key: cord-1050993-z43c7z5y
authors: Williams, Caroline G.; Jureka, Alexander S.; Silvas, Jesus A.; Nicolini, Anthony M.; Chvatal, Stacie A.; Carlson-Stevermer, Jared; Oki, Jennifer; Holden, Kevin; Basler, Christopher F.
title: Inhibitors of VPS34 and fatty-acid metabolism suppress SARS-CoV-2 replication
date: 2021-07-20
journal: Cell Rep
DOI: 10.1016/j.celrep.2021.109479
sha: f4a7a888bb25f11dbbcd6b17c535da414177ec8b
doc_id: 1050993
cord_uid: z43c7z5y

Coronaviruses rely on host membranes for entry, establishment of replication centers, and egress. Compounds targeting cellular membrane biology and lipid biosynthetic pathways have previously shown promise as antivirals and are actively being pursued as treatments for other conditions. Here, we test small molecule inhibitors that target the PI3 kinase VPS34 or fatty acid metabolism for anti-SARS-CoV-2 activity. Our studies determine that compounds targeting VPS34 are potent SARS-CoV-2 inhibitors. Mechanistic studies with compounds targeting multiple steps up- and downstream of fatty acid synthase (FASN) identify the importance of triacylglycerol production and protein palmitoylation as requirements for efficient viral RNA synthesis and infectious virus production. Further, FASN knockout results in significantly impaired SARS-CoV-2 replication that can be rescued with fatty acid supplementation. Together, these studies clarify roles for VPS34 and fatty acid metabolism in SARS-CoV-2 replication and identify promising avenues for the development of countermeasures against SARS-CoV-2.

Inhibition of VPS34 or Fatty acid metabolism No treatment This allowed us to determine if the anti-viral activity of each compound impacted the viral life 183 cycle at steps pre-or post-entry. As indicated in Figure 4A¸ three conditions were tested: 1) 184 single treatment 1 hour prior to viral infection, with compound removed prior to infection; 2) 185 dosing at 2 hpi; and 3) dosing at 4 hpi. We observed that a single 5 μM treatment of VPS34-IN1 186 or PIK-III prior to infection inhibited SARS-CoV-2 replication, and inhibition could be observed 187 even when added up to 4 hpi ( Figure 4B ). In contrast, Orlistat or Triacsin C showed minimal 188 efficacy when removed prior to infection but remained inhibitory when added up to 4hpi. 189

Altogether, these data demonstrate that the VPS34 inhibitors likely act on viral entry and at later 190 steps in the replication cycle; and inhibition by Orlistat and Triacsin C occurs post-entry. Figure 1A -B). This 215 data further supports the importance of the class III PI3 kinase during SARS-CoV-2 replication. 216

To confirm that the compounds were able to inhibit VPS34 activity, we assessed in Huh7 cells 217 the effect of the inhibitors on GFP-2xFYVE localization to endosomes, indicating a loss of 218 Given that our data suggests a role for fatty acid metabolism, we set out to further clarify the 230 enzymatic steps required for SARS-CoV-2 replication. 231

To further evaluate the importance of de novo fatty acid synthesis, TOFA, a competitive 232 inhibitor of acetyl-CoA carboxylase (ACC), the enzyme directly upstream of FASN, and C75, an 233 additional inhibitor of FASN, were used (Halvorson and McCune, 1984; Kuhajda et al., 2000) . Lastly, the importance of the terminal steps of the fatty acid metabolism pathways was assessed 252 by inhibiting neutral lipid production and lipid droplet formation using A922500, a potent 253 inhibitor of diacylglycerol acyltransferase 1 (DGAT1) (Zhao et al., 2008) . Treatment with 254 A922500 inhibited SARS-CoV-2 with an IC50 value of 4.017 M ( Figure 6A , Supplemental 255 Figure 2F ). All together, these data suggest that protein palmitoylation and neutral lipid synthesis 256 are needed for efficient SARS-CoV-2 replication. 257

As time-of-addition studies implicated post-entry steps as targets of the compounds, we 260 sought to assess the status of sites of viral RNA synthesis, which can be detected with anti- 

