(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).

(A) Infection of VSV pseudotypes of LASV, LCMV, and VSV is affected by anti-LBPA antibody. A549 cells (10⁴ cells/well) in 96 well plates 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), and rVSVΔG-VSVG (VSV), and AdV5-EGFP at 500 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 Ab, 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). (B) Anti-LBPA perturbs infection of rLCMV-LASVGP and LCMV. Monolayers of A549 cells were treated as in (A), followed by infection with rLCMV-LASVGP, LCMV, and AdV5-EGFP (AdV) at 500 PFU/well. Cells were fixed after 16 hours (rLCMV-LASVGP, LCMV, VSV, or AdV5-EGFP) and 6 hours (VSV). Infection was detected with a mAb to LCMV NP (rLCMV-LASVGP, LCMV), VSV M protein (VSV), or EGFP (AdV5-EGFP) (mean ± SD; n = 3). (C) Representative specimens of cells infected with rLCMV-LASVGP in (B) LCMV NP is in red and nuclei (DAPI) appear in blue. Bar = 50 µm. (D) Anti-LBPA treatment does not interfere with transferrin uptake. Cells treated as in (A) were incubated for 10 min at 37°C with serum-free medium containing 20 µg/ml of Alexa594-labeled human transferrin, washed with ice cold PBS, acid-stripped to remove surface-bound transferrin, fixed, and the cellular distribution of transferrin (red) examined. Cell nuclei are stained in blue with DAPI (bar = 10 µM).

(A) Over-expression of a MYC-tagged recombinant Tsg101 by transient transfection of HEK293 cells. Cells were transfected with recombinant Tsg101 (Tsg101) or empty vector (control). After 36 hours total cell lysates were probed by Western-blot with antibodies recognizing Tsg101 or the MYC epitope. The MYC-tagged form of Tsg101 runs at a slightly higher apparent molecular mass, due to the presence of the tag (*). (B) Overexpression of Tsg101 accelerates the entry kinetics of rLCMV-LASVGP. Cells transfected with recombinant Tsg101 (Tsg101) or empty vector (Control) as in (A) were cooled on ice for 30 min and rLCMV-LFVGP added at an MOI of 3. After incubation for 1 h on ice, unbound virus was washed off, and cells shifted to 37°C. At different time points, medium containing 20 mM ammonium chloride was added. After a total of 16 h, cells were fixed and LCMVNP detected by intracellular staining with mAb 113 to LCMV NP, combined with Alexa 488-labeled secondary antibody. NP positive cells were quantified by flow cytometry as in Fig. 5D. Note the delay in the fusion kinetics relative to Fig. 1A due to incubations of cells with ice cold medium after the washes. Data presented are means (n = 3+SD). (C) Entry kinetics in cells depleted of Tsg101. HEK293 cells were transfected with siRNA to Tsg101 or control siRNA as in Fig. 5A, resulting in >90% reduction of Tsg101 protein expression after 72 hours (data not shown). Cells treated with siRNA for 72 hours were cooled on ice for 30 minutes and rLCMV-LFVGP added at an MOI of 3. After removal of unbound virus entry kinetics was assessed as in (B). Data presented are means (n = 3+SD).

(A) Co-localization of LCMV WE54 with Tsg101 and Rab7. A549 cells were cooled on ice for 30 min and LCMV WE54 added at an MOI of ∼100. After incubation for 1 hour on ice, unbound virus was washed off, cells shifted to 37°C and fixed at the indicated time points. Cells were then immunostained to detect endogenous Tsg101 or Rab7 and incoming viral particles. Representative images are shown. Left: LCMV (green) and Tsg101 (red) at 20 min after temperature shift; right: LCMV (green) and Rab7 (red) at 60 min after temperature shift. Scale bar = 5 µm. (B) Quantification of co-localization. Ten randomly selected cells per time point were analyzed by confocal microscopy and the percentage of co-localizing viruses determined as described in Materials and Methods. Data presented are means ± SD (n = 10).

