SARS-CoV-2 engages replication and inflammasome activation through lipid remodeling via SREBPs

SARS-CoV-2 engages replication and inflammasome activation through lipid remodeling via SREBPs | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article SARS-CoV-2 engages replication and inflammasome activation through lipid remodeling via SREBPs Patrícia Bozza, Vinicius Soares, Suelen Dias, Julia Santos, Isaclaudia Azevedo-Quintanilha, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2338983/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract SARS-CoV-2 and other ssRNA + viruses induce major cellular lipid rearrangements, exploiting the host's metabolic pathways to replicate. Sterol regulatory-element binding proteins (SREBPs) are a family of transcription factors that control lipid metabolism. SREBP1 is associated with the regulation of fatty acid metabolism, while SREBP2 controls cholesterol metabolism, and both isoforms are associated with lipid droplet (LD) biogenesis. SARS-CoV-2 infection has been shown to increase the expression and activation of SREBPs, but the impact of this pathway on the infection outcome is still poorly explored. Here, we evaluated the effect of pharmacologic and molecular inhibition of SREBP1 and SREBP2 in a SARS-CoV-2-infected lung epithelial cell line (Calu-3). We showed that SARS-CoV-2 infection induced the expression and activation of SREBP1 and SREBP2, enzymes of lipid metabolism and LD accumulation. Partial inhibition of SARS-CoV-2 replication and cell death was observed with the genetic knockdown of SREBP1 or SREBP2, while combined SREBP1 and SREBP2 knockdown led to synergistic inhibition. Combined SREBP1 and SREBP2 knockdown inhibited DGAT-1 expression and abrogated SARS-CoV-2-triggered LD formation in Calu-3 cells. Moreover, blockage of LD biogenesis by DGAT1 siRNA inhibited SARS-CoV-2 replication and cell death. Pharmacological inhibition with the dual SREBP activation inhibitor fatostatin reduced virus replication, cell death and LD biogenesis. In addition, we demonstrated that SARS-CoV-2 induced cell death by pyroptosis, with activation of caspase-1, cleavage of gasdermin D1 and release of IL-1β and IL-18 depending on SREBP activation. Collectively, our findings help to elucidate that SREBPs are crucial host factors required for viral replication, LD biogenesis and inflammasome activation and indicate SREBP as a host target for the development of antiviral strategies. Biological sciences/Immunology/Infection Health sciences/Pathogenesis/Infection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 6 Introduction As the worldwide pandemic of coronavirus disease 2019 (COVID-19) enters its third year with more than 630 million cases worldwide, it still poses significant challenges for patients, families and health systems. SARS-CoV-2, like other viruses, are obligate intracellular pathogens that make use of the host's cellular metabolic machinery to meet their biosynthetic needs. A better understanding of the host factors and pathways used by SARS-CoV-2, potentially common to other viruses, which are essential for execution of their life cycles, could contribute to potential targets for therapeutic intervention, such as broad-spectrum antiviral agents, to the development of therapies to treat COVID-19 and increase preparedness for potential future outbreaks. Lipids are essential in viral infection, as they are the structural basis of cell membranes and viral envelopes 1 . Accordingly, SARS-CoV-2 infection triggers major lipid metabolism remodeling in human cells 2 – 5 . SREBP is a family of transcription factors associated with the regulation of lipid homeostasis, controlling the expression of a broad range of enzymes of fatty acid (SREBP1) and cholesterol (SREBP2) metabolism. Both isoforms of SREBP are found to increase during viral infections, as observed in HCV, MERS-CoV, and SARS-CoV-2 2,6−8 . Indeed, increased expression and activation of the SREBP pathway are associated with disease severity in COVID-19 patients 3 , 8 . Moreover, SREBP activation is associated with the immune response through induced assembly of the inflammasome complex with the release of IL-1β 9 . In this study, we demonstrated that SARS-CoV-2 modulates lipid metabolism in Calu-3 cells by activating the SREBP transcription factor, favoring lipid remodeling through an increase in triglycerides and cholesterol, leading to the accumulation of LDs. Furthermore, double gene knockdown and the pharmacological inhibition of SREBPs with fatostatin blocked viral replication and proinflammatory cytokines, such as IL-1β and IL-18. In addition, SREBPs inhibition reduced caspase-1 activation and prevented cell death induced by SARS-CoV-2 infection. Our results reveal new details of SARS-CoV-2 infection on lipid metabolism and may supply new insights for understanding the pathology of COVID-19. Results SARS-CoV-2 induces alterations in lipid metabolism Viruses are intracellular parasites that can alter cell metabolism to favor their own maintenance and replication. Members of the Flaviviridae family 10 – 14 and the Coronaviridae family 2 , 6 are + RNA viruses that can modify lipid metabolism in different cells, triggering lipid droplet (LD) formation, using these host organelles for different steps of their replicative cycle 2 . To evaluate the effects of SARS-CoV-2 infection on lipid metabolism, we used type II pneumocytes (Calu-3 cells) infected with SARS-CoV-2 at an MOI of 0.01. Here, we observed decreased expression of the precursor form and augmented activation (mature form) of the lipid transcription factors SREBP1 and SREBP2 24 h after infection with SARS-CoV-2 in Calu-3 cells (Fig. 1 A). SREBP1 is involved in fatty acid metabolism, while SREBP2 controls cholesterol homeostasis 15 , 16 . Accordingly, we observed that SARS-CoV-2 infection upregulated pathways of LD biogenesis and maturation, such as DGAT1 and PLIN2, as well as pathways of both fatty acid (FASN) and cholesterol synthesis (ABCA1 and SOAT1) (Fig. 1 B and C). Moreover, SARS-CoV-2 infection upregulated inflammatory pathways, with increases in IL-6, IL-1β and IL-10 (Fig. 1 C). As previously observed in other cells 2 , SARS-CoV-2 infection of Calu-3 cells induced LD biogenesis (Fig. 1 D and E), with increased cholesterol (Fig. 1 F and G) and triacylglycerol accumulation (Fig. 1 H) after 48 hours of infection. Altogether, SARS-CoV-2 infection of Calu-3 cells triggered SREBP activation along with upregulation of genes and key proteins of lipid metabolism and increased cholesterol and triacylglycerol, which accumulated in LDs (Fig. 1 I). Srebfs Are Master Regulators Of Lipid Metabolism During Sars-cov-2 Infection To investigate the functions of SREBPs in SARS-CoV-2 infection, we knocked down SREBF1 and SREBF2 with siRNA separately or in combination. The efficiency of the knockdown was confirmed by western blotting (Fig. S1A and S1B) and real-time PCR (Fig. S1D and S1E). Knockdown of SREBF1 and − 2 separately partially inhibited viral replication (Fig. 2 A) but failed to protect Calu-3 cells from death (Fig. 2 B). Combined knockdown of both SREBFs demonstrated a significant reduction in viral replication in relation to the control group with scramble (Fig. 2 A) and protected against cell death, decreasing LDH release into the supernatant (Fig. 2 B). These data suggest that both SREBPs are important for SARS-CoV-2 infection and replication in Calu-3 cells. To gain insights into the contributions and mechanisms of SREBP1 and SREBP2 during SARS-CoV-2 infection in Calu-3 cells, we analyzed the expression of different genes involved in lipid metabolism regulated during SARS-CoV-2 infection after SREBFs knockdown. SREBF1 knockdown downregulated genes related to LD formation, such as DGAT1 and FASN, and inflammatory genes, such as IL-1β (Fig. 2 C, and S2A and B). SREBF2 knockdown downregulated genes involved in cholesterol metabolism, such as SOAT1 and inflammatory genes (Fig. 2 C, and S2A and B). Moreover, the combined knockdown of SREBF1 and − 2 downregulated all the genes previously observed (Fig. 2 C, and S2A and B). Altogether, these data reinforce the concept that both genes that encode SREBPs are important and complementary to modulate the lipid and inflammatory profile during SARS-CoV-2 infection. Previous findings have established an important role for LDs in SARS-CoV-2 infection at least in part due to their roles in the biogenesis of replication organelles 2 , 17 . To evaluate the role of SREBPs in LD formation, Calu-3 cells were stained with a Lipidtox LD probe and J2 antibody for double-stranded RNA (dsRNA) labeling, and the fluorescent area of each marker was quantified. SREBF1, but not SREBF2 knockdown, was able to significantly reduce viral replication sites and LD accumulation (Fig. 2 D-F). The combined knockdown of SREBF1 and − 2 was more effective in reducing the SARS-CoV-2 replication sites and LD accumulation compared with the cells infected with scramble (Fig. 2 D-F). Moreover, knockdown of the DGAT1 enzyme was evaluated, and we observed a reduction in LD biogenesis and viral replication sites, comparable to the effects of combined knockdown for SREBF1 and SREBF2 (Fig. 2 A, B and D-F). Altogether, these results indicate that during SARS-CoV-2 infection, the genes that encode the SREBPs are master regulators of lipid metabolism and participate in different processes, such as inflammatory cytokines, cell death, viral replication, and LD accumulation, in Calu-3 cells. Indeed, the mechanisms that control DGAT1 and LD accumulation are downstream and largely dependent on the activation and transcriptional regulation of SREBPs. Fatostatin Inhibits Both Srebps Activation And Reduces The Replication Sites Of Sars-cov-2 To further analyze the role of the SREBPs during SARS-CoV-2 infection, we used fatostatin, a pharmacologic inhibitor of the ER-Golgi translocation of SREBPs through binding to their escort protein, SREBP cleavage-activating protein (SCAP) 18 . Of note, activation of all SREBP isoforms is controlled by SCAP. Fatostatin has been demonstrated to inhibit the viral replication of several viruses from the Flaviviridae family, including WNV and ZIKV 19 . First, we investigated whether fatostatin inhibits SREBPs in our model. We pretreated Calu-3 cells with fatostatin for 2 h before SARS-CoV-2 infection and maintained the treatment for all times of infection until the analyses of the experiment. Fatostatin treatment inhibited the processing and activation of both SREBP1 and SREBP2 during infection, leading to the accumulation of the precursor form of SREBPs in Calu-3 cells (Fig. 3 A and B). Furthermore, the blockage of SREBP1 and SREBP2 activation by treatment with fatostatin during SARS-CoV-2 infection reduced cholesterol accumulation (Fig. 3 C and D), as observed by filipin III labeling and triacylglycerol accumulation (Fig. 3 E). As shown in Fig. S3A, treatment with fatostatin was devoid of cytotoxicity at the doses used. To confirm that the inhibition of SREBPs by fatostatin treatment could affect LD accumulation, Calu-3 cells were pretreated with fatostatin, and LD accumulation was analyzed 48 h after infection. As shown in Fig. 3 , treatment with fatostatin reduced LD accumulation after SARS-CoV-2 infection, similar to the DGAT1 inhibitor A922500 (Fig. 3 F and G), as previously observed in other cell types 2 . Indeed, we observed a reduction in the protein expression related to the LD form and maturation (DGAT1 and PLIN2) in Calu-3 cells treated with fatostatin (Fig. 3 H and I). Thus, this confirms that SREBPs are master regulators in the process of LD accumulation in Calu-3 cells infected with SARS-CoV-2 through the increase in the DGAT1 enzyme and PLIN2, favoring LD biogenesis. SARS-CoV-2 may explore host lipid metabolism to favor its replication using LDs as an energy source for its own replication 2 , 17 . To investigate the role of SREBP in the formation of SARS-CoV-2 replication sites, we labeled Calu-3 cells with a J2 clone for dsRNA and BODIPY for LD and quantified the labeled area of each marker. First, we observed an increase in dsRNA and BODIPY during SARS-CoV-2 infection, and when we treated the cells with fatostatin or A922500, we observed a reduction in the labeled area (Fig. 4 A and B). Moreover, almost all cells were positive for dsRNA when they were infected with SARS-CoV-2, and both treatments reduced the number of dsRNA-positive cells up to 20% (Fig. 4 C). Considering the double-positive cells (dsRNA and BODIPY labeling), we observed that almost all cells infected with SARS-CoV-2 presented a close association with LDs (Fig. 4 D and E), but treatment with fatostatin or A922500 reduced the double-positive cells up to 10%, reducing the association of dsRNA with LDs (Fig. 4 D and E). We observed through electron microscopy that SARS-CoV-2-infected cells presented an increase in LDs (*), and a close association of the viral particles (arrow) with LDs (Fig. 4 F and S4B-D), as previously shown 2 , 17 . Moreover, SARS-CoV-2 infection induced a clear signal of cell injury in Calu-3 cells, as indicated by the presence of myelin figures (arrowhead) (Fig. S4B-D), in comparison with control cells (Fig. S4A). Furthermore, fatostatin or A922500 treatment reduced LD accumulation and the presence of viral particles in comparison with the cells infected and treated with vehicle (DMSO) (Fig. 4 F). Altogether, our data suggest that SARS-CoV-2 modulates the lipid metabolism of Calu-3 cells in favor of increasing the activation of SREBPs, inducing LD accumulation, and promoting a close association with dsRNA and viral replication sites. Fatostatin Protects