Similar uptake but different trafficking and escape routes of reovirus virions and infectious subvirion particles imaged in polarized Madin-Darby canine kidney cells

Abstract

Polarized epithelial cells that line the digestive, respiratory, and genitourinary tracts form a barrier that many viruses must breach to infect their hosts. Current understanding of cell entry by mammalian reovirus (MRV) virions and infectious subvirion particles (ISVPs), generated from MRV virions by extracellular proteolysis in the digestive tract, are mostly derived from in vitro studies with nonpolarized cells. Recent live-cell imaging advances allow us for the first time to visualize events at the apical surface of polarized cells. In this study, we used spinning-disk confocal fluorescence microscopy with high temporal and spatial resolution to follow the uptake and trafficking dynamics of single MRV virions and ISVPs at the apical surface of live polarized Madin-Darby canine kidney cells. Both types of particles were internalized by clathrin-mediated endocytosis, but virions and ISVPs exhibited strikingly different trafficking after uptake. While virions reached early and late endosomes, ISVPs did not and instead escaped the endocytic pathway from an earlier location. This study highlights the broad advantages of using live-cell imaging combined with single-particle tracking for identifying key steps in cell entry by viruses.

FIGURE 1:

Fluorescence labeling of MRV particles. (A) Left, AF647-labeled virions and ISVPs were analyzed by SDS–PAGE, and the gel was visualized using a TYPHOON imager to identify which MRV proteins were labeled with the fluorescent dye. Right, AF647-labeled virions (top) and ISVPs (bottom) were deposited on a glass coverslip and imaged by spinning-disk confocal microscopy. (B) The fluorescence intensities of the labeled virions (black bars) and ISVPs (gray bars) imaged on coverslips were measured by use of Slidebook5 to identify particles by intensity thresholding and to record their intensities. Fluorescence intensity per pixel (in arbitrary units) is shown in bins on the x-axis, and percentage of total virions or ISVPs falling within each bin is shown on the y-axis. A Gaussian fit of the data for each type of particle is also shown. The legend shows the mean, SD, and total number of particles analyzed for each type.

FIGURE 2:

Evidence for MDCK cell polarization and associated localization of JAM-A. (A) MDCK cells expressing AP2-GFP were plated on coverslips 1 d (top: nonpolarized) or 3–5 d (bottom: polarized) before being fixed and then immunostained for actin (blue) and tight-junction protein ZO-1 (red). Images were obtained by spinning-disk confocal microscopy, as described in Materials and Methods. A representative transversal (side) view is also shown for each panel at top right. (B) MDCK cells were seeded on Transwell supports, and the trans-epithelial resistance (TER) was measured at 1, 2, 3, and 4 d after plating. Results are shown as the mean value ± SD from three independent experiments. (C) MDCK cells were grown on Transwell supports for 4 d, and after polarization was confirmed by TER measurement, cells were fixed and then immunostained for tight-junction protein ZO-1 (left: red) and JAM-A (right: red). Nuclei were stained using 4′,6-diamidino-2-phenylindole (blue). Z-series were obtained by scanning confocal microscopy using a Zeiss LSM-780. Representative transversal (side) views are shown for each panel and correspond to the YZ view (left strip) and the XZ view (top strip). Black arrows correspond to the focal-plane display in the XY view toward the top of the cells.

FIGURE 3:

Internalization of MRV particles from the apical surface of polarized MDCK cells. (A) Polarized MDCK cells, which had been plated on coverslips 3 d previously, were pretreated with or without jasp or amiloride. Fluorescently labeled transferrin or dextran was added to the cells in the presence or absence of the inhibitors and allowed to internalize for 7 min at 37°C; this was followed by an acid wash to remove membrane-bound transferrin or dextran. Cells were then fixed, and images were obtained by laser-scanning confocal microscopy. The panels correspond to representative maximum-intensity projections. (B) Polarized MDCK cells, which had been plated on coverslips 3 d previously, were pretreated or not with the inhibitors. AF568-labeled virions or ISVPs were then allowed to attach to the cells, after which unbound particles were removed, and internalization of bound particles was measured in the presence or absence of inhibitor(s). Results are expressed as the percentage of internalized particles among total particles counted in each cell, and are shown as the mean value ± SD from at least 10 cells for each condition.

FIGURE 4:

Productive infection by MRV particles following uptake at the apical surface of polarized MDCK cells. (A) The assay procedure, as detailed in Materials and Methods, is depicted schematically. MDCK cells were seeded 5 d before infection to allow complete polarization. (B) Top, representative fields of polarized MDCK cells subjected to the infection assay with virions or ISVPs in the absence or presence of inhibitors as indicated. Immunostained μNS protein, indicative of productive infection, appears green. Bottom, the number of infected cells in the presence of each inhibitor was assessed by μNS immunostaining and normalized to the number of infected cells in mock-treated samples. Data are shown as the mean value ± SD from at least 10 fields in each of three independent experiments.

