Fig. 3.

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.)