(A, B) Depletion of Hrs, Tsg101, Vps22, Vps24, and Alix. A549 cells were subjected to siRNA knock down of Hrs, Tsg101, Vps22, Vps24, and Alix as in Figs. 4A and 8A. Depletion of Hrs, Tsg101, and Alix was verified by Western blot as in 4A and 8A. The know-down of Vps22 and Vps24 was validated by detection of mRNA using RT-qPCR as in 4B. (C) LCMV WE22 retains the low fusion pH of WE54. LCMV WE54, WE22, cl-13 and rLCMV-LASVGP (10⁷ PFU/ml) were exposed to buffer solution with the indicated pH for 15 minutes. Samples were immediately neutralized and infectious virus titers determined by immunofocus assay on fresh monolayers of A549 cells. Means of 2 independent experiments are shown. (D) Infection with LCMV WE22 depends on Hrs, Tsg101, Vps22, Vps24, and Alix. A549 cells were subjected to RNAi silencing of Hrs, Tsg101, Vps22, Vps24, and Alix as in (A) and (B) respectively. After 72 hours cells were infected with the indicated viruses (500 PFU/well) and infection assessed after 16 hours by IFA (mean ± SD; n = 3).

For details please see text.

(A) LASV and LCMV cell entry depend on microtubules. A549 cells (10⁴ cells/well) in 96 well plates were pre-treated with 10 µM nocodazole (Noc) for 1 hour, chilled on ice and incubated with rVSVΔG-LASVGP (LASVGP), rVSVΔG-LCMVGP (LCMVGP), and rVSVΔG-VSVG (VSVG) at MOI of 1 for one hour in the cold. Unbound virus was removed and cells shifted to 37°C in presence of nocodazole. To induce fusion at the plasma membrane, cells were pulsed with acidified medium (pH 5.0) for 15 minutes, followed by immediate neutralization. Cells subjected to normal infection were cultured in parallel, without applying the low pH pulse. After a total of 12 hours, infection assessed by detection of GFP expressing cells in flow cytometry as in Fig. 1A (mean ± SD; n = 3). (B) A role of PI3K in cell entry of LASV and LCMV. A549 cells were incubated with rVSVΔG-LASVGP (LASVGP), rVSVΔG-LCMVGP (LCMVGP), and rVSVΔG-VSVG (VSVG) at MOI of 1 for one hour in the cold and unbound virus removed. Cells were shifted to 37°C and fusion at the membrane was induced with a low pH pulse with acidified medium (pH 5.0) for 15 minutes. After neutralization, cells were cultured for a total of 12 hours in presence of 100 nM wortmannin. Cells subjected to normal infection were pre-incubated with virus in the cold. After removal of unbound virus, cells were rapidly shifted to 37°C for 5 minutes to allow internalization of the virus. Wortmannin was then added (100 nM) and left throughout the experiment. Infection was determined by detection of GFP expressing cells by flow cytometry as in Fig. 1A (mean ± SD; n = 3).

