Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inhibition of PIKfyve kinase prevents infection by Zaire ebolavirus and SARS-CoV-2

doi: 10.1073/pnas.2007837117

Figure Lengend Snippet: Endolysosomal traffic of VSV-MeGFP-ZEBOV in cells expressing TagRFP-Rab5c or TagRFP-Rab7a in the presence of Apilimod or Vacuolin-1 ( SI Appendix and Movies S1 and S2 ). ( A ) Schematic of live cell imaging experiment using SVG-A cells expressing fluorescently tagged TagRFP-Rab5c or TagRFP-Rab7a. Cells were infected with VSV-MeGFP, VSV-MeGFP-V269H, or VSV-MeGFP-ZEBOV (MOI = 4). Viruses trafficking (monitored with MeGFP) to the endolysosomal system (recognized by their labeling with TagRFP-Rab5c or TagRFP-Rab7a) and virus entry (established by MeGFP at the nuclear margin) were ascertained by live-cell florescence imaging using a spinning disk confocal microscope. ( B ) Visualization of VSV-MeGFP infection in TagRFP-Rab5c cells in the absence ( Left ) or presence ( Right , white arrows) of CHX using live-cell imaging. (Scale bar: 10 µm.) ( C ) Genomic PCR analysis of SVG-A cells showing biallelic integration of TagRFP into the RAB5C genomic locus by cotransfection of a plasmid coding for Cas9, a linear PCR product coding for the specific guide RNAstargeting a region near the ATG codon of Rab5c under the control of the U6 promoter, and a template plasmid containing the RFP sequence flanked by 800 base pairs upstream and downstream of the targeted region (see Materials and Methods for more details) to generate a clonal gene-edited cell line expressing TagRFP-Rab5c. ( D ) Quantification of VSV-MeGFP and VSV-MeGFP-ZEBOV colocalization with Rab5c containing endosomes in the presence of CHX together with absence or presence of 5 µM Apilimod depicted in E . Data show number of viruses that colocalized with endosomes containing or not containing Rab5c within the complete volume of the single cells depicted in E . ( E ) Representative examples of maximum-Z projection images from four optical sections spaced 0.35 µm apart of virus entry without or with IN1, Vacuolin, or Apilimod for VSV-MeGFP ( Top ), VSV-Me-GFP-V269H ( Middle ), and VSV-MeGFP-ZEBOV ( Bottom ). Each condition is in the presence of CHX. All viruses reach Rab5c-containing endosomes, but only VSV-MeGFP-ZEBOV fails to penetrate in the presence of IN1, Vacuolin-1, or Apilimod. (Scale bars: 10 µm.) Insets correspond to a single optical section. Insets (yellow boxes) correspond to a single optical section. (Scale bars: 3 µm.) ( F ) Visualization of VSV infection in TagRFP-Rab7a cells in the absence of CHX ( Left ) and entry in the presence of CHX ( Right , white arrows). (Scale bar: 10 µm.) ( G ) Genomic PCR analysis showing biallelic integration of TagRFP into the RAB7A genomic locus to generate a clonal gene-edited cell-line expressing TagRFP-Rab7a, using the same approach as used for RAB5C . ( H ) Quantification of VSV-MeGFP and VSV-MeGFP-ZEBOV colocalization with Rab7a containing endosomes in the presence of CHX with or without 5 µM Apilimod within the complete cell volumes in the images depicted in I . ( I ) Representative examples of maximum-Z projection images from four optical sections spaced 0.35 µm apart of virus entry without or with IN1, Vacuolin, or Apilimod for VSV-MeGFP ( Top ), VSV-Me-GFP-V269H ( Middle ), and VSV-MeGFP-ZEBOV ( Bottom ). All viruses reach Rab7a-containing endosomes, but only VSV-MeGFP-ZEBOV fails to penetrate in the presence of IN1, Vacuolin-1, or Apilimod. (Scale bars: 10 µm.) Insets correspond to a single optical section. Insets (yellow boxes) correspond to a single optical section. (Scale bars: 3 µm.)

Article Snippet: Vacuolin-1 ( ) was custom synthesized; Apilimod (HY-14644) was from MedChem Express, IN1 was a kind gift from N. Gray, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA , U-18666A (10009085) and Filipin III (70440) were from Cayman Chemical, Bafilomycin A1 (B1793-2UG) was from Sigma-Aldrich, Cycloheximide (239764) was from Calbiochem, and wheat germ agglutinin conjugated with Alexa Fluor-647 (W32466) was from ThermoFisher.

