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 family10–14 and the Coronaviridae family2,6 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 cycle2. 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. 1A). SREBP1 is involved in fatty acid metabolism, while SREBP2 controls cholesterol homeostasis15,16. 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. 1B and C). Moreover, SARS-CoV-2 infection upregulated inflammatory pathways, with increases in IL-6, IL-1β and IL-10 (Fig. 1C). As previously observed in other cells2, SARS-CoV-2 infection of Calu-3 cells induced LD biogenesis (Fig. 1D and E), with increased cholesterol (Fig. 1F and G) and triacylglycerol accumulation (Fig. 1H) 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. 1I).
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. 2A) but failed to protect Calu-3 cells from death (Fig. 2B). Combined knockdown of both SREBFs demonstrated a significant reduction in viral replication in relation to the control group with scramble (Fig. 2A) and protected against cell death, decreasing LDH release into the supernatant (Fig. 2B). 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. 2C, and S2A and B). SREBF2 knockdown downregulated genes involved in cholesterol metabolism, such as SOAT1 and inflammatory genes (Fig. 2C, and S2A and B). Moreover, the combined knockdown of SREBF1 and − 2 downregulated all the genes previously observed (Fig. 2C, 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 organelles2,17. 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. 2D-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. 2D-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. 2A, 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. 3A and B). Furthermore, the blockage of SREBP1 and SREBP2 activation by treatment with fatostatin during SARS-CoV-2 infection reduced cholesterol accumulation (Fig. 3C and D), as observed by filipin III labeling and triacylglycerol accumulation (Fig. 3E). 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. 3F 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. 3H 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. 4A 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. 4C). 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. 4D and E), but treatment with fatostatin or A922500 reduced the double-positive cells up to 10%, reducing the association of dsRNA with LDs (Fig. 4D 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. 4F 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. 4F).
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. 5A 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. 5A 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. 5C), 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. 5C), 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. 5D 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. 5F). 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. 5G). 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. 5G 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.