Calu-3 Cells From Death During Sars-cov-2 Infection Viral infections can cause alterations in cell homeostasis, leading the virus to use the cellular compounds for your own benefit, increasing viral replication that can induce a process of cell death using cellular resources or by heightened inflammatory response. SARS-CoV-2 infection has the capacity to induce cell death in different cells, such as human monocytes, by the liberation of LDH into the extracellular space 2 , 20 , 21 . Here, we observed the cell morphology and measured the LDH release of Calu-3 cells infected with SARS-CoV-2 after 48 h of infection. Our data showed that infection with SARS-CoV-2 was able to alter the cell monolayer, causing damage to the cellular membrane and increasing LDH release into the supernatant (Fig. 5 A and B). Treatment with fatostatin alone did not interfere with cell morphology or LDH release, while treatment during SARS-CoV-2 infection was able to decrease damage, block cell death, and reduce LDH release in Calu-3 cells (Fig. 5 A and B). As previously observed with the knockdown of both genes that encode SREBPs, treatment with fatostatin, which inhibits the activation of both SREBPs, was able to reduce viral replication by 2 logs (Fig. 5 C), presenting viral inhibition of almost 90% and with a 50% antiviral concentration (IC50) of 14.15 µM (Fig. S3A). Similarly, pretreatment with A922500 inhibited viral replication (Fig. 5 C), with viral inhibition of almost 100% and an IC50 of 3.88 µM (Fig. S3B). To analyze other inhibitors of SREBP, we evaluated two other inhibitors, betulin and AM580. Neither of the inhibitors presented any cytotoxicity in Calu-3 cells (Fig. S3C and D). To observe the effects of these inhibitors during SARS-CoV-2 infection, we analyzed the protection from cell death and viral replication. The inhibitor Betulin did not protect against cell death, as observed by LDH release, and did not affect viral replication (Fig. S3C), but the inhibitor AM580 was able to reduce viral replication without altering cell death (Fig. S3D). Srebp Inhibition Blocks Inflammasome Complex Activation And Pyroptosis Death During Sars-cov-2 Infection Several works associate SREBP activation with the inflammasome, culminating in the release of proinflammatory cytokines, such as IL-1β 9 , 22 . In previous work, we already demonstrated that human monocytes infected with SARS-CoV-2 activate caspase-1 and promote IL-1β release with activation of GSDMD1, which suggests a process of pyroptosis 20 . In addition, other works supporting these data have already demonstrated inflammasome NLRP3 assembly and activation in human primary monocytes infected with SARS-CoV-2 as well as PBMCs from COVID-19 patients 23 . To investigate the relationship between SREBP activation and the cell death process by pyroptosis during SARS-CoV-2 infection, we evaluated caspase-1 activation by staining with FAM-YVAD-FLICA in Calu-3 cells. Indeed, infected cells presented an increase in activated caspase-1 during SARS-CoV-2 infection, and when SREBPs were inhibited by treatment with fatostatin (Fig. 5 D and E) or A922500 (Fig. S5A), a reduction in activated caspase-1 was observed by fluorescence microscopy and flow cytometry, which was also confirmed by western blotting (Fig. S5B and C). Moreover, we observed that the cells infected with SARS-CoV-2 presented an increase in GSDMD1 activation, a protein related to membrane pore formation that is activated during the process of pyroptosis. Moreover, treatment with fatostatin was able to reduce the activation of GSDMD1 (Fig. 5 F). Altogether, these data suggest that activation of SREBPs during SARS-CoV-2 infection is important to promote an increase in activated caspase-1, contributing to the formation of membrane pores by GSDMD1. It is already well known that SARS-CoV-2 promotes an exacerbated inflammatory response that aggravates cell damage and consequently cell death with the release of several cytokines 2 , 20 . Here, we observed an increase in the main inflammatory cytokines produced by the activation of caspase-1, such as IL-1β and IL-18 (Fig. 5 G). We also observed an increase in other proinflammatory cytokines, such as IL-6 and TNFα, and the chemokine CxCL-10 (Fig. S5D and E). Consistent with prior data, the inhibition of SREBPs by fatostatin (Fig. 5 G and S5D) or A922500 (Fig. S5E) was able to reduce all cytokines and chemokines previously observed. Altogether, our data suggest that SREBPs participate in the inflammatory process during SARS-CoV-2 infection in Calu-3 cells. Discussion Accumulating evidence indicates that SARS-CoV-2 infection promotes major host cellular lipid metabolism reprogramming to enhance fitness and replication assembly capacity. In this context, lipid metabolism dysregulation is associated with disease severity 24 , 25 . However, the mechanisms and metabolic pathways explored by SARS-CoV-2 to support its replication within host cells are still poorly understood. The findings presented here provide direct evidence that the SREBP lipid synthesis pathway is critically required for SARS-CoV-2 infection, replication, and amplification of the inflammatory response. Here, we demonstrate that SREBP participates in SARS-CoV-2 infection at two levels of host pathogen interaction: first, they are essential transcriptional regulators of the major host metabolic pathways that support virus replication; and second, they are central in the amplification of inflammatory mediator production and cell death through activation of inflammasomes. Several viruses alter lipid metabolism through the increase in expression and/or activation of transcription factors, such as SREBP1 6,12 and SREBP2 19 . The presence of cholesterol in COVID-19 plasma patients is increased through SREBP2 activation 12 , while the accumulation of triglycerides is associated with the activation of SREBP1 6 , which has already been observed in cells infected with SARS-CoV-2 2 . In our data, SARS-CoV-2 infection in Calu-3 cells activates both SREBP isoforms, suggesting that the infection reprograms the cells toward a lipogenic phenotype, increasing the triglyceride and cholesterol pathways that were shown to be required for the SARS-CoV-2 viral cycle 2 , 26 . Accordingly, enhanced expression and/or activation of both SREBPs has been reported during infection with respiratory viruses, such as MERS-CoV, SARS-CoV, and SARS-CoV-2 2,6,12 . Consistently, targeting the lipid biosynthetic SREBPs pathways was shown to present antiviral properties 2 , 6 , 7 . Knockdown of genes that encode SREBP1 and SREBP2 proteins during SARS-CoV-2 infection downregulates lipid metabolism and directly impacts LD biogenesis and SARS-CoV-2 replication, reducing cell death. Using a double knockdown for both SREBPs, the effects were aggravated and were similar, which occurs during MERS-CoV infection 6 . The knockdown of the gene that encodes the DGAT1 protein presents effects resembling double knockdown for both SREBPs. This suggests that the SREBPs present a special contribution to viral replication, where these transcription factors are able to modulate lipid metabolism by increasing LD through the DGAT1 enzyme, altering SARS-CoV-2 replication. Once double knockdown of SREBPs was more efficient in reducing viral replication and LD biogenesis, we used the pharmacological inhibitor fatostatin, which was predicted to inhibit both SREBP isoforms 18 , to analyze the effects of these transcription factors during SARS-CoV-2 infection on lipid metabolism. Here, the double inhibition of the SREBPs activated form was confirmed by western blotting, and a downregulation in lipid metabolism was noted in cells treated with fatostatin during SARS-CoV-2 infection, demonstrating that the inhibitor not only inhibits SREBPs activation but also reduces the activation of lipid metabolism, such as triglycerides and cholesterol storage, caused by viral infection. The molecular mechanisms involved during LD biogenesis are a highly coordinated process, requiring new lipid syntesis and lipid remodeling, but these processes during inflammation and infection need to be better understood. As observed in several studies, LDs are a key organelle during the + RNA virus replicative cycle 2 , 6 , 27 , 28 . Here, we observed that treatment with fatostatin reduces LD biogenesis and protein expression related to LD maturation, such as DGAT1 and PLIN2. This suggests that the inhibition of SREBPs is important during SARS-CoV-2 infection for LD biogenesis. Furthermore, Calu-3 cells infected with SARS-CoV-2 present strong labeling for dsRNA, which seems to be correlated with LDs, as previously observed in VERO E6 cells 2 . It is important to consider that LDs may participate as replication sites, but other cellular compartments may also have an important role in viral replication. Indeed, recent studies have uncovered the mechanisms of LD recruitment to viral replication compartments with bidirectional content exchange and essential functions in replication and virus particle assembly 12 , 29 . Of note, LD accumulation was also observed in Type II pneumocytes undergoing cell death with characteristics of pyroptosis in lung tissue from autopsy of COVID deceased patients. 30 SREBPs are crucial for the replication of several viruses and have been related to the increase in LD biogenesis 6 , 28 . Moreover, pharmacological inhibition of SREBPs with betulin reduces the viral replication and LD biogenesis of MERS-CoV 6 . Here, we found that the expression of the precursor form of SREBPs is reduced and that the mature form (active), which enters the nucleus, remains highly increased during infection, were downregulated by fatostatin treatment, contributing to LD remodeling in Calu-3 cells. Inhibitors of lipid metabolism, such as fatostatin and A922500, reduced the viral replication observed by dsRNA labeling. Moreover, the treatments reduced the proximity of dsRNA to LDs. Thus, SREBPs are crucial for LD accumulation through the DGAT1 enzyme. Future studies that target proteins of LD biogenesis and specific proteins of SARS-CoV-2 would be interesting to probe and understand the role of LDs in the SARS-CoV-2 replication cycle. Under homeostatic conditions, the C-terminal domain of SREBPs binds to SREBP cleavage-activating protein (SCAP) in the endoplasmic reticulum (ER) membrane. This complex interacts with insulin-induced gene 1 protein (INSIG1) 31 , and in high levels of cholesterol, INSIG becomes stable and binds to SREBP-SCAP, creating a complex retained in the ER membrane. In contrast, when cholesterol levels are reduced, INSIG is rapidly degraded by the ubiquitin‒proteasome system, and the SREBP-SCAP complex is cleaved and directed to the nucleus for sterol regulatory elements (SREs) to modulate lipid metabolism 32 . To analyze the mechanism of SREBP activation during SARS-CoV-2, three inhibitors were used for the activation of SREBPs. Fatostatin inhibitor acts to prevent SREBPs-SCAP cleavage in the ER 18 . Betulin blocks the degradation of INSIG protein and inhibits SREBPs cleavage for SCAP in the ER, and AM580 acts as an agonist of retinoic acid, blocking the association of SREBPs with SREs in the nucleus 6 . In this context, our results suggest that SARS-CoV-2 induces SREBP through an INSIG-independent mechanism since betulin did not affect viral replication (Fig. S3C), but AM580 is able to affect viral replication (Fig. S3D), although the inhibition is more significant using fatostatin, showing a different mechanism than that observed during MERS-CoV infection 6 . In addition to lipid metabolism reprogramming, SARS-CoV-2 causes an uncontrolled inflammatory response associated with an increase in the cell death process 2 , 21 , 33 . Here, we observed an increase in proinflammatory cytokines and chemokines during SARS-CoV-2 infection in Calu-3 cells. The inhibition of SREBPs or DGAT1 activity significantly reduced the inflammatory cytokine response in epithelial cells, confirming previous data from our group that DGAT1 and LD accumulation are involved in the inflammatory response amplification during SARS-CoV-2 infection in human monocytes 2 . This finding corroborates the well-established role of LDs in inflammation and innate immunity 2 , 24 , 34 and supports a role for LD biogenesis in the heightened inflammatory production triggered by SARS-CoV-2, and drugs that target SREBPs or the DGAT1 enzyme may have beneficial effects on disease pathogenesis. Accumulating evidence indicates a central role for NLRP3 inflammasome activation during SARS-CoV-2 infection contributing to increased cytokine release and the cell death process of pyroptosis 20 , 23 . Indeed, SARS-CoV-2 infection causes caspase-1 activation with increased IL-1β and IL-18 release and cleaved GSDMD1, which causes membrane pore formation with the extravasations of intracellular content 20 , 23 . Moreover, inflammasome activation has been implicated as a major determinant associated with severity and mortality in COVID-19 patients 20 , 23 . Here, we add another layer to the mechanisms of SARS-CoV-2-induced inflammasome activation by demonstrating a required role for SREBP in this process. Accordingly, SREBP2-SCAP involvement in inflammasome activation has been previously demonstrated in macrophages by acting as a signaling hub facilitating inflammasome assembly upon nigericing stimulation of LPS-primed macrophages 22 . SREBP may participate in inflammasome activation by direct or indirect effects. Activation of inflammasomes may occur through the recognition of cholesterol crystals 35 , which presents a close relationship with LDs 36 , 37 and can represent an important