels of ACE2 protein, and vice-versa for those hits associated with higher levels of Spike-RBD binding (Fig. 2a,b). To distinguish whether hit genes affected ACE2 protein levels via transcriptional or post-transcriptional mechanisms, we performed qPCR to measure ACE2 transcript levels in these same knockdown lines. For all tested genes, we observed changes in ACE2 transcript levels that were concordant with the changes in ACE2 protein levels (Fig. 2c), indicating that they acted on the transcriptional level. Some genes, such as COMP and TRAPP, showed relatively modest effects on ACE2 transcript levels, but quite large effects on ACE2 protein levels, suggesting that these hit genes additionally affect post-transcriptional regulation of ACE2 expression.
We next determined the effect of hit gene knockdown on susceptibility to SARS-CoV-2 infection. We infected cells expressing sgRNAs against hit genes with SARS-CoV-2 and measured virus replication 24, 48 and 72 hours post-infection using RT-qPCR (Fig. 2d). Already at 24 hours post-infection, viral genome copies diverged concordantly with changes in ACE2 levels and Spike-RBD binding: sgRNAs that lowered Spike-RBD binding reduced virus replication, while sgRNAs that increased Spike-RBD binding resulted in higher virus replication. BRD2 knockdown abrogated viral replication in these cells to similar levels as ACE2 knockdown, even at 72 hours post-infection, while COMP knockdown supported an order of magnitude increase in viral titers. Focusing on these three hit genes, ACE2, BRD2 and COMP, we then quantified how gene knockdown modulates Spike-RBD binding to cells (Fig 2e). Knockdown of ACE2 and BRD2 abolished spike binding, while COMP decreased the EC50 compared to WT from 9.72 nM (95% CI: 4.37 -22.42 nM) to 1.32nM (95% CI: 0.59 -3.20 nM), by almost a full order of magnitude (Fig. 2e). We also confirmed that gene knockdown decreased SARS-CoV-2 replication by performing plaque assays on WT, BRD2 KD, and ACE2 KD cells (Fig 2f).

BRD2 inhibitors prevent SARS-CoV-2 infection of human cells
Given the stringent inhibition of SARS-CoV-2 infection achieved by BRD2 knockdown, and the fact that BRD2 is currently being evaluated as therapeutic target in cancer 13,24 , with several small molecule inhibitors in clinical trials 25 , we decided to focus on this hit gene.
We validated that CRISPRi knockdown of BRD2 robustly reduced Brd2 protein levels (Extended Data Fig. 4). Transgenic expression of full-length Brd2 restored ACE2 transcript levels ( Fig.  3a), validating that the reduction in ACE2 expression triggered by CRISPRi targeting of BRD2 was indeed due to BRD2 knockdown. Transgenic expression of truncation mutants of BRD2 did not rescue ACE2 expression (Fig. 3a), indicating that full-length Brd2 is required for ACE2 expression.
To test the potential of Brd2 as a therapeutic target for COVID-19, we treated cells with a panel of compounds targeting BRD2: two BET domain inhibitors, (JQ1 26 and ABBV-744 27 , which is currently in clinical trials NCT03360006 and NCT04454658), and three Proteolysis Targeting Chimeric (PROTAC) compounds that lead to the degradation of Brd2 (dBET-6 28 , ARV-771 29 , and BETd-260 29 ). After only 24 hours of treatment with these drugs, ACE2 mRNA levels measured by qPCR decreased roughly two-fold (Fig. 3b). This effect was magnified after treatment for 72 hours, when almost no ACE2 mRNA was detectable for any of the Brd2-targeting compounds tested, phenocopying Brd2 knockdown (Fig. 3b). Similarly, we found that BET inhibitors led to substantial decreases in ACE2 mRNA levels in primary human bronchial epithelial cells (Fig. 3c) and human iPSC-derived cardiomyocytes (Fig. 3d), two non-transformed cell types that are susceptible to SARS-CoV-2 infection 30,31 . Importantly, BET inhibitors were non-toxic to Calu-3 cell, primary human bronchial epithelial cells, and cardiomyocytes at effective concentrations (Extended Data Fig. 5).
Since pharmacological inhibition of Brd2 phenocopied BRD2 knockdown, we hypothesized that these same compounds might prevent infection of cells exposed to SARS-CoV-2. To test this, we treated Calu-3 cells for 72 hours with the BET inhibitors JQ-1 and ABBV-744, and measured SARS-CoV-2 replication at 48 hours post infection. Strikingly, we found that treated cells displayed 100-fold decreased viral replication versus untreated cells (Fig. 3e), a similar effect size compared to BRD2 or ACE2 knockdown (Fig. 2d,e).

