key: cord-0942592-4iwy1rki authors: da Silva Gomes Dias, Suelen; Soares, Vinicius Cardoso; Ferreira, André C.; Sacramento, Carolina Q.; Fintelman-Rodrigues, Natalia; Temerozo, Jairo R.; Teixeira, Lívia; Barreto, Ester; Mattos, Mayara; de Freitas, Caroline S.; Azevedo-Quintanilha, Isaclaudia G.; Manso, Pedro Paulo A.; Hottz, Eugenio D.; Pão, Camila R. R.; Bou-Habib, Dumith C.; Bozza, Fernando A.; Souza, Thiago M. L.; Bozza, Patrícia T. title: Lipid droplets fuel SARS-CoV-2 replication and production of inflammatory mediators date: 2020-08-29 journal: bioRxiv DOI: 10.1101/2020.08.22.262733 sha: 7a543da282a7244cfdd9859ccbe3a3a142d65dd0 doc_id: 942592 cord_uid: 4iwy1rki Viruses are obligate intracellular parasites that make use of the host metabolic machineries to meet their biosynthetic needs, identifying the host pathways essential for the virus replication may lead to potential targets for therapeutic intervention. The mechanisms and pathways explored by SARS-CoV-2 to support its replication within host cells are not fully known. Lipid droplets (LD) are organelles with major functions in lipid metabolism and energy homeostasis, and have multiple roles in infections and inflammation. Here we described that monocytes from COVID-19 patients have an increased LD accumulation compared to SARS-CoV-2 negative donors. In vitro, SARS-CoV-2 infection modulates pathways of lipid synthesis and uptake, including CD36, SREBP-1, PPARγ and DGAT-1 in monocytes and triggered LD formation in different human cells. LDs were found in close apposition with SARS-CoV-2 proteins and double-stranded (ds)-RNA in infected cells. Pharmacological modulation of LD formation by inhibition of DGAT-1 with A922500 significantly inhibited SARS-CoV-2 replication as well as reduced production of pro-inflammatory mediators. Taken together, we demonstrate the essential role of lipid metabolic reprograming and LD formation in SARS-CoV-2 replication and pathogenesis, opening new opportunities for therapeutic strategies to COVID-19. The coronavirus disease 2019 (COVID-19) caused by the novel severe acute 51 respiratory syndrome-coronavirus 2 (SARS-CoV-2) has rapidly spread in a pandemic, 52 representing an unprecedented health, social and economic threat worldwide (Lu et in lipid uptake such as CD36, the major transcriptional factors involved in lipogenesis, PPARγ and SREBP-1, and the enzyme DGAT-1, which is involved in triacylglycerol 114 synthesis, after 24 hours of infection. Altogether, these data suggest that SARS-CoV-2 is able to modulate multiple 116 pathways of lipid metabolism and remodeling, including in immune cells from COVID-117 19 patients, culminating in new LD assembling in human cells. During HCV infection, DGAT-1 was shown to be required for LD biogenesis, and to 123 control HCV protein trafficking to LDs (Camus et al., 2013) . Consequently, DGAT-1 124 inhibition blocks HCV use of LDs as replication platforms and inhibits viral particle 125 formation (Camus et al., 2013; Herker et al., 2010) . To assess the involvement of DGAT-126 1 in LD biogenesis during the SARS-CoV-2 infection, we treated A549 cells with 127 A922500, an inhibitor of the enzyme DGAT-1, for 2 hours at different concentrations 128 prior to SARS-CoV-2 infection and evaluated the LD biogenesis 48 hours after infection. As shown in figure 2A and B, treatment with A922500 inhibited in a dose dependent 130 manner the LD formation triggered by SARS-CoV-2 infection. Similarly, pre-treatment 131 with A922500 also blocked LD induced by SARS-CoV-2 in monocytes ( Fig. 2A and C) . For these experiments, we pre-treated the VERO cells with a range of 185 concentrations of DGAT-1 inhibitor A922500 (0.1 -50 µM) for 2 hours, followed by 186 infection with SARS-CoV-2 (MOI 0.01) for 24 hours. The supernatant was used to perform a plaque assay. Here, we observed that A922500 significantly inhibited SARS-188 CoV-2 replication in a dose dependent manner with an IC50 of 3.78 µM ( Fig. 4A and B) . 