link between cholesterol metabolism and inflammation in COVID-19 pathogenesis, but direct effects of SRBP2-SCAP on inflammasome assembly have been proposed 22 . Recently, it was demonstrated that viral NSP6 of SARS-CoV-2 induces the activation of the inflammasome complex NLRP3 through lysosome acidification that triggers ATP6AP1, causing autophagic flux stagnation 38 . Interestingly, SARS-CoV-2-derived NSP6 localizes to LDs and is involved in LD recruitment and favors viral replication 17 . An intriguing possibility is that NSP6-triggered lipid remodeling and inflammasome activation are connected through the SREBPs pathway, favoring caspase-1 activation and release of IL-1β, leading to cell death by pyroptosis. In summary, our data demonstrated that SARS-CoV-2 directly affects lipid metabolism, activates SREBPs to accumulate triglycerides in LDs, and increases intracellular cholesterol. Genetic knockdown or pharmacological inhibition of SREBPs prevents reprogramming of lipid metabolism, reducing LD biogenesis, viral replication, assembly, and establishment of the infection. Moreover, the SREBPs induce inflammasome assembly, caspase-1 activation and pore formation by GSDMD1 with the release of proinflammatory cytokines, such as IL-1β and IL-18 (Fig. 6 ). This finding supports the hypothesis that SARS-CoV-2 alters lipid metabolism through SREBP activation, favoring their fitness, replication and pathogenesis. Our findings contribute to a better understanding of how SARS-CoV-2 uses host lipid metabolism for its own benefit and contributes to the inflammatory response. Further studies are necessary to better understand the mechanisms and the importance of SREBPs during SARS-CoV-2 infection and how the crosstalk between SREBPs and the inflammasome may contribute to triggering the immune response. This study may provide new insights into lipid metabolic pathways and suggest a potential strategy to reduce viral replication and the uncontrolled inflammatory response during COVID-19 pathogenesis. Material And Methods Cells and reagents. The human lung epithelial adenocarcinoma cell line (Calu-3 - ATCC/HTB-55) and African green monkey kidney (Vero subtype E6) were cultured in high glucose DMEM supplemented with 10% fetal bovine serum (FSB, HyClone, Logan, Utah) and 100 U/mL penicillin‒streptomycin (P/S; GIBCO) and were incubated at 37°C in 5% CO 2 . Virus infection and virus titration. Nasopharyngeal swab samples were collected from confirmed cases from Rio de Janeiro/Brazil (GenBank accession no. MT710714). SARS-CoV-2 was amplified in Vero-E6 cells in high-glucose DMEM supplemented with 2% FBS for 2 to 4 days of infection and incubated at 37°C in 5% CO 2 . Virus titers were determined by the tissue culture infectious dose at 50% (TCID 50 /mL), and the virus stocks were kept in -80°C freezers. All specimens were handled under the laboratory biosafety guidance required involving SARS-CoV-2 by the World Health Organization (WHO) at biosafety level 3 (BSL3) multiuser facility from Fundação Oswaldo Cruz/Fiocruz (Rio de Janeiro, RJ, Brazil). All cells were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01 with or without pretreatment with pharmacological inhibitors of SREBP activation, such as fatostatin (Cayman 13562), AM580 (Sigma A8843) and betulin (Cayman 11041), and we used the pharmacological inhibitor of DGAT1, A922500 (Sigma A1737), for two hours and maintained the treatment after the infection. During knockdown experiments, the cells were infected at the same MOI, and 24 h prior to infection, the cells were transfected with the siRNAs for SREBF1 (s129), SREBF2 (s27), DGAT-1 (16567) and the negative control with scramble RNA (4390844) according to the manufacturers' instructions (Thermo Fisher Scientific). For virus titration, we performed a plaque-forming assay in Vero-E6 cells seeded in 96-well plates. Cell monolayers were infected with different dilutions of the supernatant containing the virus for 1 h at 37°C. The cells were overlaid with high glucose DMEM containing 2% FBS and 2.4% carboxymethylcellulose. After 3 days, the cells were fixed with 10% formaldehyde in PBS for 3 h at room temperature. Cell monolayers were stained with 0.04% crystal violet in 20% ethanol for 1 h. The viral titer was calculated from the count of plaques formed in the wells corresponding to each dilution and expressed as plaque forming units per mL (PFU/mL). Cell viability assay. Calu-3 cells were seeded in 96-well plates. Then, the cells were treated with a range of concentrations of the inhibitors for 24 h and 48 h. Then, the cells were fixed using 3.7% formaldehyde for 20 mins. Cell monolayers were stained with 1% crystal violet in 20% ethanol for 10 min. The cells were washed with water, and crystal violet was extracted using methanol. The crystal violet was read in a spectrophotometer at a wavelength of 595 nm. Ultrastructural analysis of cells by transmission electron microscopy. For ultrastructural analysis, the infected and noninfected Calu-3 cell monolayers were trypsinized at 24 and 48 hours postinfection or cultivation, respectively. Cell suspensions were fixed in 2.5% glutaraldehyde in sodium cacodylate buffer (0.2 M, pH 7.2), postfixed in 1% buffered osmium tetroxide, dehydrated in acetone, embedded in epoxy resin, and polymerized at 60°C over the course of three days 39 , 40 . Ultrathin sections (50–70 nm) were obtained from the resin blocks. The sections were picked up using copper grids (300 mesh) and observed using a Hitachi HT 7800 (Hitachi, Tokyo, Japan) transmission electron microscope. Lipid droplet staining. Calu-3 cells were seeded on coverslips. Cells infected or not infected were fixed using 3.7% formaldehyde, and the LDs were stained with 0.3% Oil Red O (diluted in 60% isopropanol) at room temperature for 2 min. The coverslips were mounted on slides using antifade mounting medium (VECTASHIELD®). DAPI staining (1 µg/mL) for 5 min was used for nuclear recognition. Fluorescence was analyzed by fluorescence microscopy with a 100x objective lens (Olympus, Tokyo, Japan). The numbers of LDs were automatically quantified from 15 aleatory fields by ImageJ software analysis. Immunofluorescence staining. Calu-3 cells were seeded on coverslips and fixed using 3.7% formaldehyde after 48 h of infection for 20 min at room temperature. Cells were rinsed three times with PBS with 0.1 M CaCl 2 and 1 M MgCl 2 (PBS/CM) and then permeabilized with 0.1% Triton X-100 plus 0.2% BSA in PBS/CM for 10 min (PBS/CM/TB). Double-RNA was labeled by the mouse monoclonal antibody J2 clone Scicons 2 , 41 at a 1:500 dilution overnight, followed by a mouse anti-IgG-Dylight 550 at a 1:1000 dilution for 1 h with 0.2 µg/mL BODIPY493/503 dye for 5 min for LD staining. In addition, mouse anti-IgG-Dylight 488 was used at a 1:1000 dilution for 1 h with LipidTox Neutral Red dye (Thermo Fisher Scientific - H34476) at a dilution of 1:1000 for 30 min for LD staining. Slides were mounted using an antifade mounting medium (VECTASHIELD®). Nuclear recognition was based on DAPI staining (1 µg/mL) for 5 min. Fluorescence microscopy was analyzed with a 100x objective lens (Olympus, Tokyo, Japan). The fluorescent area was quantified from 15 aleatory fields by ImageJ software analysis. Triglyceride and cholesterol measurements. Calu-3 cells were harvested after 48 h of SARS-CoV-2 infection using ice-cold lysis buffer pH 8.0 (1% Triton X-100, 2% SDS, 150 mM NaCl, 10 mM HEPES and 2 mM EDTA in the presence of protease inhibitor cocktail - Roche). For analysis of triglyceride levels, 80 µg of protein/sample of the cell lysates was extracted with chloroform/methanol/water 1:2:0,8 (v/v/v) in glass tubes. The samples were vortexed for 5 min and 5 min for 1 h. Then, they were centrifuged at 3 000 rpm for 20 min. The aqueous phase of each sample was collected and transferred to another glass tube, the pellet was resuspended in chloroform/methanol/water 1:2:0,8 (v/v/v), and the last steps were repeated. The aqueous phase was obtained and transferred to the same glass tubes as the first aqueous phase. Then, chloroform/water 1:1 (v/v) was added and vortexed for 10 seconds. After this step, the samples were centrifuged at 3 000 rpm for 30 min, and two phases were obtained. A lower phase was collected in a glass tube, evaporated with nitrogen gas, and resuspended in chloroform/methanol 1:2 (v/v). The triglyceride levels were quantified using Triglycerides Liquiform (87 − 2/100 Labtest kit) according to the manufacturer’s instructions. For cholesterol measurements, Calu-3 cells were seeded in coverslips and after 48 h were fixed using 3.7% formaldehyde for 20 min at room temperature. Then, the cells were washed three times with PBS and incubated with 20 µM glycine for 10 min. Next, the cells were stained with 50 µg/mL filipin III (Cayman #70440) for two hours at room temperature in the dark. For nuclear recognition, the cells were labeled with 1 µM TO-PRO-3 (Thermo Fisher Scientific - T3605) for 10 min. Slides were mounted using an antifade mounting medium (VECTASHIELD®). Fluorescence was analyzed by fluorescence microscopy with a 40x objective lens (Olympus, Tokyo, Japan), and the fluorescent area was quantified from 15 aleatory fields by ImageJ software analysis. SDS‒PAGE and Western blot. After 24 h of SARS-CoV-2 infection, Calu-3 cells were harvested using ice-cold lysis buffer pH 8.0 containing 1% Triton X-100, 2% SDS, 150 mM NaCl, 10 mM HEPES, and 2 mM EDTA in the presence of protease inhibitor cocktail (Roche). The protein levels were measured by a bicinchoninic acid assay protein kit (Thermo Fisher Scientific). 30 µg of protein/sample was heated at 100°C for 5 min in Laemmli buffer pH 6.8 (20% β-mercaptoethanol; 370 mM Tris base; 160 µM bromophenol blue; 6% glycerol; 16% SDS) and resolved by electrophoresis on an SDS-containing 10% polyacrylamide gel (SDS‒PAGE). Next, the separated proteins were transferred to nitrocellulose membranes and incubated in blocking buffer (5% nonfat milk, 50 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween 20). Membranes were probed overnight with the following antibodies: anti-SREBP1 (Proteintech 14088-1-AP), anti-SREBP2 (Proteintech 28212-1-AP), anti-DGAT1 (Proteintech 11561-1-AP), anti-PLIN2 (Proteintech 15294-1-AP), anti-GSDMD1 (Cell Signaling 97558) and anti-GAPDH (Proteintech 60004-1-1g). After washing, the membranes were incubated with IRDye - LICOR or HRP-conjugated secondary antibodies for 2 h at room temperature. All antibodies were diluted in blocking buffer. Signal detection was performed by Supersignal Chemiluminescence (GE Healthcare) or fluorescence imaging using the Odyssey system. The densitometries were analyzed using Image Studio Lite Ver 5.2 software. Quantitative real-time RT‒PCR assay. Monolayers from Calu-3 cells after 24 h of SARS-CoV-2 infection were harvested, and the total RNA from each sample was extracted using an SV total RNA isolation system kit according to the manufacturer's protocol (Promega). RNA concentration and purity were determined by a spectrophotometer (Nanodrop 2000) measuring absorbance at A260 and A280 nm, and RNA was stored at − 70°C in nuclease-free water. Total RNA (2 µg) was reverse transcribed in a 20 µl reaction mixture using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s protocol. The cDNA was amplified in 10 µl of 1× TaqMan universal PCR master mix with Predeveloped TaqMan assay primers and probes Perilipin-2 (PLIN2) Hs00605340_m1; DGAT1, Hs01020362_g1; Fatty Acid Synthase (FASN) Hs01005622_m1; SREBF1 Hs01088691_m1; SREBF2 Hs01081784_m1; patatin like phospholipase domain containing 2 (PNPLA2) Hs00386101_m1; Sterol O-Acyltransferase 1 (SOAT1) Hs00162077_m1; ATP-binding cassette transporter-1 (ABCA1) Hs01059118_m1; Interleukin-6 (IL-6) Hs00985639_m1; Interleukin-10 (IL-10) Hs00961622_m1; Interleukin-1β (IL-1β) Hs01555410_m1 and as endogenous control it was used the GAPDH Hs99999905_m1 according to manufacturer’s instruction (Thermo Fisher Scientific). Quantitative RT‒PCR was performed in a StepOne™ Real-Time PCR System (Thermo Fisher Scientific). PCR products were analyzed in a comparative manner relative to the endogenous control GAPDH (ΔΔCt). Measurements of inflammatory mediators and LDH activity. Calu-3 cell supernatants were obtained after 24 hours of SARS-CoV-2 infection with or without treatment with inhibitors. Cytokines and chemokines were measured in the supernatant by ELISA following the manufacturer's instructions (Duo set, R&D). Cell death was determined according to the activity of lactate dehydrogenase (LDH) in the culture supernatants using a CytoTox® Kit according to the manufacturer’s instructions (Promega, USA). Assessment of activated caspase-1. After 48 h of infection, Calu-3 cells were stained to detect caspase-1 activation using fluorescent-labeled inhibitors of caspase‐1 activity (FAM‐YVAD‐FMK/FLICA) according to the manufacturer’s instructions (Bio-Rad). The fluorescence of caspase-1 activity, expressed as the percentage of activated cells, was evaluated by flow cytometry (FACSCalibur), and the generated data were analyzed with FlowJo. In parallel, the cells were seeded on coverslips; after 48 h of infection, the cells were labeled with FAM‐YVAD‐FMK/FLICA according to the manufacturer’s instructions. Then, the cells were fixed with 3.7% formaldehyde for 20 min at room temperature. The nuclei were stained with DAPI (1 µg/mL) for 5 min, and the coverslips were mounted using antifade mounting medium (VECTASHIELD®). Fluorescence was analyzed by fluorescence microscopy with a 100× objective lens (Olympus, Tokyo, Japan). Statistical analysis. Data are expressed as the mean ± standard error of the mean (SEM) of three and a maximum of six independent experiments. The paired two-tailed t test was used to evaluate the significance of the two groups. Multiple comparisons among three or more groups were performed by one-way ANOVA followed by Tukey’s multiple comparison test. p values < 0.05 were considered statistically significant when comparing SARS-CoV-2 infection to the uninfected control group (*) or SARS-CoV-2 infection with inhibitors groups (pharmacological inhibitors) or knockdown groups (siSREBF1 and − 2 and DGAT1) (#). Declarations Acknowledgments The authors thank the confocal imaging and Luminex facility from the Rede de Plataformas Tecnológicas FIOCRUZ and Dra. Milene Dias Miranda for assessments related to the BSL3 facility. This work was supported by grants from Inova program Fiocruz, Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) granted for Patrícia T. Bozza, Thiago Moreno L. Souza and Debora Ferreira Barreto Vieira. 