Fig 1.
RPTECs develop a polarized morphology and functionality on cell culture inserts.
(A) Scheme of cells cultured on a permeable cell culture insert. (B and C) At 8 days post-seeding (dps), RPTECs on Falcon-inserts were fixed and stained for: (B) Na/K-ATPase (green) and acetylated α-tubulin (red), and (C) ZO-1 (green). Nuclei were stained with Draq5 (blue). Images are representative images from at least three independent experiments. Scale bar 10 μm. (D) Transepithelial electrical resistance (TEER) values of RPTECs at 2 to 14 dps. Data is derived from two to four biological replicates per timepoint. Data are shown as means and error bars represent ± standard deviation (SD). (E) Diffusion of FITC-dextran from the apical to the basolateral compartment across polarized and non-polarized RPTECs. Data is normalized to the non-polarized control, n = 3 and error bars represent ± SD. * = P < 0.05, one sample t test. (F) Accumulation of intracellular calcein AM in the presence or absence of the P-gp inhibitor Psc-833, quantified by fluorescence measurement using a plate reader. Data is normalized to the untreated control, n = 3 and error bars represent ± SD. *** = P < 0.001, one sample t test.
Fig 2.
Polarized RPTECs support BKPyV infection.
(A) Binding and internalization of BKPyV (MOI 0.1) in polarized RPTECs at 2 hours post infection (hpi) demonstrated by immunofluorescence staining against Vp1 (4942) (red) and Na/K-ATPase (green). Scale bar is 10 μm. (B) Transmission electron micrographs of polarized RPTECs that have been inoculated with BKPyV for 2 hours. Top image is an overview image with scale bar 5 μm. Bottom images are representative images from 70 nm serial sections of virions on the cell surface (black arrowhead). Scale bar 100 nm. Productive BKPyV infection in polarized RPTECs at 3 days post infection (dpi) with BKPyV (MOI 1) is demonstrated by: (C) Immunofluorescence staining against Vp1 (rabbit serum) (green) and LTag (Pab416) (red) (top image) or agnoprotein (green) and Vp1 (4942) (red) (bottom image). (D) Western blot using rabbit serums against N-terminal LTag, Vp1 and agnoprotein. Lysates of mock infected cells were used as negative control and a GAPDH antibody was used as a loading control. (E) Confocal microscopy images of BKPyV infected cells. Top images are stained with rabbit serum against N-terminal LTag (green) and an antibody against Vp1 (4942) (red). Bottom images are stained for Na/K-ATPase (green) and LTag (Pab416) (red). Mock infected cells are included as a negative control. Scale bar 10 μm. In (A), (C) and (E), nuclei are stained with Draq5 (blue) and representative images from three independent experiments are shown.
Fig 3.
BKPyV preferentially enters polarized RPTECs via the apical membrane.
(A) BKPyV infectivity following apical and basolateral infection. Apical infection was performed with MOI 0.1 while basolateral infection was done with 5.3x more virus. Data represents the number of infected cells based on immunofluorescence staining for Vp1 (4942) and agnoprotein at 3 dpi and is presented as relative infectivity normalized to the mean number of infected cells for apical infection. n = 5 and error bars represent ± SD. * = P < 0.05, two-tailed t test (B) Detection of BKPyV at 2 hours after apical and basolateral infection, respectively. Immunofluorescence staining for Vp1 (4942) was performed and followed by confocal microscopy and acquisition of z-stacks. Vp1-staining intensity was measured in sum z-projections and is represented as mean fluorescence intensity. Z-stacks of mock infected cells were used as a negative control and subtracted as background. Error bars represent ± SD and n = 3. ** = P < 0.01, two-tailed t test. (C) Representative z-slices from (B) stained for Vp1 (4942) (green) and Draq5 (blue). Scale bar 10 μm. (D) BKPyV infectivity in non-polarized RPTECs following apical or basolateral infection at 2 dps. Apical infection was performed with approximately MOI 0.1 while basolateral infection was done with 5.3x more virus. Data represents the number of infected cells based on immunofluorescence staining for Vp1 (4942) and agnoprotein at 3 dpi and is presented as relative infectivity normalized to the mean number of infected cells for apical infection. n = 4 and error bars represent ± SD. ns = P > 0.05, two-tailed t test (E) BKPyV infectivity after neuraminidase-pretreatment. Data represents the number of infected cells based on immunofluorescence staining for Vp1 (4942) and agnoprotein at 3 dpi and is presented as relative infectivity normalized to the untreated control. n = 3 and error bars represent ± SD. *** = P < 0.001, one sample t test. (F) Representative apical z-slice from a z-stack of polarized RPTECs stained with Texas Red conjugated wheat germ agglutinin (red) and Hoechst (blue). Scale bar 10 μm.
Fig 4.
BKPyV is mainly released into the apical compartment.
