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, 9 (4), e1003309

Host Cell Entry of Respiratory Syncytial Virus Involves Macropinocytosis Followed by Proteolytic Activation of the F Protein

Affiliations

Host Cell Entry of Respiratory Syncytial Virus Involves Macropinocytosis Followed by Proteolytic Activation of the F Protein

Magdalena Anna Krzyzaniak et al. PLoS Pathog.

Abstract

Respiratory Syncytial Virus (RSV) is a highly pathogenic member of the Paramyxoviridae that causes severe respiratory tract infections. Reports in the literature have indicated that to infect cells the incoming viruses either fuse their envelope directly with the plasma membrane or exploit clathrin-mediated endocytosis. To study the entry process in human tissue culture cells (HeLa, A549), we used fluorescence microscopy and developed quantitative, FACS-based assays to follow virus binding to cells, endocytosis, intracellular trafficking, membrane fusion, and infection. A variety of perturbants were employed to characterize the cellular processes involved. We found that immediately after binding to cells RSV activated a signaling cascade involving the EGF receptor, Cdc42, PAK1, and downstream effectors. This led to a series of dramatic actin rearrangements; the cells rounded up, plasma membrane blebs were formed, and there was a significant increase in fluid uptake. If these effects were inhibited using compounds targeting Na⁺/H⁺ exchangers, myosin II, PAK1, and other factors, no infection was observed. The RSV was rapidly and efficiently internalized by an actin-dependent process that had all hallmarks of macropinocytosis. Rather than fusing with the plasma membrane, the viruses thus entered Rab5-positive, fluid-filled macropinosomes, and fused with the membranes of these on the average 50 min after internalization. Rab5 was required for infection. To find an explanation for the endocytosis requirement, which is unusual among paramyxoviruses, we analyzed the fusion protein, F, and could show that, although already cleaved by a furin family protease once, it underwent a second, critical proteolytic cleavage after internalization. This cleavage by a furin-like protease removed a small peptide from the F1 subunits, and made the virus infectious.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Purified RSV is efficient in cell binding and infection.
(A). Gradient purifier RSV (∼105 particles/50 µl) was resolved on the SDS-PAGE, followed by the blue silver gel staining. (B). After binding to a glass slide, purified RSV was stained with anti-F-AF488 (green), anti-N-AF594 (red), and phalloidin-AF647 (pseudocolored white). The particles (n = 457) were imaged with a confocal microscope and analyzed for colocalization by Imaris. Arrowheads show particles with all three stains. (C). Equal volumes of the virus input (moi 10), the cell bound virus lysates, and the unbound virus (sup) were resolved by a SDS-PAGE. Western blots were developed with anti-P or anti-N RSV specific antibody. (left) Representative western blots. (right) Densitometry quantification of the P- and N- protein bands intensities for the virus input and cell bound virus samples. (D). HeLa cells were infected with RSV moi (3–10) for 1 h at 37°C. Virus inoculum was replaced with medium and the infection was carried for indicated times. The percentage of infected cells expressing GFP was measured by FACS.
Figure 2
Figure 2. RSV undergoes endocytosis.
(A). HeLa cells without (top) or with bound RSV (moi ∼0.5) at 4°C (bottom) were transferred for 30 min to 37°C before fixation. Cells were processed for confocal microscopy with anti-F-AF488 (green), anti-N-AF594 (red), and phalloidin-AF647 (blue), and Z-stack image series acquired. The orthogonal views of Z-stack projections (pseudocolored white) were generated with ImageJ. (B). RSV (moi ∼1) was bound to HeLa cells at 4°C, unbound virus was removed and cells transferred to 37°C. At indicated times cells were placed on ice and treated with PBS or trypsin for 3 min (PBS wash vs. trypsin treated). Samples were processed for confocal microscopy as in (A). (C). Quantification of the image series represented in (B). Analysis was performed with Imaris to detect spots with anti-F staining only (left) or anti-F and -N staining (right) at different times after warming the cells to 37°C.