(A) A549 cells were transfected with siRNAs specific for Hrs and Tsg101 or control siRNA and efficiency of depletion assessed after 72 hours by Western-blot. For normalization, α-tubulin (α-Tu) was used. (B) Cells were transfected with siRNAs for Vps22 and Vps24 or control siRNA and efficiency of depletion assessed after 72 hours by quantification of mRNA levels by RT-qPCR (for details see Methods). Given are normalized levels of mRNA expression. (C) Depletion of Hrs, Tsg101, Vps22, and Vps24 does not affect post-entry steps of VSV pseudotype infection. A549 cells were treated with specific siRNAs to Hrs, Tsg101, Vps22, and Vps24 and control siRNA for 48 hours. Cells were then detached and reseeded in 96 well plates (10⁴ cells/well). After 24 hours, cells were incubated with rVSVΔG-LASVGP (LASV) (4000 PFU/well), rVSVΔG-LCMVGP (LCMV) (4000 PFU/well), and rVSVΔG-VSVG (VSV) (2000 PFU/well) in the cold, followed by washing and a low pH pulse to induce fusion at the plasma membrane as in Fig. 2. Upon neutralization, cells were fixed after 16 hours and EGFP positive cells scored (mean ± SD; n = 3). (D, E) Depletion of Hrs, Tsg101, Vps22, and Vps24 affects cell entry of LASV and LCMV. Cells subjected to RNAi silencing as in (A) were infected via the normal route of entry (D) with rVSVΔG-LASVGP (LASV), rVSVΔG-LCMVGP (LCMV), rVSVΔG-VSVG (VSV), and AdV5-EGFP (AdV) (500 PFU/well) and (E) with rLCMV-LASVGP, LCMV, rVSVΔG-VSVG (VSV), and AdV5-EGFP (AdV) at 500 PFU/well. After 16 hours, cells were fixed and infection quantified by detection of EGFP positive cells (D) or LCMV NP and EGFP (E) as in Fig. 3 (mean ± SD; n = 3). (F) Representative specimens of rLCMV-LASVGP infected cells in cultures treated with the indicated siRNAs in (E). LCMV NP is stained in green and cell nuclei stained with DAPI appear in blue (bar = 20 µM). (G) Depletion of Hrs, Tsg101, Vps22, and Vps24 by RNAi does not interfere with transferrin uptake. Cells were treated as in (A, B) and transferrin uptake assessed as in Fig. 3D. Transferrin is in red and cell nuclei in blue (bar = 10 µM). (H) Depletion of Hrs, Tsg101, Vps22, and Vps24 by RNAi does not affect cell surface expression of α-DG. Cells were subjected to RNAi as in (A, B). After 72 hours cell surface staining was performed with mAb IIH6 to α-DG, combined with Alexa 594 secondary antibody. Data were acquired in a FACSCalibur flow cytometer and analyzed using Cell Quest software. In histograms, y-axis represents cell numbers and x-axis Alexa 594 fluorescence intensity. Shaded area: primary and secondary antibody, empty area: secondary antibody only. The broken line indicates the superimposition of the shaded peak (primary+secondary antibody) from cells treated with control siRNA. Note that treatment with siRNAs to Hrs, Tsg101, Vps22, and Vps24 does not significantly alter the mean fluorescence intensity of the IIH6 signal.

(A) HEK293 cells were transiently transfected with FLAG-tagged wild-type Vps4A and the DN mutant Vps4AQE and expression of the recombinant proteins detected in Western-blot. (B) Expression of DN Vps4A reduced viral infection. Cells were transiently transfected with FLAG-tagged wild-type and DN Vps4A. After 36 hours, cells were infected with rLCMV-LASVGP and LCMV (MOI = 1). After 16 hours, cells were fixed and stained for the Vps4A variants (anti-FLAG) and LCMV NP. Cells were analyzed by FACS separating transfected “expressing” from untransfected “non-expressing” cells with gating based on the intensity of the anti-FLAG signal. The percentage of cells infected within each population was quantified. (C) Quantitation of (B). All data presented are means (n = 3 ± SD).

(A) A549 cells were transfected with siRNAs specific for Alix or control siRNA and efficiency of depletion assessed after 72 hours by Western-blot. For normalization, α-tubulin (α-Tu) was used. (B, C) Depletion of Alix perturbed cell entry of LASV and LCMV. A549 cells were treated with specific siRNAs to Alix and control siRNA as in (A), followed by infection with (B) rVSVΔG-LASVGP (LASV), rVSVΔG-LCMVGP (LCMV), rVSVΔG-VSVG (VSV), and AdV5-EGFP (AdV) at 500 PFU/well or (C) rLCMV-LASVGP, LCMV, rVSVΔG-VSVG (VSV), and AdV5-EGFP (AdV) (500 PFU/well). Cells were fixed after 16 hours and infection detected by IFA as in Fig. 4 (mean ± SD; n = 3). (D) Representative specimens of rLCMV-LASVGP infected cells in cultures treated with the indicated siRNAs in (C). LCMV NP is in green and cell nuclei in blue (bar = 20 µM). (E) Depletion of Alix does not interfere with transferrin uptake. Cells treated as in (A) were subjected to transferrin uptake assay as in Fig. 3D. The cellular distribution of transferrin (red) and cell nuclei stained with DAPI in blue are shown (bar = 10 µM). (F) Depletion of Alix does not affect cell surface expression of α-DG. Cells were subjected to RNAi as in (A) and cell surface staining for α-DG performed after 72 hours as in Fig. 4H. In histograms, y-axis represents cell numbers and x-axis Alexa 594 fluorescence intensity. Shaded area: primary and secondary antibody, empty area: secondary antibody only. The broken line indicates the superimposition of the shaded peak (primary+secondary antibody) from cells treated with control siRNA.

(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 Fig. 4A. 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 Fig. 4B. (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 Fig. 2. 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).