Production and Infection of Ebola and Lassa Pseudoviruses

Virions pseudotyped with Ebola virus GP_Δmucin (EBOV GPΔ) and Lassa virus GP were produced in HEK293T/17 cells using Lipofectamine 2000 (Invitrogen). Cells grown to 60–80% confluency were transfected with 4.65-µg psPAX2 (Gag-Pol helper vector), 6.25-µg pFSW-Tat, 1-µg pHIV-Rev, and 1.5-µg of EBOV GP or LASV GP. Four to six hours post-transfection, the media were replaced with phenol red free DMEM supplemented with 10% FBS, 2-mM L-glutamine, and 1-mM sodium pyruvate. Pseudovirus-containing media were collected 48-h post-transfection and clarified by low-speed centrifugation. HIV pseudoviruses were pelleted through a 25% sucrose-HME cushion and resuspended in HME buffer without sucrose. The pseudovirus preparation was aliquoted and stored at −80°C. The concentration of HIV p24 was measured by enzyme linked immunosorbent assay (ELISA) (82 (link)).
Infection of TZM-bl cells by HIV pseudoviruses was performed as described by Sarzotti-Kelsoe et al. (83 (link)). Firefly luciferase activity was measured 2 days post-infection with BriteLite reagent (PerkinElmer Life Sciences) in a plate reader (GloMax Explorer, Promega). HIV pseudovirus preparations were diluted in Opti-MEM (Gibco) to the same concentration of p24.