CytoView-Z 96-well plate and allowed to stabilize overnight, as measured by electrical 504 resistance. A) Resistance was measured every minute over the course of 72 hours in wells that 505 were mock infected or infected with SARS-CoV-2 in 10-fold dilutions ranging from an MOI of Further information and requests for resources and reagents should be directed to and will be 593 fulfilled by the lead contact, Christopher F. Basler (cbasler@gsu.edu). 594

The Caco-2 NTC and Caco-2 FASN KO cell lines may be requested from the lead contact upon 596 request. 597

The published articled includes all datasets generated during this study. Raw data are available 599 from the lead contact upon request. This paper does not report original code. Any additional 600 information required to reanalyze the data reported in this paper is available from the lead contact or three sgRNAs are bioinformatically designed to work in a cooperative manner to generate 617 small, knockout-causing, fragment deletions in early exons. These fragment deletions are larger 618 than standard indels generated from single guides. The genomic repair patterns from a multi-619 guide approach are highly predictable based on the guide-spacing and design constraints to limit 620 off-targets, resulting in a higher probability protein knockout phenotype. 

To deliver CRISPR-Cas9 ribonucleoprotein (RNP) complexes, 10 pmol Streptococcus pyogenes 634 NLS-Sp.Cas9-NLS (SpCas9) nuclease (Aldevron Cat. #9212) was combined with 30 pmol total 635 synthetic sgRNA (Synthego,10 pmol each sgRNA) to form RNPs in 20uL total volume with SF 636

Buffer (Lonza Cat #V5SC-2002) and allowed to complex at room temperature for 10 minutes. 637

Cells were dissociated into single cells using TrypLE Express (Gibco), as described above, 638 J o u r n a l P r e -p r o o f resuspended in culture media and then counted. 100,000 cells per nucleofection reaction were 639 pelleted by centrifugation at 100 xg for 3 minutes. Following centrifugation, cells were 640 resuspended in transfection buffer according and diluted to 2*10 4 cells/µL. 5 µL of cell solution 641 was added to preformed RNP solution and gently mixed. Nucleofections were performed on a 642

Lonza 96-well nucleofector shuttle system using program CM-150. Immediately following 643 nucleofection each reaction was transferred to a tissue-culture treated 96-well plate containing 644 100µL normal culture media and seeded at a density of 50,000 cells per well. Transfected cells 645 were incubated following standard protocols. 646

Two days post-nucleofection, DNA was extracted from using DNA QuickExtract 648 (Lucigen Cat. #QE09050). Briefly, cells were lysed by removal of the spent media followed by 649 addition of 50 µL of QuickExtract solution to each well. Once the QuickExtract DNA Extraction 650

Solution was added, the cells were scraped off the plate into the buffer. Following transfer to 651 compatible plates, DNA extract was then incubated at 68C for 15 minutes followed by 95C for 652 10 minutes in a thermocycler before being stored for downstream analysis. 653

Amplicons for indel analysis were generated by PCR amplification AmpliTaq Gold 360 654 polymerase (Thermo Fisher Scientific Cat. #4398881) according to the manufacturer's protocol. 655

Primers were designed to create amplicons between 400 -800bp, with both primers at least 656 100bp distance from any of the sgRNA target sites. PCR products were cleaned-up and analyzed 657 by Sanger sequencing (Genewiz). Sanger data files and sgRNA target sequences were input into 658

Inference of CRISPR Edits (ICE) analysis (ice.synthego.com) to determine editing efficiency 659 and to quantify generated indels (Hsiau et al., 2019) . Percentage of alleles edited is expressed an 660 ice-d score. This score is a measure of how discordant the Sanger trace is before versus after the 661 edit. It is a simple and robust estimate of editing efficiency in a pool, especially suited to highly 662 disruptive editing techniques like multi-guide. Plaque assays were performed as previously described (Jureka et al., 2020) . Briefly, Vero 677 E6 cells were seeded onto 24-well plates 24 hours before infection. 100ul of SARS-CoV-2 serial 678 dilutions were added and adsorbed for 1 hour at 37°C with shaking at 15-minute intervals. After 679 the absorption period, 1 mL of 0.6% microcrystalline cellulose overlay (MCC; Sigma-Aldrich 680 435244, St. Louis, MO, USA) was added and plates were returned to the incubator for the 681 indicated amount of time. To stain plaque assays, MCC was removed by aspiration and 10% 682 neutral buffered formalin (NBF) added for one hour at room temperature. Formalin was removed 683

