Figure 1. Recombinant VSV pseudotypes adopt the cell entry characteristics of LASV and LCMV.

(A) Entry kinetics of rVSVΔG-LASVGP, rVSVΔG-LCMVGP, and rVSVΔG-VSVG. A549 cells were seeded in 96 well plates (10⁴ cells/well) and cultured for 16 hours. The resulting cell monolayers were chilled on ice and the indicated pseudotypes added at MOI of 3 for 1 hour. Unbound virus was removed and cells quickly shifted to 37°C. At the indicated time points 20 mM ammonium chloride (NH₄Cl) was added and left throughout the experiment. After a total of 12 hours, cells were detached, fixed, and infection detected by the GFP reporter in flow cytometry. Data presented show percent GFP positive cells. Means (n = 3±SD). (B) Depletion of membrane cholesterol by MβCD. Monolayers of A549 cells were incubated with the indicated concentrations of MβCD for one hour and total cholesterol determined in a colorimetric assay. Shown are two independent experiments in triplicates expressed in percent of untreated control (mean ± SD). (C) Cholesterol depletion reduces infection with rVSVΔG-LASVGP, rVSVΔG-LCMVGP, but not rVSVΔG-VSVG. A549 cells (10⁴ cells/well) were treated with the indicated concentrations of MβCD as in (B), followed by infection with 200 PFU of rVSVΔG-LASVGP (LASVGP), rVSVΔG-LCMVGP (LCMVGP), and rVSVΔG-VSVG (VSVG) per well. Infection was assessed after 12 hours by detection of GFP expressing cells in fluorescence microscopy (mean ± SD; n = 3). (D) Infection with rVSVΔG-LASVGP and rVSVΔG-LCMVGP does not depend on clathrin. A549 cells were transfected with either a pool of siRNA targeting clathrin heavy chain (CHC) (50 nM per siRNA; 200 nM total concentration) or control siRNA (200 nM). After 48 hours, cells were lysed and expression of CHC assessed by Western-blot. For normalization, α-tubulin (α-Tu) was detected. Cells transfected with siRNA to either CHC or control siRNAs (Control) for 48 hours were infected with 200 PFU of rVSVΔG-LASVGP (LASVGP), rVSVΔG-LCMVGP (LCMVGP), and rVSVΔG-VSVG (VSVG) per well. After 12 hours, cells were fixed and infection assessed by detection of GFP as in (C) (mean ± SD; n = 3). (E) Infection with arenavirus pseudotypes is not sensitive to chlorpromazine (CPZ). A549 cells were pre-treated with the indicated concentrations of CPZ for 2 hours, followed by infection with rVSVΔG-LASVGP (LASVGP), rVSVΔG-LCMVGP (LCMVGP), and rVSVΔG-VSVG (VSVG) at MOI of 3. After 12 hours, infection was assessed as in (A) (mean ± SD; n = 3). (F) Comparison of the effects of DN mutants of Eps15, dynamin II, and Rab5 on infection with VSV pseudotypes. HEK293 cells were transfected with GFP-tagged DN and wild-type (WT) Eps15, dynamin II, and Rab5 using nucleofection, resulting in >90% transfection efficiency, as assessed by detection of GFP. After 24 hours, cells were infected with rVSVΔG-LASVGP (LASVGP), rVSVΔG-LCMVGP (LCMVGP), and rVSVΔG-VSVG (VSVG) at 200 PFU/well. Infected cells were detected by IFA using a mAb to VSV M protein and a rhodamine-red-X conjugated secondary antibody. Specimens were examined using a fluorescence microscope with a narrow band-pass filter and VSV M positive cells scored (mean ± SD; n = 3).