BRD2 regulates the transcription of ACE2 and other host genes induced by SARS-CoV-2 infection
We next asked whether Brd2 controls transcription of additional genes beyond ACE2. We performed RNA sequencing of Calu-3 cells after treatment with the BET-domain inhibitors JQ-1 and ABBV-744 as well as BRD2 CRISPRi knockdown (Extended Data Table 2). We also included CRISPRi knockdown of two other validated hit genes from our screen, COMP and ACE2 as well as over-expression of the viral protein E, which had been reported to interact with Brd2 32 . RNA-seq of BRD2 knockdown and BET domain inhibitor treated cells recapitulated downregulation of ACE2 (Fig.  4a). TMPRSS2, the gene encoding a protease important for viral entry in many cell types, was not a differentially expressed gene in any condition (Extended Data Table 2). Surprisingly, BRD2 knockdown or pharmacological inhibition also resulted in marked downregulation of genes involved in the type I interferon response, while ACE2 knockdown slightly increased expression of those same genes (Fig. 4a,b). Furthermore, the genes downregulated by both BRD2 knockdown and inhibition were strongly enriched in genes induced by SARS-CoV-2 infection in patient and cultured cells (Fig.  4c).
These findings are compatible with two distinct mechanisms: Brd2 could independently regulate ACE2 and SARS-CoV-2-induced interferon response genes, or Brd2 could mediate the response to interferon, which in turn regulates ACE2 transcription. ACE2 expression has been reported to be induced by interferons in some studies 1,2 . Other studies, however, suggest that interferon suppresses ACE2 expression 3 .
In Calu-3 cells, disruption of basal interferon signaling, via knockdown of the genes essential for interferon signal transduction IRF9, STAT1, or IFNAR1, abrogated ACE2 expression (Fig. 4d, Extended Data Fig. 6). Conversely, treatment with exogenous IFNβ stimulates ACE2 expression in a concentration dependent manner. Upon BRD2 knockdown, however, this concentration-dependent increase in ACE2 expression is inhibited (Fig. 4e). Thus, BRD2 is required for interferon-induced ACE2 expression. Treatment with IFNβ similarly strongly increased ACE2 mRNA levels in primary human bronchial epithelial cells, but reduced ACE2 mRNA levels in human iPSC-derived cardiomyocytes (Fig. 4f), suggesting that the effect of interferons on ACE2 can be context-dependent.
To test if Brd2 is a direct transcriptional regulator of ACE2, we performed CUT&RUN 33 to comprehensively map genomic loci bound by Brd2 in Calu-3 cells (Extended Data Table 3). CUT&RUN is similar to ChIP-seq, as it measures the occupancy of factors bound to DNA, but has the advantage of higher sensitivity and lower requirement for cell numbers 33 . Genes adjacent to Brd2bound sites detected in our experiment showed a highly significant overlap with Brd2-bound sites previously mapped by ChIP-seq in NCI-H23 34 cells, another lung epithelium-derived cancer cell line (Fig. 5a). To further validate our CUT&RUN analysis, we performed Binding and Expression Target Analysis 35 (BETA) to uncover direct BRD2 targets that were differentially expressed upon BRD2 knockdown, and identified several interferon response genes as direct BRD2 targets that were downregulated (Fig. 5b). We verified that a previously described 34 Brd2 binding side upstream of PVT1 was also detected in our experiment (Fig. 5c). We also mapped a Brd2 binding site upstream of the several interferon-stimulated genes (ISGs), including IRF9, STAT1, and MX1 (Fig. 5d). While there was some signal in the WT background at the ACE2 locus that is decreased in BRD2 knockdown cells, there were no peaks as determined by the peak calling algorithm (Fig 5e), suggesting that Brd2 is not a direct transcriptional regulator of ACE2 expression.
We also performed CUT&RUN for histone H2A.Z, which was previously reported to modulate the magnitude of ISG expression and thus connect Brd2 activity to interferon stimulation 36 . We found decreased H2A.Z occupancy at ISGs in BRD2 knockdown cells (Fig. 5d), recapitulating the role of Brd2 as a potential chaperone of H2A.Z. These results support a model in which BRD2 controls the transcription of key interferon response genes, which can in turn induce ACE2 transcription in some cell types (   Fig 5f). Alternatively, ACE2 expression may controlled by other genes that are expressed in a Brd2-dependent, interferon-stimulated manner (Fig. 5f).