Techniques: Expressing, Live Cell Imaging, Infection, Labeling, Virus, Imaging, Microscopy, Cotransfection, Plasmid Preparation, Control, Sequencing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inhibition of PIKfyve kinase prevents infection by Zaire ebolavirus and SARS-CoV-2

doi: 10.1073/pnas.2007837117

Figure Lengend Snippet: Apilimod and Vacuolin-1 inhibit VSV-MeGFP-ZEBOV infection. ( A ) Schematic of infectivity assay, where SVG-A cells were pretreated for 1 h with 5 μM Vacuolin, 5 μM Apilimod, 5 μM IN1, or 10 nM BAF A1 and subsequently infected with VSV-MeGFP (MOI = 2), VSV-MeGFP-V269H (MOI = 1), VSV-MeGFP-RABV (MOI = 0.6), VSV-MeGFP-LASV (MOI = 0.6), VSV-MeGFP-LCMV (MOI = 0.6), or VSV-MeGFP-ZEBOV (MOI = 0.6) for 1 h in the presence of drugs. The cells were then washed to remove unbound virus and incubated for the indicated times in the presence of drugs. The cells were then fixed, and the percentage of cells expressing viral MeGFP was measured by flow cytometry. ( B ) Representative flow cytometry results of an infection assay using VSV-MeGFP-ZEBOV. ( C ) Quantification of the infectivity is shown with averages from three independent experiments per condition, each determined as a duplicate measurement (error bars show SEM). The statistical significance was determined using a one-way ANOVA and Tukey post hoc test (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001).

Article Snippet: Vacuolin-1 ( ) was custom synthesized; Apilimod (HY-14644) was from MedChem Express, IN1 was a kind gift from N. Gray, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA , U-18666A (10009085) and Filipin III (70440) were from Cayman Chemical, Bafilomycin A1 (B1793-2UG) was from Sigma-Aldrich, Cycloheximide (239764) was from Calbiochem, and wheat germ agglutinin conjugated with Alexa Fluor-647 (W32466) was from ThermoFisher.

Techniques: Infection, Virus, Incubation, Expressing, Flow Cytometry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inhibition of PIKfyve kinase prevents infection by Zaire ebolavirus and SARS-CoV-2

doi: 10.1073/pnas.2007837117

Figure Lengend Snippet: Apilimod and Vacuolin-1 inhibit VSV-MeGFP-ZEBOV. ( A ) Schematic of entry assay where SVG-A cells were infected with VSV-MeGFP (MOI = 4), VSV-MeGFP-V269H (MOI = 4), or VSV-MeGFP-ZEBOV (MOI = 4). Experiments were performed in the presence of 5 µg/mL cycloheximide (CHX) to prevent protein synthesis. Entry assay was based on the appearance of MeGFP fluorescence on the nuclear margin, on a per cell basis, of fixed infected cells visualized by fluorescence microscopy. Staining the fixed cells with Alexa647-labeled wheat germ agglutinin identified the plasma membrane of each cell (dashed outlines in C ). ( B ) Virus infection in the absence of CHX ( Left ) resulted in the appearance of MeGFP fluorescence throughout the cell volume. The presence of CHX resulted in virus entry being observed by MeGFP fluorescence at the nuclear margin, which was released from incoming viral particles ( Right , white arrows). (Scale bar: 10 µm.) ( C ) Representative examples of maximum-Z projections images from the whole-cell volume obtained with optical sections separated by 0.3 µm using spinning disk confocal microscopy. MeGFP fluorescence at the nuclear margin released from incoming viral particles is highlighted (white arrows). (Scale bar: 10 µm.) ( D ) Quantification of the number of cells with nuclear margin labeling from three independent experiments, each determined from fields containing 59 to 90 cells (error bars show SEM). The statistical significance of the entry data was analyzed for statistical significance by one-way ANOVA and Tukey post hoc test (*** P ≤ 0.001).

Article Snippet: Vacuolin-1 ( ) was custom synthesized; Apilimod (HY-14644) was from MedChem Express, IN1 was a kind gift from N. Gray, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA , U-18666A (10009085) and Filipin III (70440) were from Cayman Chemical, Bafilomycin A1 (B1793-2UG) was from Sigma-Aldrich, Cycloheximide (239764) was from Calbiochem, and wheat germ agglutinin conjugated with Alexa Fluor-647 (W32466) was from ThermoFisher.

Techniques: Infection, Fluorescence, Microscopy, Staining, Labeling, Membrane, Virus, Confocal Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inhibition of PIKfyve kinase prevents infection by Zaire ebolavirus and SARS-CoV-2

doi: 10.1073/pnas.2007837117

Figure Lengend Snippet: Apilimod and Vacuolin-1 inhibit infection of VSV-eGFP-SARS-CoV-2. ( A ) Schematic of infectivity assay of VSV-eGFP, VSV-eGFP-ZEBOV, and VSV-eGFP-SARS-CoV-2 in MA104 cells. MA104 cells were pretreated for 1 h with the indicated concentration of Apilimod. Pretreated cells were inoculated with the indicated virus (MOI = 1) for 1 h at 37 °C. At 6 h postinfection cells were harvested, and the fraction of cell expressing eGFP cells was quantified by flow cytometry. ( B ) Quantification of the infectivity is shown with averages ± SEM from three independent experiments. Statistical significance was determined using a t test (* P ≤ 0.05; ** P ≤ 0.01).

Article Snippet: Vacuolin-1 ( ) was custom synthesized; Apilimod (HY-14644) was from MedChem Express, IN1 was a kind gift from N. Gray, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA , U-18666A (10009085) and Filipin III (70440) were from Cayman Chemical, Bafilomycin A1 (B1793-2UG) was from Sigma-Aldrich, Cycloheximide (239764) was from Calbiochem, and wheat germ agglutinin conjugated with Alexa Fluor-647 (W32466) was from ThermoFisher.

Techniques: Infection, Concentration Assay, Virus, Expressing, Flow Cytometry