Figure 3
Figure 3. Rapid RSV endocytosis is followed by the intracellular fusion.
(A). HeLa cells were incubated with RSV-DiOC (moi ∼3) for 30 min at 37°C. Single focal plane image of live cell samples was acquired with a confocal microscope before (green) and after TB addition (pseudocolored red/white). Arrowheads indicate virus spots that were not quenched after TB addition. (B). RSV-DiOC (moi ∼1–5) was bound to versene detached HeLa cells at 4°C for 1 h, unbound virus was washed away and cells were incubation at 37°C. (top) At the indicated times, cells were fixed and the mean fluorescence intensity (MFI) of DiOC measured by FACS in the presence of TB. (bottom) In parallel experiments at indicated times cells were trypsinized on ice for 10 min, washed, permeabilized, stained with anti-N-AF647 or anti-F-AF647 and the MFI of AF647 measured by FACS. (C). RSV-R18/DiOC (moi ∼5) was bound to HeLa cells at 4°C, unbound virus was removed and cells were incubated at 37°C. At indicated times, single focal planes of live cell samples were acquired with confocal microscope. (D). RSV-R18/DiOC (moi ∼5) was bound to versene detached HeLa cells at 4°C, unbound virus was washed away and cells were incubated at 37°C. At indicated times cells were fixed and the MFI of DiOC measured by FACS with or without TB. (E). RSV (moi ∼1 to 30) was bound to HeLa cells at 4°C for 1 h, unbound viruses were removed, and cells warmed to 37°C. At indicated times, samples were placed on ice and trypsinized for 10 min to remove virus remaining on the cell surface. Cells were washed and re-plated for up to 10 h before the percent of GFP expressing cells determined by FACS.
Figure 4
Figure 4. RSV endocytosis and infection are clathrin-, dynamin- and pH-independent.
In A and B, HeLa cells were pretreated with solvent (MOCK), chlorpromazine (Chlp), pitstop-2, dynasore, dyngo-4a or dynol-34-2 at indicated concentrations and each inhibitor was continuously present during following steps of the experiment: (A) RSV (moi ∼3) or transferrin AF-488 (Trf-AF488) (2 µg/ml) were bound to cells at 4°C. After internalization at 37°C, RSV (1 h) or Trf-AF488 (20 min) cells were treated with trypsin on ice for 10 min before fixation. RSV infected cells were additionally stained by IIF with anti-N-AF488 antibody and the MFI of AF-488 measured by FACS. (B). Cells were infected with RSV (moi ∼3) or SFV-ZsGreen (moi ∼0.5) for up to 6 h before FACS analysis of GFP expressing cells. (C). HeLa cells were pretreated with bafilomycin A (BafA), ammonium chloride (NH4Cl), or monensin (Mon) at indicated concentrations and infected with RSV (moi ∼3) or ZsGreen-SFV (moi ∼0.5) in the continued presence of the compounds for up to 6 h before FACS analysis of GFP expressing cells.
Figure 5
Figure 5. RSV infection induces actin rearrangement.
(A). RSV (moi ∼0.5) was bound to HeLa cells at 4°C, unbound virus was removed and cells warmed to 37°C, fixed at indicated times, and stained with phalloidin-AF488 (pseudocolored white) and anti-F-AF647 (red) antibody. Images represent Z-stack projections acquired with a confocal microscope. Arrowheads show actin blebs formed at the cell surface. (B). RSV (moi ∼30) was incubated with HeLa cells for 30 or 120 min at 37°C. Samples were processed according to the kit manufacturer's protocol (Cytoskeleton Inc.). Controls included mock-treated cells and cells either treated with F actin enhancer or F actin depolymerizing agent. (left) The F and G actin fractions were resolved by SDS-PAGE and western blots probed with anti-actin antibody. (right) Quantification of actin protein bands intensities by densitometry. (C–F). HeLa cells were pretreated with solvent (MOCK), cytochalasin D (CytoD), latrunculin A (LatA), jasplakinolide (Jas), nocodazole (Noc), taxol (Tax), NCS23766, pirl1, IPA-3, wiskostatin (Wisko), CK-869, CT04, Y24632 at indicated concentrations and each inhibitor was continuously present during following steps of the experiment: (C, E). Cells where infected with RSV (moi ∼3) or SFV-ZsGreen (moi ∼0.5) for up to 6 hours before FACS analysis of GFP expressing cells. (D, F). RSV (moi ∼3) was bound to the cells at 4°C followed by 1 h of internalization at 37°C. Cells were trypsinized, fixed and stained with anti-N-AF488 antibody, and the MFI of AF-488 measured by FACS.
