Cell entry of LASV and LCMV into human macrophages depends on the MVB and ESCRT proteins.

<p>(A) Differentiation of THP-1 cells into macrophages. THP-1 cells were treated with 50 ng/ml PMA for 48 hours and changes in cell morphology assessed by differential interference contrast microscopy (bar = 20 µM). (B) THP1 cells were seeded in 96 well plates (2×10⁴ cells/well) and differentiated into macrophage-like cells as in (A). Cells were then transfected with siRNAs for Hrs, Tsg101, Alix, or control siRNA and efficiency of depletion assessed after 48 hours by Western-blot as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002232#ppat-1002232-g004" target="_blank">Fig. 4A</a>. For normalization, α-tubulin (α-Tu) was used. (C) Cells were transfected with siRNAs for Vps22 and Vps24 or control siRNA and efficiency of depletion assessed after 48 hours by quantification of mRNA levels by RT-qPCR as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002232#ppat-1002232-g004" target="_blank">Fig. 4B</a>. (D) Cell entry of LASV and LCMV into human macrophage-like cells depends on PI3K and microtubules. THP1-derived macrophage-like cells generated as in (A) were treated with the indicated concentrations of wortmannin and nocodazole. Cells were then infected with rVSVΔG-LASVGP (LASV), rVSVΔG-LCMVGP (LCMV), and rVSVΔG-VSVG (VSV) either via fusion at the plasma membrane (2000 PFU/well) or via the normal route of infection (200 PFU/well) as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002232#ppat-1002232-g002" target="_blank">Fig. 2</a>. Infection was detected by scoring EGFP positive cells (mean ± SD; n = 3). (E) Infection of VSV pseudotypes of LASV, LCMV, and VSV is perturbed by anti-LBPA treatment. THP-1-derived macrophage-like cells were pre-incubated with no antibody (1), 50 µg/ml mAb anti-LBPA (2) or isotype antibody control (3) for 14 hours. Cells were then infected with rVSVΔG-LASVGP (LASV), rVSVΔG-LCMVGP (LCMV), rVSVΔG-VSVG (VSV), and AdV5-EGFP at 300 PFU/well. In specimens subjected to pretreatment only (4), the cells were incubated for 1 h at 4°C with viruses in presence of the antibody, unbound virus washed out, and cell incubated at 37°C in normal medium. Cells were fixed after 16 hours and EGFP positive cells counted (mean ± SD; n = 3). (F) Cell entry of LASV and LCMV into human macrophages depends on Tsg101, Vps22, Vps24, and Alix, but not Hrs. THP-1-derived macrophage-like cells were subjected to RNAi silencing of Hrs, Tsg101, Vps22, Vps24, and Alix as in (B) and (C). After 48 hours cells were infected with rVSVΔG-LASVGP (VSV-LASV), rVSVΔG-LCMVGP (VSV-LCMV), rVSVΔG-VSVG (VSV-VSV), rLCMV-LASVGP, LCMV, and AdV5-EGFP (AdV) at 300 PFU/well. Infection was assessed after 16 hours by IFA (mean ± SD; n = 3).