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Nucleic Acids Res 19 , 2993–3000 (1991). https://doi.org:10.1093/nar/19.11.2993 Additional Declarations (Not answered) Supplementary Files UncutWBSREBPpaper.pptx SupplementaryFigure2.jpg S2 Figure. Double knockdown of both SREBPs reduces lipid metabolism and inflammatory genes during SARS-CoV-2 infection. Calu-3 cells were treated with the siRNAs for single or double knockdown of SREBF1 and SREBF2, DGAT1 and the scramble RNA for the negative control 24 h before SARS-CoV-2 infection at an MOI of 0.01 for 48 h. The mRNA expression of key genes associated with (A) lipid metabolism and (B) inflammatory cytokines after 24 h of SARS-CoV-2 infection normalized to the scramble control. Data are presented as the mean ± SEM obtained in three independent experiments. *p<0.05 versus uninfected cells and #p <0.05 versus infected cells. SupplementaryFigure3.jpg S3 Figure. Effects of SREBPs and DGAT1 inhibitors on cell death and SARS-CoV-2 replication. Calu-3 cells were treated with a range of concentrations of the inhibitors for 2 h before infection with SARS-CoV-2 at an MOI of 0.01 for 48 h in the presence of the inhibitors. Cell death was measured by LDH activity, and viral replication was determined by plaque assay after treatment with (A) fatostatin, (B) A922500, (C) betulin and (D) AM580. Data are expressed as the mean ± SEM obtained in three independent experiments. *p<0.05 versus uninfected cells and #p <0.05 versus infected cells. SupplementaryFigure4.jpg S4 Figure. Ultrastructural analysis of Calu-3 cells after SARS-CoV-2 infection. Calu-3 cells were infected with SARS-CoV-2 at an MOI of 0.01 for 48 h. (A) uninfected Calu-3 cells and (B-D) Calu-3 cells infected with SARS-CoV-2 at an MOI of 0.01. Lipid droplets (asterisk), nucleus (N), myelin figures (arrowhead) and SARS-CoV-2 particles (arrow). SupplementaryFigure5.jpg S5 Figure. Fatostatin and A922500 inhibitors decrease the proinflammatory profile of SARS-CoV-2 infection in Calu-3 cells. Calu-3 cells were pretreated with the SREBPs inhibitor fatostatin (20 μM) or the DGAT-1 inhibitor A922500 (20 μM) for 2 h before infection with SARS-CoV-2 at an MOI of 0.01 for 24 h in the presence of the inhibitors. (A) After 48 h of infection, Calu-3 cells were treated with A922500 and stained with FAM-YVAD-FLICA to determine caspase-1 activity by flow cytometry. (B) Protein expression in cell lysates was evaluated after 24 h of infection by western blot for total (p45) and cleaved (p10) caspase-1. GAPDH was used as a control for protein loading. (C) The densitometries are representative of western blot images. The inflammatory cytokines were measured in the supernatants by ELISA after treatment with (D) fatostatin and (E) A922500. Data are expressed as the mean ± SEM obtained in three independent experiments for ELISA or western blot and four independent experiments for flow cytometry. *p<0.05 versus uninfected cells and #p <0.05 versus infected cells. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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Cruz","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Debora","middleName":"","lastName":"Barreto-Vieira","suffix":""},{"id":159402692,"identity":"5f057a14-0930-412d-b586-e8d89918efc2","order_by":11,"name":"Thiago Souza","email":"","orcid":"https://orcid.org/0000-0003-2212-3899","institution":"Oswaldo Cruz Foundation","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Thiago","middleName":"","lastName":"Souza","suffix":""}],"badges":[],"createdAt":"2022-12-02 22:55:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2338983/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2338983/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":30445692,"identity":"d5e050f6-d6b6-47bb-af51-8673e4be5461","added_by":"auto","created_at":"2022-12-16 18:34:17","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":170403,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSARS-CoV-2 infection induces alterations in lipid metabolism in Calu-3 cells. \u003c/strong\u003eCells were infected with SARS-CoV-2 at an MOI of 0.01 for 24 h or 48 h. Protein expression in cell lysates was evaluated after 24 h of infection by western blotting for (A) total and cleaved transcription factors SREBP1 and SREBP2 and (B) the expression of proteins associated with lipid droplet (LD) maturation, such as DGAT1 and PLIN2. GAPDH was used as a control for protein loading, and the densitometries are presented below the western blot images. (C) The mRNA expression heatmap of genes related to lipid metabolism and inflammatory cytokines after 24 h of SARS-CoV-2 infection. (D) LDs were stained with Oil Red O (red), and nuclei were stained with DAPI (blue) after 24 h and 48 h of infection and observed by fluorescence microscopy; scale bar, 10 μm. (E) LDs were quantified using ImageJ software analysis by measuring the fluorescent area of the LDs. (F) Cholesterol was stained with Filipin III (blue), and nuclei were stained with To-Pro-3 (Fucsia) in the cells 48 h after infection. Scale bar, 50 μm. (G) The fluorescence area was quantified by ImageJ software analysis. (H) The triglyceride levels were analyzed in lipids extracted from cells infected after 48 h. (I) A representative scheme of the increase in the activation of SREBPs by SARS-CoV-2 infection can regulate the proteins associated with LD maturation and contribute to cholesterol metabolism and LD formation in Calu-3 cells. Data are expressed as the mean ± SEM obtained in three independent experiments and five independent experiments for cholesterol fluorescence analyses. *p\u0026lt;0.05 versus uninfected cells and #p \u0026lt;0.05 versus infected cells.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2338983/v1/6f91a2aa4fc80eab1fbff77c.jpg"},{"id":30445183,"identity":"6349a357-1ed8-4c13-bb4c-7d17aacb43a9","added_by":"auto","created_at":"2022-12-16 18:26:17","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":121610,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSREBPs control lipid metabolism and reduce viral replication in Calu-3 cells infected with SARS-CoV-2. \u003c/strong\u003eCalu-3 cells were treated with siRNAs for single or double knockdown of SREBF1 and SREBF2, DGAT1 and scramble RNA for the negative control 24 h before SARS-CoV-2 infection at an MOI of 0.01. (A) Viral replication was performed 48 h after infection by plaque assay. (B) Cell death was measured in the supernatant after 48 h of infection by LDH fold change in relation to the scramble uninfected cell. (C) The mRNA expression heatmap of key genes associated with lipid metabolism and inflammatory cytokines after 24 h of SARS-CoV-2 infection normalized to the scramble control. (D) Double strain RNA (dsRNA) was detected after 48 h of infection by indirect immunofluorescence with a J2 antibody (green), LDs were stained with LipidTox (red), and nuclei were stained with DAPI (blue); scale bar, 10 μm. (E) dsRNA was quantified by ImageJ software analysis using the measurement of the fluorescent area. (F) LDs were evaluated by ImageJ software analysis by measuring the fluorescent area of LDs. Data are presented as the mean ± SEM obtained in three independent experiments and four independent experiments for viral replication and cell death analyses. *p\u0026lt;0.05 versus uninfected cells and #p \u0026lt;0.05 versus infected cells.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2338983/v1/f43f7ed20adde16fec114945.jpg"},{"id":30445845,"identity":"80cc2163-959e-484f-ae65-42b3f4b04764","added_by":"auto","created_at":"2022-12-16 18:42:17","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":153894,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePharmacological inhibition with fatostatin blocks the activation of both SREBPs during SARS-CoV-2 infection. \u003c/strong\u003eCalu-3 cells were treated with fatostatin (20 µM) or vehicle (DMSO) for 2 h before SARS-CoV-2 infection at an MOI of 0.01 for 24 h in the presence of the inhibitor. Cells were lysed, and the expression and activation of (A) SREBP1 and SREBP2 were determined by western blotting. GAPDH was used as a control for protein loading. (B) The densitometries represent the western blot images. (C) Cholesterol was stained with filipin III (blue), and nuclei were stained with To-Pro-3 (Fucsia) in the cells 48 h after infection; scale bar, 50 μm. (D) Cholesterol was evaluated using ImageJ software analysis by measuring the fluorescent area. (E) The triglyceride levels were analyzed in lipids extracted from cells infected for 48 h. (F) LDs were stained with Oil Red O (red), and nuclei were stained with DAPI (blue) and observed by fluorescence microscopy; scale bar, 10 μm. (G) LDs were evaluated by ImageJ software analysis by measuring the fluorescent area of LDs. (H) Representative western blot images of DGAT1 and PLIN2. GAPDH was used as a control for protein loading. (I) Densitometry evaluation of data panel 4H. Data are expressed as the mean ± SEM obtained in three independent experiments and four independent experiments for cholesterol fluorescence analyses. *p\u0026lt;0.05 versus uninfected cells and #p \u0026lt;0.05 versus infected cells.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2338983/v1/c960bc1e637103ae908e5034.jpg"},{"id":30443535,"identity":"83bdc358-82b7-44a0-9e29-5cb909984ce7","added_by":"auto","created_at":"2022-12-16 18:10:17","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":225117,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePharmacological inhibition of SREBPs reduces the association with the SARS-CoV-2 replication complex. \u003c/strong\u003eCalu-3 cells were treated with fatostatin (20 µM), A922500 (20 µM) or vehicle (DMSO) for 2 h before SARS-CoV-2 infection at an MOI of 0.01 for 48 h in the presence of the inhibitors. (A) dsRNA was detected by indirect immunofluorescence with a J2 antibody (red), LDs were stained with BODIPY 493/503 (green), and nuclei were stained with DAPI (blue); scale bar, 20 μm. (B) Quantification of dsRNA using ImageJ software analysis by the measurement of the fluorescent area. Percentage of (C) dsRNA-positive cells and (D) double-positive cells for dsRNA and BODIPY. (E) Representative magnified images of immunofluorescence by J2 antibody (red). The LDs were stained with BODIPY 493/503 (green), and nuclei were stained with DAPI (blue). Scale bar, 10 μm. (F) Ultrastructural analysis of Calu-3 cells treated with vehicle (DMSO), fatostatin or A922500 and infected with SARS-CoV-2 (48 h). SARS-CoV-2 particles (arrow) near LDs (asterisk) and nuclear alteration (N). Scale bar, 2 μm. Data are expressed as the mean ± SEM obtained in three independent experiments and four independent experiments for microscopy fluorescence analyses. *p\u0026lt;0.05 versus uninfected cells and #p \u0026lt;0.05 versus infected cells.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2338983/v1/995d06e1c65f8a0f4b02f101.jpg"},{"id":30444439,"identity":"3a663ba8-945d-4722-b29e-3251ca42109c","added_by":"auto","created_at":"2022-12-16 18:18:17","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":69364,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConclusion figure. \u003c/strong\u003eSARS-CoV-2 infection is able to increase the activation of important transcription factors, SREBP1 and SREBP2, leading to increased levels of DGAT1, PLIN2 and cholesterol synthesis, inducing LD maturation and biogenesis and supporting SARS-CoV-2 replication. In addition, SARS-CoV-2 infection induced cell death by pyroptosis, with activation of caspase-1, cleavage of GSDMD and release of IL-1β and IL-18 depending on SREBP activation. Pharmacological inhibition and genetic knockdown of SREBPs and DGAT1 reduce SARS-CoV-2 replication, cell death and LD biogenesis. Altogether, our data suggest that SREBPs are key players in the replication of SARS-CoV-2, LD biogenesis and inflammasome activation, participating in SARS-CoV-2 pathogenesis.