Figure 6
Figure 6. RSV induces bulk fluid phase uptake.
(A). Serum starved HeLa cells were incubated with 20% FCS or purified RSV (moi ∼10, 30), at 4°C for 1 h. The inoculum was replaced with medium containing 10 kDa dextran-AF488 and transferred for 15 min to 37°C before fixation. The MFI of AF-488 measured by FACS. (B). Purified RSV (moi ∼10) was bound at 4°C to serum-starved HeLa cells. The input virus was replaced with medium containing 10 kDa dextran-AF488 (green) and transferred to 37°C. At indicated times cells were fixed, permeabilized and stained with anti-F-AF594 (red) and anti-P-AF647 (blue) antibody. Images represent a Z-stack projection acquired with the same confocal microscope settings. (C). HeLa cells were pretreated with solvent (MOCK) or EIPA at indicated concentration. (left) Cells were infected with RSV (moi ∼3) at 37°C for 6 hours before FACS analysis of GFP expressing cells. (right) RSV (moi ∼3) was bound to the cells at 4°C followed by 1 h of internalization at 37°C. Cells were trypsinized, fixed and stained with anti-N-AF488 antibody, and the MFI of AF-488 measured by FACS. (D). Serum starved HeLa cells were pretreated with EIPA at indicated concentration and incubated with purified RSV (moi ∼30) or no virus control at 4°C. The inoculum was replaced with medium containing 10 kDa dextran-AF488 and EIPA and transferred for 15 min to 37°C. Cells were fixed and the MFI of AF-488 measured by FACS.
Figure 7
Figure 7. Cellular requirements for RSV endocytosis.
(A). Purified RSV (moi ∼30) or mock that did not contain virus was added to serum-starved HeLa cells for 15 min at 37°C before cells were processed according to the kit manufacturer's protocol (R&D systems). (left) Representative array blots. (right) Quantification of phospho-EGFR signal intensity. (B). HeLa cells were revers transfected with scrambled siRNA (siCtrl) or siRNA against EGFR (siRNA_1, siRNA_2). After 72 hours cells were infected with RSV (moi ∼0.3) for 18 hours, before fixation and image based analysis. (left) Western blot validation of the siRNA EGFR depletion. (right) Image based quantification of RSV infection in cells with EGFR depletion. (C–E). HeLa cells were pretreated with solvent (MOCK) or (C) genistein (Gen), CAS879127-07-8, iressa, wortmannin (Wort), Ly294002 or PI-103 (D) staurosporine (Stau), rottlerin (Rott) or calphostine C (CalphC), (E) blebbistatin (Bleb), ML7 at indicated concentrations and each inhibitor was continuously present during following steps of the experiment. (C–E top) Cells were infected with RSV (moi ∼3) or ZsGreen-SFV (moi ∼0.5) for up to 6 hours, before FACS analysis of GFP expressing cells. (C–E bottom) RSV (moi ∼3) was bound to cells at 4°C followed by 1 h internalization at 37°C. Cells were trypsinized, fixed and stained with anti-N-AF488 antibody, and the MFI of AF-488 measured by FACS.
Figure 8
Figure 8. RSV virions traffic through Rab5 positive vesicles.
(A–B). HeLa cells were transiently transfected with a GFP expressing constructs of Rab5 WT, -Rab5 Q79L (C/A), -Rab5 S34N (D/N), -Rab7 WT, -Rab7 Q67L (C/A), -Rab7 T22N (D/N) for 12 h. (A). RSV (moi ∼3) was bound to HeLa cells at 4°C, unbound virus was washed away and cells warmed at 37°C. At indicated time cells were fixed and stained with anti-F-AF594 (red) and anti-P-AF647 (blue) antibody. Images represent a single 0.37 µm thick focal planes acquired with the same confocal microscope settings. Arrowheads point the discussed in text phenotype of RSV and Rab colocalization. (B). Cells were infected with RSV-A2 (moi ∼0.5) for 16 hours before fixation and staining with anti-N-AF647. The number of AF647 positive cells among the population of GFP expressing cells was measured FACS. (C). HeLa cells were pretreated with 30 µM PIKfyve inhibitor (CAS 371942-96-7) or a solvent control (MOCK) and infected with RSV (moi ∼3) or ZsGreen-SFV (moi ∼0.5) for up to 6 hours before FACS analysis of GFP expressing cells.
