Virus-like-particle production and viral entry assays.

EBOV virus-like-particles (VLPs) were produced by cotransfecting HEK293T cells with plasmids encoding EBOV NP, EBOV VP40 fused to β-lactamase, or GFP (kind gifts from Lijun Rong, University of Illinois) and the viral envelope protein (mucin-deleted [Δmuc] EBOV GP, EBOV/Bundibugyo ebolavirus [BUDV]/Sudan ebolavirus [SUDV]/Taï Forest ebolavirus [TAFV]/Reston ebolavirus [RESTV] GP, or the glycoproteins of Marburg virus [MARV], VSV, or Junin virus [JUNV]) (all plasmids were kind gifts from James Cunningham, Brigham and Women’s Hospital) using JetPRIME transfection reagent at a 1:1:1.15 ratio. Virus-containing supernatants were collected and concentrated by ultracentrifugation (20,000 rpm, 1.5 h, 4°C; Beckman Coulter Optima XPN-100, SW32Ti rotor) through a 20% (wt/vol) sucrose cushion. Viruses were resuspended in phosphate-buffered saline (PBS) and stored at −80°C.

Entry assays were performed by seeding HT1080 cells in 48-well plates at approximately 90% confluence. VLPs were added, and plates were centrifuged at 200 × g for 30 min at 4°C and then incubated at 37°C for 3 h. Cells were washed with serum-free DMEM and loaded with CCF2-AM (Invitrogen) according to kit instructions for 1 h at room temperature. The staining solution was supplemented with 250 μM probenecid (Sigma). Following staining, cells were washed once in PBS and trypsinized, and cleavage of CCF2 was analyzed using a FACSCelesta flow cytometer (BD Biosciences). Stained cells were defined as cells that were positively shifted compared to unstained cells (emission in a 525/50 filter), and β-lactamase-positive (successful reporter release post-VLP fusion) cells were defined as cells that were positively shifted (emission in a 450/50 filter) compared to stained, uninfected cells.