Anion homeostasis is important for non-lytic release of BK polyomavirus from infected cells

Abstract

BK polyomavirus (BKPyV) is a member of a family of potentially oncogenic viruses, whose reactivation can cause severe pathological conditions in transplant patients, leading to graft rejection. As with many non-enveloped viruses, it is assumed that virus release occurs through lysis of the host cell. We now show the first evidence for a non-lytic release pathway for BKPyV and that this pathway can be blocked by the anion channel inhibitor DIDS. Our data show a dose-dependent effect of DIDS on the release of BKPyV virions. We also observed an accumulation of viral capsids in large LAMP-1-positive acidic organelles within the cytoplasm of cells upon DIDS treatment, suggesting potential late endosome or lysosome-related compartments are involved in non-lytic BKPyV release. These data highlight a novel mechanism by which polyomaviruses can be released from infected cells in an active and non-lytic manner, and that anion homeostasis regulation is important in this pathway.

Keywords: BKPyV; DIDS; anion homeostasis; polyomavirus; virus release.

Figure 1.

DIDS blocks release of infectious virions in the presence of DIDS. RPTE cells were infected at 1 IU cell⁻¹ and treated with DIDS (or DMSO as a control) after 24 h. After 48 h, the media and cells were harvested separately. (a) A fluorescent focus assay was run to determine the IU ml⁻¹ of BKPyV in the cells and supernatant. The percentage release was calculated under each condition and shown in table 1. The control, 50 and 100 µM DIDS were repeated four times, the percentage release calculated and the mean determined. (b) One-way ANOVA was run and the data found to be highly significant (p ≤ 0.0001). RPTE cells were treated with or without DIDS for 24 h, and MQAE added to cell for the last hour of incubation. (c) After washing with PBS, cells were imaged using a 10× lens on a wide-field fluorescence microscope.

Figure 2.

Analysis of released capsid proteins and viral genome support DIDS blocking viral release. (a–b) Effect of DIDS on viral capsid protein levels (a) in the cell and (b) released into the supernatant. RPTE cells were infected with BK-Dunlop at 1 IU cell⁻¹ and had DIDS added 24 h post-infection. The virus was harvested at 48 h. The controls are uninfected and untreated RPTE cells; uninfected cells treated with 50 µM DIDS and infected cells treated with DMSO instead of DIDS. Tubulin was detected in the cell-associated sample as a loading control. (c) Quantification of the western blot data was performed using the Li-Cor Odyssey software on the VP1 bands present from the cell-associated virus and the virus released into the supernatant from two independent experiments. Error bars represent the distribution of the two datasets. (d) Genome levels were detected from the cell and supernatant samples using qPCR and analysed using Rotor-Gene.

Figure 3.

Effect on localization of viral proteins by DIDS. RPTE cells were left uninfected or infected at 1 IU cell⁻¹ and were treated with DIDS or DMSO as a control after 24 h. Cells were fixed 48 h post-infection and stained for either VP1 (PAb597) or VP2/3 (red). DAPI staining is shown in blue, as is the auto-fluorescence of DIDS. Images are single z-slices acquired using confocal microscopy.

Figure 4.

VP2 and 3 co-localize with LAMP-1 and LysoTracker in the presence of DIDS. RPTE cells were infected at 1 IU cell⁻¹ and were treated with DIDS or DMSO as a control after 24 h. (a,b) Cells were fixed 48 h post-infection and stained for VP2 and VP3 (green) and LAMP-1 (b), or treated with LysoTracker 2 h before fixing (a) (red). DAPI staining is shown in blue, as is the auto-fluorescence of DIDS. Images are single z-slices acquired using confocal microscopy.

Figure 5.

Transmission electron microscopy images of RPTE cells infected with BK-Dunlop and treated with DIDS. (a,b) RPTE cells were infected at 3 IU cell⁻¹ for 48 h, with (b) DIDS or (a) a DMSO control added 24 h post-infection. (ii),(iii) Close ups from (i) in both conditions. (ii) Large numbers of BKPyV arranged in their nuclear factories and were used to determine whether the cell was infected. (iii) Compartments within the cytosol containing BKPyV particles (45 nm in size).

Figure 6.

Quantification of TEM data. The number of BKPyV capsids counted in each compartment structure and calculated as number of capsids nm⁻². RPTE cells were infected at 3 IU cell⁻¹ and then fixed at 36 (a), 48 (b) or 60 h (c). A total of 50 µM DIDS or DMSO as a control was added 24 h before fixing. The Mann–Whitney test was used to determine significance, with values of p ≤ 0.0001 for all time points.