189 To gain insights on the interaction of the SARS-CoV-2 with LDs we labeled the 190 virus using immune serum from a convalescent COVID-19 patient that exhibit high anti- Collectively, our data suggest that SARS-CoV-2 uses LDs as a replication 211 platform, and establish that pharmacological targeting of LD formation inhibit SARS- CoV-2 replication, emerging as a potential strategy for antiviral development. Supplementary Figure 1 Herpes simplex type 1 activates glycolysis through engagement of the enzyme 6-515 phosphofructo-1-kinase (PFK-1) Differential 518 TLR2 downstream signaling regulates lipid metabolism and cytokine production triggered by 519 Mycobacterium bovis BCG infection CD36-specific antibodies block 522 release of HIV-1 from infected primary macrophages and its transmission to T cells Disrupting the association of hepatitis 525 C virus core protein with lipid droplets correlates with a loss in production of infectious virus Lipid body 528 function in eicosanoid synthesis: an update Lipid droplets in inflammation and cancer Diacylglycerol acyltransferase-1 localizes hepatitis C virus NS5A protein to lipid droplets and 534 enhances NS5A interaction with the viral capsid core Dengue 537 virus capsid protein binding to hepatic lipid droplets (LD) is potassium ion dependent and is 538 mediated by LD surface proteins Centers for Disease Control and Prevention Virus entry, assembly, budding, and membrane rafts CD36 is a co-receptor for hepatitis C virus E1 protein attachment Rotaviruses associate with cellular lipid 548 droplet components to replicate in viroplasms, and compounds disrupting or blocking lipid 549 droplets inhibit viroplasm formation and viral replication Protects Adipocytes from Lipid-Induced ER Stress during Lipolysis Elevated 556 Glucose Levels Favor SARS-CoV-2 Infection and Monocyte Response through a HIF-557 1α/Glycolysis-Dependent Axis Reovirus 559 outer capsid protein micro1 induces apoptosis and associates with lipid droplets, endoplasmic 560 reticulum, and mitochondria The cytokine storm 562 in COVID-19: An overview of the involvement of the chemokine/chemokine-receptor system CD36: a class B scavenger receptor 565 involved in angiogenesis, atherosclerosis, inflammation, and lipid metabolism Hepatitis C virus and lipid droplets: finding a niche Atazanavir 571 inhibits SARS-CoV-2 replication and pro-inflammatory cytokine production. bioRxiv Cytokine and chemokine profiles in lung 575 tissues from fatal cases of 2009 pandemic influenza A (H1N1): role of the host immune response 576 in pathogenesis Efficient hepatitis C virus particle formation requires diacylglycerol 579 acyltransferase-1 Emerging role of lipid droplets in host/pathogen interactions Viral Generated Inter-Organelle Contacts 583 Redirect Lipid Flux for Genome Replication Spatiotemporal Coupling of the Hepatitis 586 C Virus Replication Cycle by Creating a Lipid Droplet-Proximal Membranous Replication 587 Compartment Genomic characterisation and epidemiology of 2019 novel coronavirus: implications 590 for virus origins and receptor binding Bidirectional lipid 592 droplet velocities are controlled by differential binding strengths of HCV core DII protein West Nile virus replication requires fatty acid synthesis but is independent on 596 phosphatidylinositol-4-phosphate lipids Increased CD36 expression in vertically human immunodeficiency virus-infected 599 children unrelated to antiretroviral therapy The lipid droplet is an important organelle 602 for hepatitis C virus production Virus 604 factories: associations of cell organelles for viral replication and morphogenesis Fat, fight, and 607 beyond: The multiple roles of lipid droplets in infections and inflammation Role of macrophage cytokines in 610 influenza A virus infections Dengue virus capsid protein usurps lipid droplets 613 for viral particle formation Monoclonal antibodies to double-stranded RNA as probes of RNA structure in crude nucleic acid 616 extracts Proteomic and Metabolomic Characterization of COVID-19 Mesenchymal Stromal Cells Modulate Lipid Metabolism and Lipid Droplet Biogenesis via 622 AKT/mTOR -PPARγ Signalling in Macrophages Hepatitis C virus hijacks host lipid metabolism Function of membrane rafts in viral lifecycles and host 626 cellular response The 629 neuropeptides VIP and PACAP inhibit SARS-CoV-2 replication in monocytes and lung epithelial 630 cells, decrease the production of proinflammatory cytokines, and VIP levels are associated with 631 survival in severe Covid-19 patients. bioRxiv Phospholipid synthesis fueled by lipid droplets drives the structural development of poliovirus 634 replication organelles Targeting host lipid 636 synthesis and metabolism to inhibit dengue and hepatitis C viruses A new coronavirus associated with human respiratory disease in China Fatty acid synthase is up-regulated during hepatitis C virus infection and regulates 642 hepatitis C virus entry and production SREBP-dependent lipidomic reprogramming as a broad-spectrum antiviral 645 target Modulation of Lipid Droplet Metabolism-A Potential 647 Target for Therapeutic Intervention in Clinical course and risk factors for mortality of adult inpatients with China: a retrospective cohort study A pneumonia outbreak associated with a new coronavirus of probable bat 653 origin A Novel Coronavirus from Patients with Pneumonia in China