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2338983/v1/1ffded9ea68232c6bd2c0724.jpg"},{"id":31937894,"identity":"9883d813-1ea5-490c-b87f-6e4bd62f769e","added_by":"auto","created_at":"2023-01-23 11:42:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1224783,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2338983/v1/bd840587-877a-49ee-ae36-a521d9c73e3a.pdf"},{"id":30443546,"identity":"2a8fe8d2-5b06-4af4-be96-422546e7e1fa","added_by":"auto","created_at":"2022-12-16 18:10:18","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11906475,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"UncutWBSREBPpaper.pptx","url":"https://assets-eu.researchsquare.com/files/rs-2338983/v1/c192954b859ea18e23763833.pptx"},{"id":30444434,"identity":"9a8670a6-a000-43f8-b1c6-0dd1de37a685","added_by":"auto","created_at":"2022-12-16 18:18:17","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":147100,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eS2 Figure. Double knockdown of both SREBPs reduces lipid metabolism and inflammatory genes during SARS-CoV-2 infection. \u003c/strong\u003eCalu-3 cells were treated with the siRNAs for single or double knockdown of SREBF1 and SREBF2, DGAT1 and the scramble RNA for the negative control 24 h before SARS-CoV-2 infection at an MOI of 0.01 for 48 h. The mRNA expression of key genes associated with (A) lipid metabolism and (B) inflammatory cytokines after 24 h of SARS-CoV-2 infection normalized to the scramble control. Data are presented as the mean ± SEM obtained in three independent experiments. *p\u0026lt;0.05 versus uninfected cells and #p \u0026lt;0.05 versus infected cells.\u003c/p\u003e","description":"","filename":"SupplementaryFigure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2338983/v1/3d75ead0071489a5e2ba136e.jpg"},{"id":30443544,"identity":"1337e6d1-4a2c-431b-94c7-7572357acf56","added_by":"auto","created_at":"2022-12-16 18:10:17","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":128139,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eS3 Figure. Effects of SREBPs and DGAT1 inhibitors on cell death and SARS-CoV-2 replication. \u003c/strong\u003eCalu-3 cells were treated with a range of concentrations of the inhibitors for 2 h before infection with SARS-CoV-2 at an MOI of 0.01 for 48 h in the presence of the inhibitors. Cell death was measured by LDH activity, and viral replication was determined by plaque assay after treatment with (A) fatostatin, (B) A922500, (C) betulin and (D) AM580. Data are expressed as the mean ± SEM obtained in three independent experiments. *p\u0026lt;0.05 versus uninfected cells and #p \u0026lt;0.05 versus infected cells.\u003c/p\u003e","description":"","filename":"SupplementaryFigure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2338983/v1/954b45b5aae5d0fed7d8d6c9.jpg"},{"id":30445697,"identity":"9cd3e6d7-1379-4ff5-9749-7418bea5f57e","added_by":"auto","created_at":"2022-12-16 18:34:18","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":262971,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eS4 Figure. Ultrastructural analysis of Calu-3 cells after SARS-CoV-2 infection. \u003c/strong\u003eCalu-3 cells were infected with SARS-CoV-2 at an MOI of 0.01 for 48 h. (A) uninfected Calu-3 cells and (B-D) Calu-3 cells infected with SARS-CoV-2 at an MOI of 0.01. Lipid droplets (asterisk), nucleus (N), myelin figures (arrowhead) and SARS-CoV-2 particles (arrow).\u003c/p\u003e","description":"","filename":"SupplementaryFigure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2338983/v1/93d09fe4a1c22536e4521901.jpg"},{"id":30445694,"identity":"7f83b92d-3cb4-448f-8f15-6e70f479eb10","added_by":"auto","created_at":"2022-12-16 18:34:17","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":122365,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eS5 Figure. Fatostatin and A922500 inhibitors decrease the proinflammatory profile of SARS-CoV-2 infection in Calu-3 cells. \u003c/strong\u003eCalu-3 cells were pretreated with the SREBPs inhibitor fatostatin (20 μM) or the DGAT-1 inhibitor A922500 (20 μM) for 2 h before infection with SARS-CoV-2 at an MOI of 0.01 for 24 h in the presence of the inhibitors. (A) After 48 h of infection, Calu-3 cells were treated with A922500 and stained with FAM-YVAD-FLICA to determine caspase-1 activity by flow cytometry. (B) Protein expression in cell lysates was evaluated after 24 h of infection by western blot for total (p45) and cleaved (p10) caspase-1. GAPDH was used as a control for protein loading. (C) The densitometries are representative of western blot images. The inflammatory cytokines were measured in the supernatants by ELISA after treatment with (D) fatostatin and (E) A922500. Data are expressed as the mean ± SEM obtained in three independent experiments for ELISA or western blot and four independent experiments for flow cytometry. *p\u0026lt;0.05 versus uninfected cells and #p \u0026lt;0.05 versus infected cells.\u003c/p\u003e","description":"","filename":"SupplementaryFigure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2338983/v1/014e3b5c65b3103f28b3255a.jpg"}],"financialInterests":"(Not answered)","formattedTitle":"SARS-CoV-2 engages replication and inflammasome activation through lipid remodeling via SREBPs","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAs the worldwide pandemic of coronavirus disease 2019 (COVID-19) enters its third year with more than 630\u0026nbsp;million cases worldwide, it still poses significant challenges for patients, families and health systems. SARS-CoV-2, like other viruses, are obligate intracellular pathogens that make use of the host's cellular metabolic machinery to meet their biosynthetic needs. A better understanding of the host factors and pathways used by SARS-CoV-2, potentially common to other viruses, which are essential for execution of their life cycles, could contribute to potential targets for therapeutic intervention, such as broad-spectrum antiviral agents, to the development of therapies to treat COVID-19 and increase preparedness for potential future outbreaks.\u003c/p\u003e \u003cp\u003eLipids are essential in viral infection, as they are the structural basis of cell membranes and viral envelopes\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Accordingly, SARS-CoV-2 infection triggers major lipid metabolism remodeling in human cells\u003csup\u003e\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. SREBP is a family of transcription factors associated with the regulation of lipid homeostasis, controlling the expression of a broad range of enzymes of fatty acid (SREBP1) and cholesterol (SREBP2) metabolism. Both isoforms of SREBP are found to increase during viral infections, as observed in HCV, MERS-CoV, and SARS-CoV-2\u003csup\u003e2,6\u0026minus;8\u003c/sup\u003e. Indeed, increased expression and activation of the SREBP pathway are associated with disease severity in COVID-19 patients\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Moreover, SREBP activation is associated with the immune response through induced assembly of the inflammasome complex with the release of IL-1β\u003csup\u003e9\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we demonstrated that SARS-CoV-2 modulates lipid metabolism in Calu-3 cells by activating the SREBP transcription factor, favoring lipid remodeling through an increase in triglycerides and cholesterol, leading to the accumulation of LDs. Furthermore, double gene knockdown and the pharmacological inhibition of SREBPs with fatostatin blocked viral replication and proinflammatory cytokines, such as IL-1β and IL-18. In addition, SREBPs inhibition reduced caspase-1 activation and prevented cell death induced by SARS-CoV-2 infection. Our results reveal new details of SARS-CoV-2 infection on lipid metabolism and may supply new insights for understanding the pathology of COVID-19.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSARS-CoV-2 induces alterations in lipid metabolism\u003c/h2\u003e \u003cp\u003eViruses are intracellular parasites that can alter cell metabolism to favor their own maintenance and replication. Members of the Flaviviridae family\u003csup\u003e\u003cspan additionalcitationids=\"CR11 CR12 CR13\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e and the Coronaviridae family\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e are +\u0026thinsp;RNA viruses that can modify lipid metabolism in different cells, triggering lipid droplet (LD) formation, using these host organelles for different steps of their replicative cycle\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. To evaluate the effects of SARS-CoV-2 infection on lipid metabolism, we used type II pneumocytes (Calu-3 cells) infected with SARS-CoV-2 at an MOI of 0.01.\u003c/p\u003e \u003cp\u003eHere, we observed decreased expression of the precursor form and augmented activation (mature form) of the lipid transcription factors SREBP1 and SREBP2 24 h after infection with SARS-CoV-2 in Calu-3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). SREBP1 is involved in fatty acid metabolism, while SREBP2 controls cholesterol homeostasis\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Accordingly, we observed that SARS-CoV-2 infection upregulated pathways of LD biogenesis and maturation, such as DGAT1 and PLIN2, as well as pathways of both fatty acid (FASN) and cholesterol synthesis (ABCA1 and SOAT1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and C). Moreover, SARS-CoV-2 infection upregulated inflammatory pathways, with increases in IL-6, IL-1β and IL-10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). As previously observed in other cells\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, SARS-CoV-2 infection of Calu-3 cells induced LD biogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and E), with increased cholesterol (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF and G) and triacylglycerol accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH) after 48 hours of infection. Altogether, SARS-CoV-2 infection of Calu-3 cells triggered SREBP activation along with upregulation of genes and key proteins of lipid metabolism and increased cholesterol and triacylglycerol, which accumulated in LDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSrebfs Are Master Regulators Of Lipid Metabolism During Sars-cov-2 Infection\u003c/h3\u003e\n\u003cp\u003eTo investigate the functions of SREBPs in SARS-CoV-2 infection, we knocked down SREBF1 and SREBF2 with siRNA separately or in combination. The efficiency of the knockdown was confirmed by western blotting (Fig. S1A and S1B) and real-time PCR (Fig. S1D and S1E). Knockdown of SREBF1 and \u0026minus;\u0026thinsp;2 separately partially inhibited viral replication (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) but failed to protect Calu-3 cells from death (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Combined knockdown of both SREBFs demonstrated a significant reduction in viral replication in relation to the control group with scramble (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and protected against cell death, decreasing LDH release into the supernatant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). These data suggest that both SREBPs are important for SARS-CoV-2 infection and replication in Calu-3 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo gain insights into the contributions and mechanisms of SREBP1 and SREBP2 during SARS-CoV-2 infection in Calu-3 cells, we analyzed the expression of different genes involved in lipid metabolism regulated during SARS-CoV-2 infection after SREBFs knockdown. SREBF1 knockdown downregulated genes related to LD formation, such as DGAT1 and FASN, and inflammatory genes, such as IL-1β (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, and S2A and B). SREBF2 knockdown downregulated genes involved in cholesterol metabolism, such as SOAT1 and inflammatory genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, and S2A and B). Moreover, the combined knockdown of SREBF1 and \u0026minus;\u0026thinsp;2 downregulated all the genes previously observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, and S2A and B). Altogether, these data reinforce the concept that both genes that encode SREBPs are important and complementary to modulate the lipid and inflammatory profile during SARS-CoV-2 infection.\u003c/p\u003e \u003cp\u003ePrevious findings have established an important role for LDs in SARS-CoV-2 infection at least in part due to their roles in the biogenesis of replication organelles\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. To evaluate the role of SREBPs in LD formation, Calu-3 cells were stained with a Lipidtox LD probe and J2 antibody for double-stranded RNA (dsRNA) labeling, and the fluorescent area of each marker was quantified. SREBF1, but not SREBF2 knockdown, was able to significantly reduce viral replication sites and LD accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-F). The combined knockdown of SREBF1 and \u0026minus;\u0026thinsp;2 was more effective in reducing the SARS-CoV-2 replication sites and LD accumulation compared with the cells infected with scramble (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-F). Moreover, knockdown of the DGAT1 enzyme was evaluated, and we observed a reduction in LD biogenesis and viral replication sites, comparable to the effects of combined knockdown for SREBF1 and SREBF2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B and D-F).\u003c/p\u003e \u003cp\u003eAltogether, these results indicate that during SARS-CoV-2 infection, the genes that encode the SREBPs are master regulators of lipid metabolism and participate in different processes, such as inflammatory cytokines, cell death, viral replication, and LD accumulation, in Calu-3 cells. Indeed, the mechanisms that control DGAT1 and LD accumulation are downstream and largely dependent on the activation and transcriptional regulation of SREBPs.