key: cord-0909312-uu883ofc authors: Kang, Yuan-Lin; Chou, Yi-Ying; Rothlauf, Paul W.; Liu, Zhuoming; Soh, Timothy K.; Cureton, David; Case, James Brett; Chen, Rita E.; Diamond, Michael S.; Whelan, Sean P. J.; Kirchhausen, Tom title: Inhibition of PIKfyve kinase prevents infection by Zaire ebolavirus and SARS-CoV-2 date: 2020-06-15 journal: bioRxiv DOI: 10.1101/2020.04.21.053058 sha: 8a8188c6578944389667d06617980d37c68f9921 doc_id: 909312 cord_uid: uu883ofc Virus entry is a multistep process. It initiates when the virus attaches to the host cell and ends when the viral contents reach the cytosol. Genetically unrelated viruses can subvert analogous subcellular mechanisms and use similar trafficking pathways for successful entry. Antiviral strategies targeting early steps of infection are therefore appealing, particularly when the probability for successful interference through a common step is highest. We describe here potent inhibitory effects on content release and infection by chimeric VSV containing the envelope proteins of Zaire ebolavirus (VSV-ZEBOV) or SARS-CoV-2 (VSV-SARS-CoV-2) elicited by Apilimod and Vacuolin-1, small molecule inhibitors of the main endosomal Phosphatidylinositol-3-Phosphate/Phosphatidylinositol 5-Kinase, PIKfyve. We also describe potent inhibition of SARS-CoV-2 strain 2019-nCoV/USA-WA1/2020 by Apilimod. These results define new tools for studying the intracellular trafficking of pathogens elicited by inhibition of PIKfyve kinase and suggest the potential for targeting this kinase in developing small-molecule antivirals against SARS-CoV-2. Transporter 1) -the late endosomal-lysosomal receptor protein (2). Proteolytic 67 processing is also required for severe acute respiratory syndrome coronavirus (SARS-68 CoV) (3, 4), and for the current pandemic SARS-CoV-2 (5). Lassa fever virus (LASV) 69 uses a different mechanism, binding alpha-dystroglycan at the plasma membrane (6), 70 for internalization with a subsequent pH-regulated switch that leads to engagement of 71 lysosomal associated membrane protein 1 (LAMP1) for membrane fusion (7). 72 Lymphocytic choriomeningitis virus (LCMV) also uses alpha-dystroglycan (6) and is 73 internalized in a manner that depends on endosomal sorting complexes required for also interfered with VSV-MeGFP-LCMV and VSV-MeGFP-ZEBOV infection (Fig. 1C) . 143 All of these viruses require low pH to trigger viral membrane fusion with the endosomal 144 membranes, and as expected, infection was fully blocked by Bafilomycin A1, which 145 inhibits the vacuolar type H + -ATPase (V-ATPase) acidification activity (Fig. 1C) . Using live-cell spinning disk confocal microscopy ( Fig. 3, 4) , we monitored the presence Table I; primers used for 525 screening are listed in Table II . Endosomal proteolysis of the Ebola virus glycoprotein is necessary for infection Ebola virus entry requires the cholesterol transporter 645 SARS coronavirus, but not human coronavirus NL63, 647 utilizes cathepsin L to infect ACE2-expressing cells Inhibitors of cathepsin L prevent severe acute 650 respiratory syndrome coronavirus entry Characterization of spike glycoprotein of SARS-CoV-2 on virus 653 entry and its immune cross-reactivity with SARS-CoV Identification of alpha-dystroglycan as a receptor for 655 lymphocytic choriomeningitis virus and Lassa fever virus Virus entry. Lassa virus entry requires a trigger-induced 658 receptor switch Old world 660 arenaviruses enter the host cell via the multivesicular body and depend on the 661 endosomal sorting complex required for transport Coincidence detection in phosphoinositide signaling PIKfyve, a mammalian ortholog of 665 yeast Fab1p lipid kinase, synthesizes 5-phosphoinositides. Effect of insulin Cloning, characterization, and expression of a novel Zn2+-binding FYVE finger-containing phosphoinositide 669 kinase in insulin-sensitive cells Mammalian cell morphology and 671 endocytic membrane homeostasis require enzymatically active phosphoinositide 672 5-kinase PIKfyve The mammalian phosphatidylinositol 3-phosphate 5-674 kinase (PIKfyve) regulates endosome-to-TGN retrograde transport Core protein machinery for mammalian 677 phosphatidylinositol 3,5-bisphosphate synthesis and turnover that regulates the 678 progression of endosomal transport. Novel Sac phosphatase joins the ArPIKfyve-679 PIKfyve complex Functional dissection of lipid and protein kinase 681 signals of PIKfyve reveals the