Figure 9
Figure 9. RSV F requires post endocytic activation.
(A). HeLa cells were pretreated with dec-RVKR-CMK, α -PDX or leupeptin at indicated concentration for 1 h before experiment and inhibitors were continuously present during following steps of the experiment. Cells were infected with RSV or RSVΔGΔSH for 6 h before FACS analysis of GFP expressing cells. (B). HeLa cells were infected with RSV or RSVΔGΔSH for 1 h. Virus inoculum was replaced with medium containing 100 µM dec-RVKR-CMK and incubated for 6 before FACS analysis of GFP expressing cells. (C). Versene detached HeLa cells were pretreated with solvent (MOCK), dec-RVKR-CMK or EIPA at indicated concentrations and inhibitors were continuously present during following steps of the experiment. RSV-R18/DiOC or RSVΔGΔSH-R18/DiOC (moi ∼5) was bound to cells at 4°C. Unbound virus was removed and cells were incubated at 37°C for 2 h in the presence of inhibitor. Cells were fixed and the MFI of DiOC fluorescence measured by FACS and normalized to mock no inhibitor controls. (D). RSV F protein (574 aa) is proteolytically processed by furin like protease at the two sites (A aa-106 and B aa-136) to generate disulfide bonds linked F1+F2 and small peptide p27 (aa sequence depicted above). At the N-terminus of F1 is a FP (fusion peptide) and at the C-terminus TM (transmembrane domain), numbers indicate aa position and red underlines specify peptide sequences detected in mass spectrometry. (E). Proteomic analysis of HEp-2 cells and purified RSV particles. The N-terminal sequence of the p27 peptide (F protein) was quantified by a targeted mass spectrometry based on the selected reaction monitoring (SRM). Representative SRM peaks of peptides (left) FMNYTLNNAKKTNVTLSK 3+ and (right) ELPRFMNYTLNNAK 3+ peptides, corresponding to the aa 113–131 and 109–123 of F protein, respectively. Different SRM transitions for a peptide shown in different colors (see supporting information Table S1). The bar graphs show the results of the targeted peptide quantitation, presented as the sum of the areas of all the SRM peaks for a given peptide. Where no peptide peak was detectable, noise values were reported as a reference. RT retention time, and Cps counts per second. (F). HeLa cells were pretreated or MOCK treated with dec-RVKR-CMK, α-PDX, or leupeptin at indicated concentration. RSV (input control in the first line) was bound for 1 h in cold (B-binding) or after wash-away unbound virus was internalized for 1.5 h at 37°C before processing (I-internalized). Lysed cell samples were resolved by SDS-PAGE and blots were probed with anti-F1 antibody. (G). RSV was bound for 1 h in cold to HeLa cells; unbound virus wash away and cells were placed at 37°C for indicated times before, lysis, SDS-PAGE, and processing for western blot probed with anti-F1 antibody.
Figure 10
Figure 10. RSV enters human bronchial epithelial cells by macropinocytosis.
(A). Human bronchial epithelial cells (16HBE14o) were polarized for 9 days and then infected with rgRSV for 20 h. After fixation cells were stained with anti-ZO-1-AF594 antibody (red) and TO-PRO-3 nuclear dye (blue) to examine polarization of the cell monolayer. RSV infection was visualized by GFP expression (green). (B) Polarized 16HBE14o cells were pretreated with solvent (MOCK), low or high concentrations of dynasore (10, 20 µM), cytochalasin D (CytoD 1.5, 3 µM), jasplakinolide (Jas 0.5, 1 µM), IPA-3 (40, 80 µM), genistein (Gen 50, 100 µM), iressa (10, 20 µM), Ly294002, EIPA (40, 80 µM), and dec-RVKR-CMK (50, 100 µM). The cells were infected with RSV for 20 h in the presence of the inhibitors, fixed and counterstained with DAPI. RSV infection was quantified by an image-based approach and normalized to mock infected controls.

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Grant support

This work was supported by the ERC grant (VIRNA 2-73905-09) awarded to AH and the EMBO fellowship (ALTF 349-2010) awarded to MAK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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