Brd2 inhibitors rescue cytotoxicity and reduce SARS-CoV-2 infection in human nasal epithelia and inhibit SARS-CoV-2 infection in Syrian hamsters
We then tested if ABBV-744, a Bromodomain inhibitor in clinical trials, could reduce SARS-CoV-2 infection and infection-associated phenotypes in more physiological models.
First, we investigated a human nasal epithelial model 37 . We treated reconstituted nasal epithelia maintained in air/liquid interphase conditions with 100 nM and 300 nM ABBV-744 and performed SARS-CoV-2 or mock infections (Fig. 6a). First, we found that ABBV-744 treatment reduced ACE2 levels in these conditions (Fig. 6b). Apical supernatants did not show significant changes in viral RNA concentrations at two or four days post-infection (Extended Data Figure 7). Intracellular viral RNA concentrations, however, were significantly decreased in the ABBV-744 conditions (Fig. 6c). Furthermore, epithelial barrier integrity, as measured by trans-epithelial electrical resistance (Fig. 6d), and cytotoxicity (Fig. 6e), were rescued in infected cells treated with ABBV-744. Thus, ABBV-744 partially inhibited SARS-CoV-2 replication and fully rescued epithelial barrier integrity in a primary human nasal epithelial model.
Next, we tested if ABBV-744 could reduce SARS-CoV-2 infection in golden Syrian hamsters. Syrian hamsters provide a physiologically relevant model for SARS-CoV-2 infection, with high viral replication and signs of lung involvement [38][39][40][41] . After 24-hour treatment with ABBV-744 or vehicle, hamsters were infected with SARS-CoV-2 ( Fig. 6f) and treated daily with ABBV-744 or vehicle. Three days post-infection, the lungs of hamsters were harvested and subjected to RNA-seq. Infected, but untreated, hamsters showed marked up-regulation of a number of genes including ISGs when compared to uninfected controls (Fig. 6g). In contrast, infected hamsters treated with ABBV-744 showed a down-regulation of ISG ( Fig. 6h) levels relative to vehicle-treated infected hamsters, confirming ABBV-744 activity. Remarkably, viral RNA counts were reduced by about five orders of magnitude in the ABBV-744 treated hamsters versus those treated with vehicle controls (Fig. 6i). Thus, Brd2 inhibition can dramatically decrease SARS-CoV-2 infection in Syrian hamsters.