role of PtdIns 3,5-P2 production for endomembrane 682 integrity A selective PIKfyve inhibitor blocks PtdIns(3,5)P(2) 684 production and disrupts endomembrane transport and retroviral budding Vacuolin-1 inhibits autophagy by impairing lysosomal 687 maturation via PIKfyve inhibition The small chemical vacuolin-1 inhibits Ca(2+)-dependent 689 lysosomal exocytosis but not cell resealing PIKfyve, a class III PI kinase, is the target of the small 691 molecular IL-12/IL-23 inhibitor apilimod and a player in Toll-like receptor signaling A family of PIKFYVE inhibitors with therapeutic potential 694 against autophagy-dependent cancer cells disrupt multiple events in lysosome 695 homeostasis Active PIKfyve associates with and promotes the membrane attachment of the late endosome-to-trans-Golgi network transport 698 factor Rab9 effector p40 Identification of Novel Vacuolin-1 Analogues as Inhibitors by Virtual Drug Screening and Chemical Synthesis Vacuolin-1 potently and reversibly inhibits autophagosome-703 lysosome fusion by activating RAB5A Randomized, double-blind, placebo-controlled trial of the 705 oral interleukin-12/23 inhibitor apilimod mesylate for treatment of active Crohn's 706 disease Apilimod inhibits the production of IL-12 and IL-23 and 708 reduces dendritic cell infiltration in psoriasis The phosphatidylinositol-3-phosphate 5-kinase inhibitor 710 apilimod blocks filoviral entry and infection Ebola virus requires phosphatidylinositol (3,5) bisphosphate 712 production for efficient viral entry Visualization of Ebola Virus Fusion Triggering in the Endocytic Pathway A phase 1/2A trial of STA 5326 inhibitor, in patients with active moderate to severe Crohn's disease Brief report: a phase IIa, randomized, double-blind, 720 placebo-controlled trial of apilimod mesylate patients with rheumatoid arthritis Arbidol and Other Low-Molecular-Weight Drugs That 723 Inhibit Lassa and Ebola Viruses Identification of Combinations of Approved Drugs With Synergistic Activity Against Ebola Virus in Cell Cultures Characterization of VPS34-IN1, a selective inhibitor of 728 Vps34, reveals that the phosphatidylinositol 3-phosphate-binding SGK3 protein 729 kinase is a downstream target of class III phosphoinositide 3-kinase Evidence that TMPRSS2 activates the severe acute 732 respiratory syndrome coronavirus spike protein for membrane fusion and reduces 733 viral control by the humoral immune response A transmembrane serine protease is linked to the severe 735 acute respiratory syndrome coronavirus receptor and activates virus entry Efficient activation of the severe acute respiratory 738 syndrome coronavirus spike protein by the transmembrane protease TMPRSS2 SARS-CoV-2 Cell Entry Depends on ACE2 and 741 TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor Characterization of severe acute respiratory syndrome-744 associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entry Identification of apilimod as a first-in-class PIKfyve kinase 747 inhibitor for treatment of B-cell non-Hodgkin lymphoma The VP1 750 subunit of JC polyomavirus recapitulates early events in viral trafficking and is a 751 novel tool to study polyomavirus entry The Phosphoinositide Kinase PIKfyve Promotes 753 Small molecule inhibitors reveal Niemann-Pick C1 is 756 essential for Ebola virus infection Endosome-to-cytosol transport of viral nucleocapsids Protease inhibitors targeting coronavirus and filovirus entry Tracking the Fate of Genetically Distinct Vesicular 762 Stomatitis Virus Matrix Proteins Highlights the Role for Late Domains in 763 Assembly Uptake of rabies virus into 765 epithelial cells by clathrin-mediated endocytosis depends upon actin A 768 forward genetic strategy reveals destabilizing mutations in the Ebolavirus 769 glycoprotein that alter its protease dependence during cell entry Efficient recovery of infectious 772 vesicular stomatitis virus entirely from cDNA clones Genome engineering using the CRISPR-Cas9 system Identification and characterization of a novel broad 777 spectrum virus entry inhibitor A highly conserved cryptic epitope in the receptor-binding 779 domains of SARS-CoV-2 and SARS-CoV The first five seconds in the 781 life of a clathrin-coated pit 614 We thank Walter J. Atwood for providing the parental SVG-A cells, Eric Marino, Justin 615 H. Houser and Tegy John Vadakkan for maintaining the spinning disc confocal *** ** ** *** *** *** *** A Table I Table II