\u003c/p\u003e\n\u003ch3\u003eFatostatin Inhibits Both Srebps Activation And Reduces The Replication Sites Of Sars-cov-2\u003c/h3\u003e\n\u003cp\u003eTo further analyze the role of the SREBPs during SARS-CoV-2 infection, we used fatostatin, a pharmacologic inhibitor of the ER-Golgi translocation of SREBPs through binding to their escort protein, SREBP cleavage-activating protein (SCAP) \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Of note, activation of all SREBP isoforms is controlled by SCAP. Fatostatin has been demonstrated to inhibit the viral replication of several viruses from the Flaviviridae family, including WNV and ZIKV \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFirst, we investigated whether fatostatin inhibits SREBPs in our model. We pretreated Calu-3 cells with fatostatin for 2 h before SARS-CoV-2 infection and maintained the treatment for all times of infection until the analyses of the experiment. Fatostatin treatment inhibited the processing and activation of both SREBP1 and SREBP2 during infection, leading to the accumulation of the precursor form of SREBPs in Calu-3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). Furthermore, the blockage of SREBP1 and SREBP2 activation by treatment with fatostatin during SARS-CoV-2 infection reduced cholesterol accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and D), as observed by filipin III labeling and triacylglycerol accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). As shown in Fig. S3A, treatment with fatostatin was devoid of cytotoxicity at the doses used.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo confirm that the inhibition of SREBPs by fatostatin treatment could affect LD accumulation, Calu-3 cells were pretreated with fatostatin, and LD accumulation was analyzed 48 h after infection. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, treatment with fatostatin reduced LD accumulation after SARS-CoV-2 infection, similar to the DGAT1 inhibitor A922500 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF and G), as previously observed in other cell types \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Indeed, we observed a reduction in the protein expression related to the LD form and maturation (DGAT1 and PLIN2) in Calu-3 cells treated with fatostatin (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH and I). Thus, this confirms that SREBPs are master regulators in the process of LD accumulation in Calu-3 cells infected with SARS-CoV-2 through the increase in the DGAT1 enzyme and PLIN2, favoring LD biogenesis.\u003c/p\u003e \u003cp\u003eSARS-CoV-2 may explore host lipid metabolism to favor its replication using LDs as an energy source for its own replication \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. To investigate the role of SREBP in the formation of SARS-CoV-2 replication sites, we labeled Calu-3 cells with a J2 clone for dsRNA and BODIPY for LD and quantified the labeled area of each marker. First, we observed an increase in dsRNA and BODIPY during SARS-CoV-2 infection, and when we treated the cells with fatostatin or A922500, we observed a reduction in the labeled area (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B). Moreover, almost all cells were positive for dsRNA when they were infected with SARS-CoV-2, and both treatments reduced the number of dsRNA-positive cells up to 20% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Considering the double-positive cells (dsRNA and BODIPY labeling), we observed that almost all cells infected with SARS-CoV-2 presented a close association with LDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD and E), but treatment with fatostatin or A922500 reduced the double-positive cells up to 10%, reducing the association of dsRNA with LDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD and E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe observed through electron microscopy that SARS-CoV-2-infected cells presented an increase in LDs (*), and a close association of the viral particles (arrow) with LDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF and S4B-D), as previously shown \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Moreover, SARS-CoV-2 infection induced a clear signal of cell injury in Calu-3 cells, as indicated by the presence of myelin figures (arrowhead) (Fig. S4B-D), in comparison with control cells (Fig. S4A). Furthermore, fatostatin or A922500 treatment reduced LD accumulation and the presence of viral particles in comparison with the cells infected and treated with vehicle (DMSO) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eAltogether, our data suggest that SARS-CoV-2 modulates the lipid metabolism of Calu-3 cells in favor of increasing the activation of SREBPs, inducing LD accumulation, and promoting a close association with dsRNA and viral replication sites.\u003c/p\u003e\n\u003ch3\u003eFatostatin Protects Calu-3 Cells From Death During Sars-cov-2 Infection\u003c/h3\u003e\n\u003cp\u003eViral infections can cause alterations in cell homeostasis, leading the virus to use the cellular compounds for your own benefit, increasing viral replication that can induce a process of cell death using cellular resources or by heightened inflammatory response. SARS-CoV-2 infection has the capacity to induce cell death in different cells, such as human monocytes, by the liberation of LDH into the extracellular space \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Here, we observed the cell morphology and measured the LDH release of Calu-3 cells infected with SARS-CoV-2 after 48 h of infection. Our data showed that infection with SARS-CoV-2 was able to alter the cell monolayer, causing damage to the cellular membrane and increasing LDH release into the supernatant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and B). Treatment with fatostatin alone did not interfere with cell morphology or LDH release, while treatment during SARS-CoV-2 infection was able to decrease damage, block cell death, and reduce LDH release in Calu-3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs previously observed with the knockdown of both genes that encode SREBPs, treatment with fatostatin, which inhibits the activation of both SREBPs, was able to reduce viral replication by 2 logs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), presenting viral inhibition of almost 90% and with a 50% antiviral concentration (IC50) of 14.15 \u0026micro;M (Fig. S3A). Similarly, pretreatment with A922500 inhibited viral replication (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), with viral inhibition of almost 100% and an IC50 of 3.88 \u0026micro;M (Fig. S3B).\u003c/p\u003e \u003cp\u003eTo analyze other inhibitors of SREBP, we evaluated two other inhibitors, betulin and AM580. Neither of the inhibitors presented any cytotoxicity in Calu-3 cells (Fig. S3C and D). To observe the effects of these inhibitors during SARS-CoV-2 infection, we analyzed the protection from cell death and viral replication. The inhibitor Betulin did not protect against cell death, as observed by LDH release, and did not affect viral replication (Fig. S3C), but the inhibitor AM580 was able to reduce viral replication without altering cell death (Fig. S3D).\u003c/p\u003e\n\u003ch3\u003eSrebp Inhibition Blocks Inflammasome Complex Activation And Pyroptosis Death During Sars-cov-2 Infection\u003c/h3\u003e\n\u003cp\u003eSeveral works associate SREBP activation with the inflammasome, culminating in the release of proinflammatory cytokines, such as IL-1β \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In previous work, we already demonstrated that human monocytes infected with SARS-CoV-2 activate caspase-1 and promote IL-1β release with activation of GSDMD1, which suggests a process of pyroptosis \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In addition, other works supporting these data have already demonstrated inflammasome NLRP3 assembly and activation in human primary monocytes infected with SARS-CoV-2 as well as PBMCs from COVID-19 patients \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo investigate the relationship between SREBP activation and the cell death process by pyroptosis during SARS-CoV-2 infection, we evaluated caspase-1 activation by staining with FAM-YVAD-FLICA in Calu-3 cells. Indeed, infected cells presented an increase in activated caspase-1 during SARS-CoV-2 infection, and when SREBPs were inhibited by treatment with fatostatin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and E) or A922500 (Fig. S5A), a reduction in activated caspase-1 was observed by fluorescence microscopy and flow cytometry, which was also confirmed by western blotting (Fig. S5B and C).\u003c/p\u003e \u003cp\u003eMoreover, we observed that the cells infected with SARS-CoV-2 presented an increase in GSDMD1 activation, a protein related to membrane pore formation that is activated during the process of pyroptosis. Moreover, treatment with fatostatin was able to reduce the activation of GSDMD1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Altogether, these data suggest that activation of SREBPs during SARS-CoV-2 infection is important to promote an increase in activated caspase-1, contributing to the formation of membrane pores by GSDMD1.\u003c/p\u003e \u003cp\u003eIt is already well known that SARS-CoV-2 promotes an exacerbated inflammatory response that aggravates cell damage and consequently cell death with the release of several cytokines \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Here, we observed an increase in the main inflammatory cytokines produced by the activation of caspase-1, such as IL-1β and IL-18 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). We also observed an increase in other proinflammatory cytokines, such as IL-6 and TNFα, and the chemokine CxCL-10 (Fig. S5D and E). Consistent with prior data, the inhibition of SREBPs by fatostatin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG and S5D) or A922500 (Fig. S5E) was able to reduce all cytokines and chemokines previously observed. Altogether, our data suggest that SREBPs participate in the inflammatory process during SARS-CoV-2 infection in Calu-3 cells.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAccumulating evidence indicates that SARS-CoV-2 infection promotes major host cellular lipid metabolism reprogramming to enhance fitness and replication assembly capacity. In this context, lipid metabolism dysregulation is associated with disease severity \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. However, the mechanisms and metabolic pathways explored by SARS-CoV-2 to support its replication within host cells are still poorly understood. The findings presented here provide direct evidence that the SREBP lipid synthesis pathway is critically required for SARS-CoV-2 infection, replication, and amplification of the inflammatory response. Here, we demonstrate that SREBP participates in SARS-CoV-2 infection at two levels of host pathogen interaction: first, they are essential transcriptional regulators of the major host metabolic pathways that support virus replication; and second, they are central in the amplification of inflammatory mediator production and cell death through activation of inflammasomes.\u003c/p\u003e \u003cp\u003eSeveral viruses alter lipid metabolism through the increase in expression and/or activation of transcription factors, such as SREBP1 \u003csup\u003e6,12\u003c/sup\u003e and SREBP2 \u003csup\u003e19\u003c/sup\u003e. The presence of cholesterol in COVID-19 plasma patients is increased through SREBP2 activation \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, while the accumulation of triglycerides is associated with the activation of SREBP1 \u003csup\u003e6\u003c/sup\u003e, which has already been observed in cells infected with SARS-CoV-2 \u003csup\u003e2\u003c/sup\u003e. In our data, SARS-CoV-2 infection in Calu-3 cells activates both SREBP isoforms, suggesting that the infection reprograms the cells toward a lipogenic phenotype, increasing the triglyceride and cholesterol pathways that were shown to be required for the SARS-CoV-2 viral cycle \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Accordingly, enhanced expression and/or activation of both SREBPs has been reported during infection with respiratory viruses, such as MERS-CoV, SARS-CoV, and SARS-CoV-2 \u003csup\u003e2,6,12\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eConsistently, targeting the lipid biosynthetic SREBPs pathways was shown to present antiviral properties \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Knockdown of genes that encode SREBP1 and SREBP2 proteins during SARS-CoV-2 infection downregulates lipid metabolism and directly impacts LD biogenesis and SARS-CoV-2 replication, reducing cell death. Using a double knockdown for both SREBPs, the effects were aggravated and were similar, which occurs during MERS-CoV infection \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The knockdown of the gene that encodes the DGAT1 protein presents effects resembling double knockdown for both SREBPs. This suggests that the SREBPs present a special contribution to viral replication, where these transcription factors are able to modulate lipid metabolism by increasing LD through the DGAT1 enzyme, altering SARS-CoV-2 replication.\u003c/p\u003e \u003cp\u003eOnce double knockdown of SREBPs was more efficient in reducing viral replication and LD biogenesis, we used the pharmacological inhibitor fatostatin, which was predicted to inhibit both SREBP isoforms \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, to analyze the effects of these transcription factors during SARS-CoV-2 infection on lipid metabolism. Here, the double inhibition of the SREBPs activated form was confirmed by western blotting, and a downregulation in lipid metabolism was noted in cells treated with fatostatin during SARS-CoV-2 infection, demonstrating that the inhibitor not only inhibits SREBPs activation but also reduces the activation of lipid metabolism, such as triglycerides and cholesterol storage, caused by viral infection.