Discussion
Here, we demonstrate that Brd2 is necessary for ACE2 expression in a number of different SARS-CoV-2 relevant systems. We also found that treatment with ABBV-744, a bromodomain inhibitor, can reduce SARS-CoV-2 viral RNA concentrations in primary human nasal epithelial cells and Syrian hamsters. These findings suggest that pharmacological BRD2 inhibitors may be of therapeutic benefit to prevent or reduce the impact of SARS-CoV-2 infection.
Our data suggest that Brd2 is an indirect regulator of ACE2 transcription in COVID-19-relevant cell types. Our data show that Brd2 is required for interferon-mediated stimulation of ACE2 expression, as both exogenous interferon stimulation and basal interferon stimulation of ACE2 expression is blocked upon BRD2 knockdown or pharmacological inhibition (Fig. 6i). This does not, however, preclude a more direct, and interferon-independent, regulatory mode. Our data also show that Brd2 activity is essential for the transcription of ISGs in cell culture and in Syrian hamsters. Based on our findings and the previous literature 36 , Brd2 regulation of ISG transcription is likely mediated by a reduction in Histone H2A.Z occupancy at these promoters. Taken together, this indicates that BRD2 could be a key regulator of the host response to SARS-CoV-2 infection.
The previously described 42 interaction between the SARS-CoV-2 E protein and BRD2 might have evolved to manipulate gene expression during infection, including the expression of ACE2. In isolation, however, protein E overexpression in Calu-3 cells did not recapitulate expression changes resulting from BRD2 knockdown or inhibition (Fig 4a). These data suggest that there is no direct effect of Protein E on BRD2 function, or that other viral or host factors expressed during SARS-CoV-2 infection are required to modulate BRD2 function. Further studies are needed to define the function of the protein E-BRD2 interaction.
Several previous CRISPR screens aiming to uncover strategies to inhibit SARS-CoV-2 infection were carried out in cell lines in which an ACE2 transgene was overexpressed 9,10 ; these screens therefore failed to uncover BRD2 as a regulator of endogenous ACE2 expression. BRD2 did show a phenotype, however, in a CRISPR screen carried out in Vero-E6 cells (which express ACE2 endogenously) 12 , although it was not further characterized in that study. These differences highlight the importance of conducting CRISPR-based screens in disease-relevant cell types.
There is a growing literature about the relationship between COVID-19 disease severity, ACE2 expression, and interferon regulation 1-6 . Since ACE2 is known to promote recovery after lung injury and that SARS-CoV-2 manipulates the host interferon response [43][44][45] , the mis-regulation of these two pathways may play a major role in enhancing the severity of COVID-19. Our data suggest that Brd2 is central to this regulatory network and, therefore, pharmacological targeting of Brd2 may be a promising therapeutic strategy for the treatment of COVID-19: Brd2 inhibition could both block viral entry, through ACE2 downregulation, and act as an "emergency-brake" for mis-regulated patient immune responses to COVID-19, via down-regulation of ISGs.

Cell Culture
Calu HEK293 cell culture and production of lentivirus was performed as previously described 46 .
A vial of STR authenticated Caco-2 cells was obtained from the UCSF Cell and Genome Engineering Core (CGEC). Caco-2 cells were cultured in EMEM (ATCC,   Human iPSC-derived cardiomyocytes were generated and cultured as previously described 30 , from AICS90 iPSCs (Allen Institute Cell Catalog). Drugs were added on day 69 of differentiation, and cardiomyocytes were harvested for analysis on day 72.

Generation of the Calu-3 ACE2 knockout line
The polyclonal ACE2 knockout Calu-3 cell line was generated using the Gene KO kit V2 from Synthego, using three sgRNAs targeting ACE2 with the following protospacer sequences sRNA1: 5'-GACAUUCUCUUCAGUAAUAU-3', sgRNA2: 5'-AAACUUGUCCAAAAAUGUCU-3' and sgRNA3: 5'-UUACAGCAACAAGGCUGAGA-3'. Single guide RNAs (sgRNAs) were designed according to Synthego's multiguide gene knockout kit 47 . Briefly, two or three sgRNAs are bioinformatically designed to work in a cooperative manner to generate small, knockout causing, fragment deletions in early exons. These fragment deletions are larger than standard indels generated from single guides. The genomic repair patterns from a multiguide approach are highly predictable on the basis of the guide spacing and design constraints to limit off-targets, resulting in a higher probability protein knockout phenotype.
The ribonucleoprotein (RNP) complex with a ratio of 4.5 to 1 between sgRNA and Cas9 was delivered following the protocol of the SE Cell Line 4D-NucleofectorTM X Kit (Lonza, V4XC-1012), using the nucleofection program DS-130 on the Lonza 4D X unit. 72 hours post transfection, genomic DNA was extracted to serve as the template for PCR amplification of the region that covers the sites targeted by the sgRNAs with the following two primers: ACE2-F: 5'-CTGGGACTCCAAAATCAGGGA-3' and ACE2-R: 5'-CGCCCAACCCAAGTTCAAAG-3'. Sanger sequencing reactions using the sequencing primer ACE2-seq: 5'-CAAAATCAGGGATATGGAGGCAAACATC-3' were then performed, and the knockout efficiency was determined to be 80% via ICE software from Synthego 48 (https://ice.synthego.com/#/).