(A) Polarized RPTECs were apically infected (MOI 0.3) and supernatants were collected at the indicated timepoints for BKPyV-DNA load (log 10 copies/ml) determination by qPCR. Data was generated from at least three independent experiments, except the 96 hpi timepoint which was derived from two independent experiments. (B) Polarized RPTECs were apically infected (MOI 1) and supernatants were collected at the indicated timepoints for determination of BKPyV infectious load (log 10 FFU/ml) by infectivity assay. Infectious load at 24 hpi was defined as input and subtracted as background. Data was generated from six independent experiments. Error bars represent ± SD for (A) and (B).
Fig 5.
BKPyV induced cytopathic effects and cell death in polarized RPTECs.
(A) Phase-contrast images of mock infected and BKPyV infected RPTECs (MOI 3) from 24 to 120 hpi. Representative images from two independent experiments are shown. Scale bar 100 μm. (B) Widefield microscopy of mock infected and BKPyV infected RPTECs incubated with CellTox dye. Data is presented as CellTox-ratio (mean total fluorescence from infected inserts/mean total fluorescence from mock infected inserts). Error bars represent ± SD and data is derived from at least three independent experiments. (C) Release of LDH into apical supernatants as measured by Promega LDH-Glo Cytotoxicity assay. Data is presented as LDH-ratio (infected-RLU/mock-RLU). Error bars represent ± SD and data is derived from at least three independent experiments.
Fig 6.
Time-lapse imaging and TEER-monitoring throughout BKPyV replication.
(A) Time-lapse imaging of mock infected and BKPyV infected (MOI 1) polarized RPTECs. Nuclei of permeabilized cells are stained with CellTox dye. Representative images from two independent experiments at 24, 48, 60, 72, 96 and 120 hpi are shown. Scale bar 50 μm. (B) Quantitation of cell permeabilization in (A). Presented is the number of new CellTox-positive cells from 36 hpi. Error bars represent ± SD, n = 2. (C) TEER-values of mock infected and BKPyV infected (MOI 0.1 and 10) polarized RPTECs from 0 to 7 dpi. Data is derived from three to six biological replicates and error bars represent ± SD.
Fig 7.
BKPyV replication leads to shedding of infectious decoy cells.
Widefield microscopy of detached cells harvested from supernatants of mock infected and BKPyV infected (MOI 1) polarized RPTECs at 5 dpi. Harvested cells were imaged with phase-contrast and fluorescence microscopy. All images are representative images from two independent experiments, except (B) which is derived from one experiment. (A) Detached cells incubated with CellTox dye (green) and imaged with a combination of phase-contrast and fluorescence microscopy. Scale bar 100 μm. (B) Detached cells after Papanicolaou staining. (C) Immunofluorescence staining of detached cells from mock infected and BKPyV infected (MOI 1) polarized RPTECs using rabbit serums against Vp1 and agnoprotein. (D) Western blot of lysates of detached RPTECs harvested at 5 dpi from BKPyV infected (MOI 1) RPTECs. Mock infected RPTEC-lysate was used as the negative control. The membrane was probed with rabbit serums against N-terminal LTag, Vp1 and agnoprotein and an antibody against GAPDH. A representative blot from two experiments is shown. (E) Immunofluorescence staining of non-polarized RPTECs after infection with decoy cells harvested from BKPyV infected polarized RPTECs. A rabbit serum against agnoprotein (green) and an antibody against Vp1 (4942) (red) were used. Prior to infection, the decoy cells were washed and centrifuged five times. The supernatant from the last centrifugation was used as a control. Images are representative images from two independents experiments.
Fig 8.
Cell shedding mainly occurs via extrusion.
(A and B) Representative time-lapses with oblique contrast and fluorescence microscopy of a cell undergoing extrusion (A) or sloughing off (B). Membranes are stained with CellMask (orange) and the nuclei of permeabilized cells are stained with CellTox dye (green). Scale bar 10 μm. Images are representative time-lapses from two independent experiments. (C) Quantification of cell fate of CellTox-positive cells at 4 dpi. Data is derived from two independent experiments where 653 cells have been examined. Error bars represent ± SD. (D) Classification of the morphology of detachment for CellTox-positive cells at 4 dpi (extrusion vs. slough off). Data is derived from two independent experiments and 71 examined cells. Error bars represent ± SD. (E) Neutralizing antibodies undergo transepithelial transport and inhibit spread of BKPyV infection. Polarized RPTECs were infected with a low MOI (0.1) and at 24 hpi, a BKPyV-specific neutralizing antibody or control antibody was added to the basolateral compartment. At 5 dpi, cells were stained for LTag with a mouse antibody (Pab416) and a C-terminal LTag rabbit serum and the number of infected cells were counted. Data is derived from two independent experiments and error bars represent ± SD.