\u003c/p\u003e \u003cp\u003eThe molecular mechanisms involved during LD biogenesis are a highly coordinated process, requiring new lipid syntesis and lipid remodeling, but these processes during inflammation and infection need to be better understood. As observed in several studies, LDs are a key organelle during the +\u0026thinsp;RNA virus replicative cycle \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Here, we observed that treatment with fatostatin reduces LD biogenesis and protein expression related to LD maturation, such as DGAT1 and PLIN2. This suggests that the inhibition of SREBPs is important during SARS-CoV-2 infection for LD biogenesis. Furthermore, Calu-3 cells infected with SARS-CoV-2 present strong labeling for dsRNA, which seems to be correlated with LDs, as previously observed in VERO E6 cells \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. It is important to consider that LDs may participate as replication sites, but other cellular compartments may also have an important role in viral replication. Indeed, recent studies have uncovered the mechanisms of LD recruitment to viral replication compartments with bidirectional content exchange and essential functions in replication and virus particle assembly \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Of note, LD accumulation was also observed in Type II pneumocytes undergoing cell death with characteristics of pyroptosis in lung tissue from autopsy of COVID deceased patients. \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eSREBPs are crucial for the replication of several viruses and have been related to the increase in LD biogenesis \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Moreover, pharmacological inhibition of SREBPs with betulin reduces the viral replication and LD biogenesis of MERS-CoV \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Here, we found that the expression of the precursor form of SREBPs is reduced and that the mature form (active), which enters the nucleus, remains highly increased during infection, were downregulated by fatostatin treatment, contributing to LD remodeling in Calu-3 cells. Inhibitors of lipid metabolism, such as fatostatin and A922500, reduced the viral replication observed by dsRNA labeling. Moreover, the treatments reduced the proximity of dsRNA to LDs. Thus, SREBPs are crucial for LD accumulation through the DGAT1 enzyme. Future studies that target proteins of LD biogenesis and specific proteins of SARS-CoV-2 would be interesting to probe and understand the role of LDs in the SARS-CoV-2 replication cycle.\u003c/p\u003e \u003cp\u003eUnder homeostatic conditions, the C-terminal domain of SREBPs binds to SREBP cleavage-activating protein (SCAP) in the endoplasmic reticulum (ER) membrane. This complex interacts with insulin-induced gene 1 protein (INSIG1) \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, and in high levels of cholesterol, INSIG becomes stable and binds to SREBP-SCAP, creating a complex retained in the ER membrane. In contrast, when cholesterol levels are reduced, INSIG is rapidly degraded by the ubiquitin‒proteasome system, and the SREBP-SCAP complex is cleaved and directed to the nucleus for sterol regulatory elements (SREs) to modulate lipid metabolism \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo analyze the mechanism of SREBP activation during SARS-CoV-2, three inhibitors were used for the activation of SREBPs. Fatostatin inhibitor acts to prevent SREBPs-SCAP cleavage in the ER \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Betulin blocks the degradation of INSIG protein and inhibits SREBPs cleavage for SCAP in the ER, and AM580 acts as an agonist of retinoic acid, blocking the association of SREBPs with SREs in the nucleus \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. In this context, our results suggest that SARS-CoV-2 induces SREBP through an INSIG-independent mechanism since betulin did not affect viral replication (Fig. S3C), but AM580 is able to affect viral replication (Fig. S3D), although the inhibition is more significant using fatostatin, showing a different mechanism than that observed during MERS-CoV infection \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn addition to lipid metabolism reprogramming, SARS-CoV-2 causes an uncontrolled inflammatory response associated with an increase in the cell death process \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Here, we observed an increase in proinflammatory cytokines and chemokines during SARS-CoV-2 infection in Calu-3 cells. The inhibition of SREBPs or DGAT1 activity significantly reduced the inflammatory cytokine response in epithelial cells, confirming previous data from our group that DGAT1 and LD accumulation are involved in the inflammatory response amplification during SARS-CoV-2 infection in human monocytes \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. This finding corroborates the well-established role of LDs in inflammation and innate immunity \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e and supports a role for LD biogenesis in the heightened inflammatory production triggered by SARS-CoV-2, and drugs that target SREBPs or the DGAT1 enzyme may have beneficial effects on disease pathogenesis.\u003c/p\u003e \u003cp\u003eAccumulating evidence indicates a central role for NLRP3 inflammasome activation during SARS-CoV-2 infection contributing to increased cytokine release and the cell death process of pyroptosis \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Indeed, SARS-CoV-2 infection causes caspase-1 activation with increased IL-1β and IL-18 release and cleaved GSDMD1, which causes membrane pore formation with the extravasations of intracellular content \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Moreover, inflammasome activation has been implicated as a major determinant associated with severity and mortality in COVID-19 patients \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere, we add another layer to the mechanisms of SARS-CoV-2-induced inflammasome activation by demonstrating a required role for SREBP in this process. Accordingly, SREBP2-SCAP involvement in inflammasome activation has been previously demonstrated in macrophages by acting as a signaling hub facilitating inflammasome assembly upon nigericing stimulation of LPS-primed macrophages \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. SREBP may participate in inflammasome activation by direct or indirect effects. Activation of inflammasomes may occur through the recognition of cholesterol crystals \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, which presents a close relationship with LDs \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e and can represent an important link between cholesterol metabolism and inflammation in COVID-19 pathogenesis, but direct effects of SRBP2-SCAP on inflammasome assembly have been proposed \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Recently, it was demonstrated that viral NSP6 of SARS-CoV-2 induces the activation of the inflammasome complex NLRP3 through lysosome acidification that triggers ATP6AP1, causing autophagic flux stagnation \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Interestingly, SARS-CoV-2-derived NSP6 localizes to LDs and is involved in LD recruitment and favors viral replication \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. An intriguing possibility is that NSP6-triggered lipid remodeling and inflammasome activation are connected through the SREBPs pathway, favoring caspase-1 activation and release of IL-1β, leading to cell death by pyroptosis.\u003c/p\u003e \u003cp\u003eIn summary, our data demonstrated that SARS-CoV-2 directly affects lipid metabolism, activates SREBPs to accumulate triglycerides in LDs, and increases intracellular cholesterol. Genetic knockdown or pharmacological inhibition of SREBPs prevents reprogramming of lipid metabolism, reducing LD biogenesis, viral replication, assembly, and establishment of the infection. Moreover, the SREBPs induce inflammasome assembly, caspase-1 activation and pore formation by GSDMD1 with the release of proinflammatory cytokines, such as IL-1β and IL-18 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This finding supports the hypothesis that SARS-CoV-2 alters lipid metabolism through SREBP activation, favoring their fitness, replication and pathogenesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOur findings contribute to a better understanding of how SARS-CoV-2 uses host lipid metabolism for its own benefit and contributes to the inflammatory response. Further studies are necessary to better understand the mechanisms and the importance of SREBPs during SARS-CoV-2 infection and how the crosstalk between SREBPs and the inflammasome may contribute to triggering the immune response. This study may provide new insights into lipid metabolic pathways and suggest a potential strategy to reduce viral replication and the uncontrolled inflammatory response during COVID-19 pathogenesis.\u003c/p\u003e"},{"header":"Material And Methods","content":"\u003cp\u003e \u003cb\u003eCells and reagents.\u003c/b\u003e The human lung epithelial adenocarcinoma cell line (Calu-3 - ATCC/HTB-55) and African green monkey kidney (Vero subtype E6) were cultured in high glucose DMEM supplemented with 10% fetal bovine serum (FSB, HyClone, Logan, Utah) and 100 U/mL penicillin‒streptomycin (P/S; GIBCO) and were incubated at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eVirus infection and virus titration.\u003c/b\u003e Nasopharyngeal swab samples were collected from confirmed cases from Rio de Janeiro/Brazil (GenBank accession no. MT710714). SARS-CoV-2 was amplified in Vero-E6 cells in high-glucose DMEM supplemented with 2% FBS for 2 to 4 days of infection and incubated at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e. Virus titers were determined by the tissue culture infectious dose at 50% (TCID\u003csub\u003e50\u003c/sub\u003e/mL), and the virus stocks were kept in -80\u0026deg;C freezers. All specimens were handled under the laboratory biosafety guidance required involving SARS-CoV-2 by the World Health Organization (WHO) at biosafety level 3 (BSL3) multiuser facility from Funda\u0026ccedil;\u0026atilde;o Oswaldo Cruz/Fiocruz (Rio de Janeiro, RJ, Brazil).\u003c/p\u003e \u003cp\u003eAll cells were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01 with or without pretreatment with pharmacological inhibitors of SREBP activation, such as fatostatin (Cayman 13562), AM580 (Sigma A8843) and betulin (Cayman 11041), and we used the pharmacological inhibitor of DGAT1, A922500 (Sigma A1737), for two hours and maintained the treatment after the infection. During knockdown experiments, the cells were infected at the same MOI, and 24 h prior to infection, the cells were transfected with the siRNAs for SREBF1 (s129), SREBF2 (s27), DGAT-1 (16567) and the negative control with scramble RNA (4390844) according to the manufacturers' instructions (Thermo Fisher Scientific). For virus titration, we performed a plaque-forming assay in Vero-E6 cells seeded in 96-well plates. Cell monolayers were infected with different dilutions of the supernatant containing the virus for 1 h at 37\u0026deg;C. The cells were overlaid with high glucose DMEM containing 2% FBS and 2.4% carboxymethylcellulose. After 3 days, the cells were fixed with 10% formaldehyde in PBS for 3 h at room temperature. Cell monolayers were stained with 0.04% crystal violet in 20% ethanol for 1 h. The viral titer was calculated from the count of plaques formed in the wells corresponding to each dilution and expressed as plaque forming units per mL (PFU/mL).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell viability assay.\u003c/b\u003e Calu-3 cells were seeded in 96-well plates. Then, the cells were treated with a range of concentrations of the inhibitors for 24 h and 48 h. Then, the cells were fixed using 3.7% formaldehyde for 20 mins. Cell monolayers were stained with 1% crystal violet in 20% ethanol for 10 min. The cells were washed with water, and crystal violet was extracted using methanol. The crystal violet was read in a spectrophotometer at a wavelength of 595 nm.\u003c/p\u003e \u003cp\u003e \u003cb\u003eUltrastructural analysis of cells by transmission electron microscopy.\u003c/b\u003e For ultrastructural analysis, the infected and noninfected Calu-3 cell monolayers were trypsinized at 24 and 48 hours postinfection or cultivation, respectively. Cell suspensions were fixed in 2.5% glutaraldehyde in sodium cacodylate buffer (0.2 M, pH 7.2), postfixed in 1% buffered osmium tetroxide, dehydrated in acetone, embedded in epoxy resin, and polymerized at 60\u0026deg;C over the course of three days \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Ultrathin sections (50\u0026ndash;70 nm) were obtained from the resin blocks. The sections were picked up using copper grids (300 mesh) and observed using a Hitachi HT 7800 (Hitachi, Tokyo, Japan) transmission electron microscope.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLipid droplet staining.\u003c/b\u003e Calu-3 cells were seeded on coverslips. Cells infected or not infected were fixed using 3.7% formaldehyde, and the LDs were stained with 0.3% Oil Red O (diluted in 60% isopropanol) at room temperature for 2 min. The coverslips were mounted on slides using antifade mounting medium (VECTASHIELD\u0026reg;). DAPI staining (1 \u0026micro;g/mL) for 5 min was used for nuclear recognition. Fluorescence was analyzed by fluorescence microscopy with a 100x objective lens (Olympus, Tokyo, Japan). The numbers of LDs were automatically quantified from 15 aleatory fields by ImageJ software analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunofluorescence staining.