Spike-RBD binding assay
Recombinant biotinylated SARS-CoV-2 spike Spike-receptor-binding domain with a Cterminal human IgG Fc domain fusion (referred to as Spike-RBD) was prepared as previously described 50 . Calu-3 cells were grown in 96-well flat bottom plates until >50% confluent. Media was aspirated and cells were washed once with PBS. Cells were then treated with TrypLE to release them from the plate, RPMI 1640 media was added to dilute TrypLE, and cells were pelleted by centrifugation at 200xg for five minutes. From this point on, all steps were carried out on ice. Cells were incubated in 3% BSA (Sigma Aldrich A7030) in DPBS (Sigma-Aldrich D8537) for 15 minutes to block and washed twice in 3% BSA in DPBS by centrifugation at 200xg for five minutes in v-bottom plates, followed by resuspension. Spike-RBD was diluted in 3% BSA to appropriate concentrations and incubated with cells for 30 minutes on ice. Cells were then washed twice with 3% BSA in DPBS and incubated with Anti-Strep PE-Cy7 (Thermofisher SA1012) at 5 µg/mL. Cells were washed twice and subjected to flow cytometry on a FACS Celesta in HTS mode. Cells were gated to exclude doublets and the median PE-Cy7 signal was calculated for each sample. The gating strategy is shown in Supplemental Figure 2. EC 50 values and their 95% confidence intervals were calculated by fitting the RBD binding data into a Sigmoidal, 4PL model in Prism 6.

CRISPRi Screen
Calu-3 cells were infected with the H1 CRISPRi sgRNA library 22 as described 46 and selected using treatment with 1 µg/mL puromycin for 3 days. After selection, cells were stained with 10 nM Spike-RBD as described above or for TFRC as previously described 46 and subjected to FACS, where cells were sorted into top 30% and bottom 30% based on high and low expression of TFRC or Spike-RBD. Because of viability and stickiness known for Calu-3 cells, coverage was lower than optimal, at 200-fold over the library diversity. Sorted populations were spun down at 200xg for five minutes and genomic DNA was isolated as described 46 . sgRNA cassettes were amplified by PCR and sequencing and analysis was performed as described 46 but with an FDR of 0.1 rather than 0.05 or 0.01 due to noise.

Validation of screening hits
Individual sgRNAs were selected based on phenotypes in the primary screens and cloned into a lentiviral expression vector as described 46 . Protospacer sequences of these sgRNAs are provided in Extended Data Table 5. Cells expressing sgRNAs were selected using treatment with 1 µg/mL puromycin for 3-7 days.

Drug treatments
Drugs (ABBV-744 Selleckchem S8723, JQ1 -Sigma Aldrich SML1524, dBET6 -Selleckchem S8762) were dissolved in DMSO or water as per manufacturer's instructions. Cells were treated with drugs for 72 hours with media changes performed every 24 hours with media containing fresh drug.