FIGURE 5:

Live-cell imaging and single-particle tracking of single MRV virion and ISVP particles during their association with endocytic clathrin-coated pits and vesicles at the apical surface of polarized MDCK cells. Polarized MDCK cells stably expressing σ2-GFP and transiently expressing CLCa-TOM were inoculated with fluorescent virus particles, and images were acquired by 4D spinning-disk confocal microscopy. (A) A series of 50 images acquired at 3-s intervals trace a representative example of the clathrin/AP2-associated uptake of a single MRV virion: green, AP2-GFP; red, CLCa-TOM; and blue, virion. Phases of the uptake process as described in the text are labeled. (B) Left, representative images depicting virions (top) or ISVPs (bottom) colocalizing with clathrin/AP2-coated pits on the apical surface of polarized MDCK cells. Selected examples of colocalization are highlighted by arrowheads. Right, fractions of virus particles undergoing uptake by clathrin-mediated endocytosis in polarized (plated 5 d before infection) or nonpolarized (plated 1 d before infection) MDCK cells quantified by counting. Data are shown as the mean value ± SD from at least five independent experiments.

FIGURE 6:

Characteristics of clathrin-coated pits associated with entry of MRV virion and ISVP particles at the apical surface of polarized MDCK cells. Data were acquired by 4D spinning-disk confocal microscopy, as indicated for Figure 5. (A) Kinetic intensity profiles of single, representative coated pits: one empty, that is, not containing a fluorescent virus particle; one containing a fluorescent virion; and one containing a fluorescent ISVP. The AP2-GFP fluorescence intensity for each time point has been normalized to the maximum AP2-GFP fluorescence intensity reached during formation of the clathrin-coated pit in each example. (B) Scatter plot of the lifetimes of coated pits lacking or containing an MRV particle. Data are shown as the mean value ± SD from three cells for pits with each type of cargo; n = number of pits analyzed. Statistical significance values for the observed differences in pit lifetimes are shown. (C) Scatter plot of the maximum AP2-GFP fluorescence intensities of coated pits lacking or containing an MRV particle. The maximum fluorescence intensity of each pit during the course of uptake has been normalized to the average maximum fluorescence intensity of the empty pits. Data are shown as the mean value ± SD from three cells for pits with each type of cargo; n = number of pits analyzed. No statistically significant differences were found.

FIGURE 7:

Displacement of clathrin-coated vesicles mediating uptake of MRV virion and ISVP particles at the apical surface of polarized MDCK cells. Fluorescent virions or ISVPs were added to polarized MDCK cells stably expressing AP2-GFP, and their uptake was imaged by 4D live-cell spinning-disk confocal microscopy, as described for Figure 5. (A) Kinetic data for a single, representative virion-uptake event. The fluorescence intensity of AP2-GFP associated with the clathrin-coated pit is tracked in green, the Z-displacement of the virion is tracked in red, and the velocity of X/Y-displacement of the virion is tracked in cyan. (B) Kinetic data for single, representative uptake events involving an empty pit (open circles), a virion-containing pit (black circles), or an ISVP-containing pit (gray circles). The Z-displacement of each pit is tracked relative to its original position on the cell surface. Inset shows scatter plot of the total Z-displacement of pits with each different cargo. In all cases, the Z-position was defined by the AP2-GFP signal. No statistically significant differences were found.

FIGURE 8:

Endosomal association of MRV virion and ISVP particles after cell entry. Cells transiently expressing Rab5-GFP or Rab7-GFP for 16 h were inoculated with fluorescent virus particles, and Z-series images were acquired from individual cells at different times postinoculation by 4D spinning-disk confocal microscopy, as described in Materials and Methods. (A) Maximum-intensity projection images of MDCK cells expressing early endosome marker Rab5-GFP (left panels) or late endosome marker Rab7-GFP (right panels) at 30 min postinoculation with both AF563-labeled virions (red) and AF647-labeled ISVPs (blue). White boxes correspond to the magnified insets. (B) Quantification of the fraction of virions and ISVPs (same particles as shown in A) colocalizing with Rab5-GFP (left panel) and Rab7-GFP (right panel). The number of virions or ISVPs in these endosomal compartments at different times postinoculation was normalized to the total number of virions or ISVPs in those cells at each respective time. The histogram represents ± SD for 10 cells analyzed. (C) Similar experiment as described in (B), but this time inoculating the cells with AF647-labeled ISVPs (blue) and AF563-labeled μ1(N42A)-ISVPs (magenta). The histogram represents the mean ± SD for 10 cells analyzed.