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and monolayers were washed with water and stained with 0.4% crystal violet. Plaques were 684 quantified and recorded as plaque forming units (PFU)/mL. 685Focus forming assays were performed as previously described (Gordon et al., 2020b; 686 Jureka et al., 2020) . Briefly, Vero E6 cells were pre-seeded in 96-well plates and grown to 687 confluency. 50 L of SARS-CoV-2 serial dilutions were added and adsorbed for 1 hour at 37°C. 688Post-adsorption, 50 L of 2.4% microcrystalline cellulose overlay (MCC; Sigma-Aldrich 689 435244, St. Louis, MO, USA) was added and plates were returned to the incubator for 24 hours. 690Cell monolayers were inactivated with 10% NBF for one hour at room temperature, washed with 691 deionized water and fixed/permeabilized with ice cold methanol containing 0.3% hydrogen 692 peroxide for 10 minutes at -20°C followed by 20 minutes at room temperature. Prior to primary 693 antibody addition, plates were washed with PBS and blocked for one hour at room temperature 694 with 5% milk. Primary antibody detection with SARS N diluted in milk occurred overnight at 695 4°C. The next day, plates were washed with PBS and anti-rabbit-HRP diluted in milk was added 696for 1 hour at room temperature. After final washing, plates were developed using TrueBlue HRP 697 substrate (SeraCare, Milford, MA, USA). Foci were quantified and graphed as focus forming 698 units (FFU)/mL. 699

Prior to cell plating, CytoView-Z 96-well electrode plates (Axion BioSystems, Atlanta, 701 GA, USA) were coated with 5 μg/mL human fibronectin (Corning, Tewksbury, MA, USA) for 1 702 hr at 37°C. After coating, fibronectin was removed and 100 μL of DMEM containing 10% FBS 703 was added to each well. The plate was then docked into the Maestro Z instrument to measure 704 impedance electrode baseline. Vero E6 cells were then plated to confluency (~75,000 cells/well) 705in the coated CytoView-Z plates and left at room temperature for 1 hour to ensure even coverage 706 J o u r n a l P r e -p r o o f of the well. Plates containing Vero E6 cells were docked into the Maestro Z for 24 hours at 37°C 707 with 5% CO 2 to allow the cells to attach and the monolayer to stabilize, as measured by 708 resistance, a component of impedance. The Maestro Z was used to monitor the resistance of the 709 monolayer as it formed, very similar to transepithelial electrical resistance (TEER) (Benson et 710 al., 2013) . In this study, resistance was measured at 10 kHz, which reflects both cell coverage 

For confocal microscopy analysis, cells were pre-seeded in 24-well plates on fibronectin 749 coated glass coverslips and allowed to grow to confluency. Cells were infected with SARS-CoV-750 2 at an MOI of 1 and media containing compound treatments was added 2 hours post infection. Figure 6 . Calu-3 cells were pre-seeded in 24-well format, allowed to grow to confluency, and infected at an MOI of 0.01. 2 hours post-infection cells were treated with VPS34-IN1 (5 µM), Orlistat (500 µM), Triacsin C (5 µM), TOFA (50 µM), 2-bromopalmitate (50 µM), A922500 (30 µM), Remdesivir (1 µM), or DMSO. At 4, 10, and 24 hpi total RNA was extracted from the cell monolayers, and 24 hpi supernatants were harvested for viral titers. Virus titers were determined by plaque assay (A). Levels of genomic RNA, subgenomic N RNA, and NSP14 RNA were quantified via qPCR (see materials and methods). Data are represented as fold change of RNA levels in infected compound treated samples versus infected DMSO treated samples (B-D). The virus titer at 24 hpi for compound treated cells is plotted alongside the qPCR data and presented as fold-change compared to titers from DMSO treated cells (orange bars). Data is representative of the mean and standard error of three replicates.