Western Blotting
Cells from one confluent well of a six-well plate were lysed in RIPA buffer plus c0mplete EDTA-free protease inhibitor tablets (Roche 11873580001) and spun for 10 minutes at 21,000xg at 4ºC. The pellet was removed and a BCA assay (Thermofisher 23225) was performed on the remaining supernatant. Lysate volumes with equivalent protein content were diluted with SDS-PAGE loading dye and subjected to gel electrophoresis on 4-12% BisTris SDS-PAGE gels (Life Technologies NP0322) . Gels were then transferred and blocked in 5% NFDM for 1 hour at RT. Antibodies in fresh 5% NFDM were added (Mouse monoclonal GAPDH 1:10,000; Goat polyclonal ACE2 [R&D  Figure 1.

Virus
The SARS-CoV-2 strain used (BetaCoV/France/IDF0372/2020 strain) was propagated once in Vero-E6 cells and is a kind gift from the National Reference Centre for Respiratory Viruses at Institut Pasteur, Paris, originally supplied through the European Virus Archive goes Global platform.

Cytotoxicity measurements of Calu3 cells
30,000 Calu-3 cells per well were seeded into Greiner 96-well white bottom plates and incubated for 48 hours at 37°C, 5% CO 2 . Then, cells were treated with identical drug concentrations as in the infection assays for 5 days by refreshing the media with 100µL per well fresh drug-containing media every 24 hours. Cell viability was then assayed by adding 100µL per well of CellTiter-Glo 2.0 (Promega) and incubated for 10 minutes at room temperature. Luminescence was recorded with an Infinite 200 Pro plate reader (Tecan) using an integration time of 1s.

Virus infection assays
30,000 Calu-3 cells per well were seeded into 96-well plates and incubated for 48 hours at 37°C, 5% CO 2 . At the time of infection, the media was replaced with virus inoculum (MOI 0.1 PFU/cell) and incubated for one hour at 37°C, 5% CO 2 . Following the one-hour adsorption period, the inoculum was removed, replaced with fresh media, and cells incubated at 37°C, 5% CO 2 . 24h, 48h and 72h post infection, the cell culture supernatant was harvested, and viral load assessed by RT-qPCR as described previously 42 . Briefly, the cell culture supernatant was collected, heat inactivated at 95°C for 5 minutes and used for RT-qPCR analysis. SARS-CoV-2 specific primers targeting the N gene region: 5′-TAATCAGACAAGGAACTGATTA-3′ (Forward) and 5′-CGAAGGTGTGACTTCCATG-3′ (Reverse) were used with the Luna Universal One-Step RT-qPCR Kit (New England Biolabs) in an Applied Biosystems QuantStudio 6 thermocycler or an Applied Biosystems StepOnePlus system, with the following cycling conditions: 55°C for 10 min, 95°C for 1 minute, and 40 cycles of 95°C for 10 seconds, followed by 60°C for 1 minute. The number of viral genomes is expressed as PFU equivalents/mL, and was calculated by performing a standard curve with RNA derived from a viral stock with a known viral titer.

Plaque assays
Viruses were quantified by plaque-forming assays. For this, Vero E6 cells were seeded in 24well plates at a concentration of 1 × 10 cells per well. The following day, tenfold serial dilutions of individual virus samples in serum-free DMEM medium were added to infect the cells at 37 °C for 1 h. After the adsorption time, a solid agarose overlay (DMEM, 10% (v/v) PBS and 0.8% agarose) was added. The cells were incubated for a further 3 days prior to fixation with 4% formalin and visualization using crystal violet solution.

CUT&RUN
CUT&RUN was performed with 1 million Calu-3 cells. Cells were removed from the plate by treatment with Versene (Life Technologies 15040066) for 20 minutes and resuspended in fresh media. They were spun down and washed twice with DPBS before proceeding with the CUTANA CUT&RUN kit (Epicypher 14-0050). The experiment was performed with the included IgG and H3K4Me control antibodies and the BRD2 antibody (abcam 197865) as well as E.coli spike-in DNA according to the kit protocol.