\u003c/b\u003e Calu-3 cells were seeded on coverslips and fixed using 3.7% formaldehyde after 48 h of infection for 20 min at room temperature. Cells were rinsed three times with PBS with 0.1 M CaCl\u003csub\u003e2\u003c/sub\u003e and 1 M MgCl\u003csub\u003e2\u003c/sub\u003e (PBS/CM) and then permeabilized with 0.1% Triton X-100 plus 0.2% BSA in PBS/CM for 10 min (PBS/CM/TB). Double-RNA was labeled by the mouse monoclonal antibody J2 clone Scicons \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e at a 1:500 dilution overnight, followed by a mouse anti-IgG-Dylight 550 at a 1:1000 dilution for 1 h with 0.2 \u0026micro;g/mL BODIPY493/503 dye for 5 min for LD staining. In addition, mouse anti-IgG-Dylight 488 was used at a 1:1000 dilution for 1 h with LipidTox Neutral Red dye (Thermo Fisher Scientific - H34476) at a dilution of 1:1000 for 30 min for LD staining. Slides were mounted using an antifade mounting medium (VECTASHIELD\u0026reg;). Nuclear recognition was based on DAPI staining (1 \u0026micro;g/mL) for 5 min. Fluorescence microscopy was analyzed with a 100x objective lens (Olympus, Tokyo, Japan). The fluorescent area was quantified from 15 aleatory fields by ImageJ software analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTriglyceride and cholesterol measurements.\u003c/b\u003e Calu-3 cells were harvested after 48 h of SARS-CoV-2 infection using ice-cold lysis buffer pH 8.0 (1% Triton X-100, 2% SDS, 150 mM NaCl, 10 mM HEPES and 2 mM EDTA in the presence of protease inhibitor cocktail - Roche). For analysis of triglyceride levels, 80 \u0026micro;g of protein/sample of the cell lysates was extracted with chloroform/methanol/water 1:2:0,8 (v/v/v) in glass tubes. The samples were vortexed for 5 min and 5 min for 1 h. Then, they were centrifuged at 3 000 rpm for 20 min. The aqueous phase of each sample was collected and transferred to another glass tube, the pellet was resuspended in chloroform/methanol/water 1:2:0,8 (v/v/v), and the last steps were repeated. The aqueous phase was obtained and transferred to the same glass tubes as the first aqueous phase. Then, chloroform/water 1:1 (v/v) was added and vortexed for 10 seconds. After this step, the samples were centrifuged at 3 000 rpm for 30 min, and two phases were obtained. A lower phase was collected in a glass tube, evaporated with nitrogen gas, and resuspended in chloroform/methanol 1:2 (v/v). The triglyceride levels were quantified using Triglycerides Liquiform (87\u0026thinsp;\u0026minus;\u0026thinsp;2/100 Labtest kit) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cp\u003eFor cholesterol measurements, Calu-3 cells were seeded in coverslips and after 48 h were fixed using 3.7% formaldehyde for 20 min at room temperature. Then, the cells were washed three times with PBS and incubated with 20 \u0026micro;M glycine for 10 min. Next, the cells were stained with 50 \u0026micro;g/mL filipin III (Cayman #70440) for two hours at room temperature in the dark. For nuclear recognition, the cells were labeled with 1 \u0026micro;M TO-PRO-3 (Thermo Fisher Scientific - T3605) for 10 min. Slides were mounted using an antifade mounting medium (VECTASHIELD\u0026reg;). Fluorescence was analyzed by fluorescence microscopy with a 40x objective lens (Olympus, Tokyo, Japan), and the fluorescent area was quantified from 15 aleatory fields by ImageJ software analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSDS‒PAGE and Western blot.\u003c/b\u003e After 24 h of SARS-CoV-2 infection, Calu-3 cells were harvested using ice-cold lysis buffer pH 8.0 containing 1% Triton X-100, 2% SDS, 150 mM NaCl, 10 mM HEPES, and 2 mM EDTA in the presence of protease inhibitor cocktail (Roche). The protein levels were measured by a bicinchoninic acid assay protein kit (Thermo Fisher Scientific). 30 \u0026micro;g of protein/sample was heated at 100\u0026deg;C for 5 min in Laemmli buffer pH 6.8 (20% β-mercaptoethanol; 370 mM Tris base; 160 \u0026micro;M bromophenol blue; 6% glycerol; 16% SDS) and resolved by electrophoresis on an SDS-containing 10% polyacrylamide gel (SDS‒PAGE). Next, the separated proteins were transferred to nitrocellulose membranes and incubated in blocking buffer (5% nonfat milk, 50 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween 20). Membranes were probed overnight with the following antibodies: anti-SREBP1 (Proteintech 14088-1-AP), anti-SREBP2 (Proteintech 28212-1-AP), anti-DGAT1 (Proteintech 11561-1-AP), anti-PLIN2 (Proteintech 15294-1-AP), anti-GSDMD1 (Cell Signaling 97558) and anti-GAPDH (Proteintech 60004-1-1g). After washing, the membranes were incubated with IRDye - LICOR or HRP-conjugated secondary antibodies for 2 h at room temperature. All antibodies were diluted in blocking buffer. Signal detection was performed by Supersignal Chemiluminescence (GE Healthcare) or fluorescence imaging using the Odyssey system. The densitometries were analyzed using Image Studio Lite Ver 5.2 software.\u003c/p\u003e \u003cp\u003e \u003cb\u003eQuantitative real-time RT‒PCR assay.\u003c/b\u003e Monolayers from Calu-3 cells after 24 h of SARS-CoV-2 infection were harvested, and the total RNA from each sample was extracted using an SV total RNA isolation system kit according to the manufacturer's protocol (Promega). RNA concentration and purity were determined by a spectrophotometer (Nanodrop 2000) measuring absorbance at A260 and A280 nm, and RNA was stored at \u0026minus;\u0026thinsp;70\u0026deg;C in nuclease-free water. Total RNA (2 \u0026micro;g) was reverse transcribed in a 20 \u0026micro;l reaction mixture using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA) according to the manufacturer\u0026rsquo;s protocol. The cDNA was amplified in 10 \u0026micro;l of 1\u0026times; TaqMan universal PCR master mix with Predeveloped TaqMan assay primers and probes Perilipin-2 (PLIN2) Hs00605340_m1; DGAT1, Hs01020362_g1; Fatty Acid Synthase (FASN) Hs01005622_m1; SREBF1 Hs01088691_m1; SREBF2 Hs01081784_m1; patatin like phospholipase domain containing 2 (PNPLA2) Hs00386101_m1; Sterol O-Acyltransferase 1 (SOAT1) Hs00162077_m1; ATP-binding cassette transporter-1 (ABCA1) Hs01059118_m1; Interleukin-6 (IL-6) Hs00985639_m1; Interleukin-10 (IL-10) Hs00961622_m1; Interleukin-1β (IL-1β) Hs01555410_m1 and as endogenous control it was used the GAPDH Hs99999905_m1 according to manufacturer\u0026rsquo;s instruction (Thermo Fisher Scientific). Quantitative RT‒PCR was performed in a StepOne\u0026trade; Real-Time PCR System (Thermo Fisher Scientific). PCR products were analyzed in a comparative manner relative to the endogenous control GAPDH (ΔΔCt).\u003c/p\u003e \u003cp\u003e \u003cb\u003eMeasurements of inflammatory mediators and LDH activity.\u003c/b\u003e Calu-3 cell supernatants were obtained after 24 hours of SARS-CoV-2 infection with or without treatment with inhibitors. Cytokines and chemokines were measured in the supernatant by ELISA following the manufacturer's instructions (Duo set, R\u0026amp;D). Cell death was determined according to the activity of lactate dehydrogenase (LDH) in the culture supernatants using a CytoTox\u0026reg; Kit according to the manufacturer\u0026rsquo;s instructions (Promega, USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAssessment of activated caspase-1.\u003c/b\u003e After 48 h of infection, Calu-3 cells were stained to detect caspase-1 activation using fluorescent-labeled inhibitors of caspase‐1 activity (FAM‐YVAD‐FMK/FLICA) according to the manufacturer\u0026rsquo;s instructions (Bio-Rad). The fluorescence of caspase-1 activity, expressed as the percentage of activated cells, was evaluated by flow cytometry (FACSCalibur), and the generated data were analyzed with FlowJo. In parallel, the cells were seeded on coverslips; after 48 h of infection, the cells were labeled with FAM‐YVAD‐FMK/FLICA according to the manufacturer\u0026rsquo;s instructions. Then, the cells were fixed with 3.7% formaldehyde for 20 min at room temperature. The nuclei were stained with DAPI (1 \u0026micro;g/mL) for 5 min, and the coverslips were mounted using antifade mounting medium (VECTASHIELD\u0026reg;). Fluorescence was analyzed by fluorescence microscopy with a 100\u0026times; objective lens (Olympus, Tokyo, Japan).\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis.\u003c/b\u003e Data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM) of three and a maximum of six independent experiments. The paired two-tailed \u003cem\u003et\u003c/em\u003e test was used to evaluate the significance of the two groups. Multiple comparisons among three or more groups were performed by one-way ANOVA followed by Tukey\u0026rsquo;s multiple comparison test. p values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant when comparing SARS-CoV-2 infection to the uninfected control group (*) or SARS-CoV-2 infection with inhibitors groups (pharmacological inhibitors) or knockdown groups (siSREBF1 and \u0026minus;\u0026thinsp;2 and DGAT1) (#).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe authors thank the confocal imaging and Luminex\u0026nbsp;facility from the Rede de Plataformas Tecnol\u0026oacute;gicas FIOCRUZ and Dra. Milene Dias Miranda for assessments related to the BSL3 facility.\u0026nbsp;This work was supported by grants from Inova program Fiocruz, Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq) and Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior (CAPES) granted for Patr\u0026iacute;cia T. Bozza, Thiago Moreno L. Souza and Debora Ferreira Barreto Vieira.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceived the study: VCS, SSGD, JCS, PTB;\u003c/p\u003e\n\u003cp\u003eDesigned the experiments: VCS, SSGD, JCS, PTB;\u003c/p\u003e\n\u003cp\u003ePerformed the experiments: VCS, SSGD, JCS, IGAQ, IBGM, CQS, NFR, JRT, DFBV, MANS;\u003c/p\u003e\n\u003cp\u003eAnalyzed the data: VCS, SSGD, JCS, IBGM, DFBV, TMLS, PTB;\u003c/p\u003e\n\u003cp\u003eWrote the paper: VCS, SSGD, JCS, PTB.\u003c/p\u003e\n\u003cp\u003eAll authors reviewed and approved the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGirdhar, K. \u003cem\u003eet al.\u003c/em\u003e Viruses and Metabolism: The Effects of Viral Infections and Viral Insulins on Host Metabolism. 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Nucleic Acids Res \u003cb\u003e19\u003c/b\u003e, 2993\u0026ndash;3000 (1991). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1093/nar/19.11.2993\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1093/nar/19.11.2993\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2338983/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2338983/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSARS-CoV-2 and other ssRNA\u0026thinsp;+\u0026thinsp;viruses induce major cellular lipid rearrangements, exploiting the host's metabolic pathways to replicate. Sterol regulatory-element binding proteins (SREBPs) are a family of transcription factors that control lipid metabolism. SREBP1 is associated with the regulation of fatty acid metabolism, while SREBP2 controls cholesterol metabolism, and both isoforms are associated with lipid droplet (LD) biogenesis. SARS-CoV-2 infection has been shown to increase the expression and activation of SREBPs, but the impact of this pathway on the infection outcome is still poorly explored. Here, we evaluated the effect of pharmacologic and molecular inhibition of SREBP1 and SREBP2 in a SARS-CoV-2-infected lung epithelial cell line (Calu-3). We showed that SARS-CoV-2 infection induced the expression and activation of SREBP1 and SREBP2, enzymes of lipid metabolism and LD accumulation. Partial inhibition of SARS-CoV-2 replication and cell death was observed with the genetic knockdown of SREBP1 or SREBP2, while combined SREBP1 and SREBP2 knockdown led to synergistic inhibition. Combined SREBP1 and SREBP2 knockdown inhibited DGAT-1 expression and abrogated SARS-CoV-2-triggered LD formation in Calu-3 cells. Moreover, blockage of LD biogenesis by DGAT1 siRNA inhibited SARS-CoV-2 replication and cell death. Pharmacological inhibition with the dual SREBP activation inhibitor fatostatin reduced virus replication, cell death and LD biogenesis. In addition, we demonstrated that SARS-CoV-2 induced cell death by pyroptosis, with activation of caspase-1, cleavage of gasdermin D1 and release of IL-1β and IL-18 depending on SREBP activation. Collectively, our findings help to elucidate that SREBPs are crucial host factors required for viral replication, LD biogenesis and inflammasome activation and indicate SREBP as a host target for the development of antiviral strategies.\u003c/p\u003e","manuscriptTitle":"SARS-CoV-2 engages replication and inflammasome activation through lipid remodeling via SREBPs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-12-16 18:10:12","doi":"10.21203/rs.3.rs-2338983/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cddbdaea-7df2-4f8d-aeef-d87b414c61bd","owner":[],"postedDate":"December 16th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":17639475,"name":"Biological sciences/Immunology/Infection"},{"id":17639476,"name":"Health sciences/Pathogenesis/Infection"}],"tags":[],"updatedAt":"2023-01-23T11:41:51+00:00","versionOfRecord":[],"versionCreatedAt":"2022-12-16 18:10:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2338983","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2338983","identity":"rs-2338983","version":["v1"]},"buildId":"ApUGefWb6u5IBVtyqm6d5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}