SARS-CoV-2 infection of reconstructed human nasal epithelia
MucilAir TM , corresponding to reconstructed human nasal epithelium cultures differentiated in vitro for at least 4 weeks, were purchased from Epithelix (Saint-Julien-en-Genevois, France). The cultures were generated from pooled nasal tissues obtained from 14 human adult donors. Cultures were maintained in air/liquid interface (ALI) conditions in transwells with 700 µL of MucilAir TM medium (Epithelix) in the basal compartment, and kept at 37°C under a 5% CO2 atmosphere. SARS-CoV-2 infection was performed as previously described 37 . Briefly, the apical side of ALI cultures was washed 20 min at 37°C in Mucilair TM medium (+/-drug) to remove mucus. Cells were then incubated with 10 4 plaque-forming units (pfu) of the isolate BetaCoV/France/IDF00372/2020 (EVAg collection, Ref-SKU: 014V-03890; kindly provided by S. Van der Werf). The viral input was diluted in DMEM medium (+/-drug) to a final volume 100 µL, and left on the apical side for 4 h at 37°C. Control wells were mock-treated with DMEM medium (Gibco) for the same duration. Viral inputs were removed by washing twice with 200 µL of PBS (5 min at 37°C) and once with 200 µL Mucilair TM medium (20 min at 37°C). The basal medium was replaced every 2-3 days. Apical supernatants were harvested every 2-3 days by adding 200 µL of Mucilair TM medium on the apical side, with an incubation of 20 min at 37°C prior to collection.
For ABBV-774 treatment, cultures were pretreated for 4 days with 100 nM or 300 nM of the drug. For this pretreatment, ABBV-744 was added to an apical wash at day -4, and to the basal compartment from day -4 to day 0. The drug was then added on the apical side during viral adsorption at day 0, and then every 2-3 days to both the apical wash and the basal compartment throughout the infection.

Transepithelial electrical resistance (TEER) measurement
The apical side of transwell cultures was washed for 20 min at 37°C in Mucilair TM medium. Transwell were then transferred in a new 24-well plate and DMEM medium was added to both the apical (200 µL) and basal (700 µL) sides. The TEER was then measured using an Evom3 ohmmeter (World Precision Instruments).

Viral RNA quantification
Apical supernatants were stored at -80°C until thawing and were diluted 4-fold in PBS for quantification in a 96-well PCR plate. Supernatants were then inactivated for 20 min at 80°C. One µL of supernatant was directly added to 4µL of PCR reaction mix for SARS-CoV-2 RNA quantification.  4) intraperitoneally, and once anesthetized they were cervically dislocated and lung lobes collected in 1 mL of Trizol reagent. Tissues were homogenized in a Tissue lyser for 2 cycles of 40 seconds, spun down for 5 minutes at 8000g and supernatants stored at -80C for plaque assay or RNA extraction.

RNA extraction
RNA extraction was performed following instructions from the manufacturer of TRIzol reagent (Invitrogen). Briefly, 1/5 volume of chloroform was added to the lung supernatants in TRIzol, phases were separated by centrifugation and RNA was precipitated by overnight incubation with isopropanol at -20C. The RNA pellet was washed with ethanol 70% and resuspended in RNase-free water. RNA was quantified by nanodrop and resuspended to a final concentration of 100 ng/ul in water.

Data availability statement:
Source data for immunoblots are provided in Supplementary Fig. 1. Gating strategies for flow cytometry experiments are provided in Supplementary Fig. 2. Sequencing data are provided available on NCBI Gene Expression Omnibus (GEO) with the following accession numbers: GSE165025 (RNA sequencing data associated with Fig. 4), GSE182993 (CUT&RUN data associated with Fig. 5), and GSE182994 (RNA sequencing data associated with Fig. 6f-h. There are no restrictions on data availability.

Code availability statement:
Analysis of CRISPRi screen results was carried out using custom code (MAGeCK-iNC) developed in the Kampmann lab, which was previously described 46 and is freely available at https://kampmannlab.ucsf.edu/mageck-inc.   was performed to identify direct BRD2 targets that were differentially expressed upon BRD2 knockdown. Many interferon response genes were identified as direct BRD2 targets. Direct BRD2 targets that were downregulated upon BRD2 knockdown were analyzed by ENRICHR for enriched