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. 2014 May:456-457:87-95.
doi: 10.1016/j.virol.2014.03.009. Epub 2014 Apr 2.

Host cell autophagy promotes BK virus infection

Stephanie J Bouley  1 , Melissa S Maginnis  2 , Aaron Derdowski  2 , Gretchen V Gee  2 , Bethany A O'Hara  2 , Christian D Nelson  2 , Anne M Bara  1 , Walter J Atwood  2 , Aisling S Dugan  3
Affiliations

Host cell autophagy promotes BK virus infection

Stephanie J Bouley et al. Virology. 2014 May.

Abstract

Autophagy is important for a variety for virus life cycles. We sought to determine the role of autophagy in human BK polyomavirus (BKPyV) infection. The addition excess amino acids during viral infection reduced BKPyV infection. Perturbing autophagy levels using inhibitors, 3-MA, bafilomycin A1, and spautin-1, also reduced infection, while rapamycin treatment of host cells increased infection. siRNA knockdown of autophagy genes, ATG7 and Beclin-1, corresponded to a decrease in BKPyV infection. BKPyV infection not only correlated with autophagosome formation, but also virus particles localized to autophagy-specific compartments early in infection. These data support a novel role for autophagy in the promotion of BKPyV infection.

Keywords: Autophagosome; Autophagy; BK; BKPyV; BKV; Nephropathy; Polyomavirus.

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Figures

Fig. 1
Fig. 1
Amino acid supplementation decreases BKPyV Infection. (A) Vero cells were challenged with BKPyV in EMEM media with 5% fetal bovine serum with and without additional essential amino acids. EMEM without additional supplementation is labeled 0×. After infection, the cells were replaced with EMEM media with 5% fetal bovine serum with or without the addition of amino acids and left for duration of infection. Cells were fixed at 72 h post infection using paraformaldehyde and permeabilized with Triton X-100. Infection was determined by using an antibody (PAB597) specific to the viral protein VP1 and then scoring the number of VP1+ cells using indirect immunofluorescence. (B) Cell death was evaluated 24 h following amino acid supplementation by scoring the number of cells excluding trypan blue and graphing the percentage of cells that excluded the trypan blue dye. To measure cell proliferation a MTS assay was used. Vero cells in 96 well plate were incubated with amino acids for 24 h after which 20 μl of CellTiter 96 AQueous One Solution Reagent – MTS (Promega) reagent was added directly to cells and media for 2 h, and absorbance was measure at 450 nm. The absorbance of 0× was used as a control for cell viability. (C) Vero cells were transfected with a plasmid expressing LC3-GFP and incubated for 24 h. Cells were treated with different concentrations of amino acid for 24 h in the presence of 100 nM rapamycin. LC3-GFP distribution was observed using immunofluorescence. The average number of LC3-GFP+ punctae per cell for 80 cells was scored. Each graph represents the average of three or more experiments. Error bars on bar graphs represent the SEM. ⁎⁎p-value<0.02.
Fig. 2
Fig. 2
Autophagy affects BKPyV infection. (A) Vero cells were pretreated with rapamycin or an equal volume of DMSO for 24 h, infected with BKPyV, and rapamycin added back for 24 h. Cells were fixed and stained for VP1 at 72 h post infection as in Fig. 1. (B) Vero cells were pretreated with 3-MA for 3 h, infected with BKPyV in the absence of 3-MA, and then media containing 3-MA was added back to cells for 24 h. Cells were fixed and stained for VP1 at 72 h. (C) Vero cells were pretreated with 100 nM of rapamycin for 24 h and 5 mM of 3-MA for 3 h where indicated, infected with BKPyV, and drugs added back for 24 h. After 72 h cells were stained for VP1 expression. (D) After Vero cells were infected with BKPyV, cells were treated with spautin-1 or DMSO for 72 h after which cells were fixed and stained from VP1 protein. (E) Vero cells were infected with BKPyV in the absence of drug. After infection, cells were treated the indicated concentrations of bafilomycin A1 or the DMSO control for 24 h. Cells were fixed and stained for VP1 at 72 h (F) Representative images of indirect immunofluorescence staining of VP1 (BKPyV infected cells) in Vero cells treated with drugs from 0 to 24 h at 72 h post infection. (G) Vero cells were transfected with a plasmid expressing LC3-GFP and incubated for 24 h. Cells were treated with media alone (no drug), serum free media for 3 h, 100 nM rapamycin for 24 h, 5 uM spautin-1 for 24 h, 5 mM 3-MA for 3 h, DMSO for 24 h and fixed then with paraformaldehyde. LC3-GFP distribution was observed using immunofluorescence. A representative image of an LC3-GFP+ expressing cell after treatment shows the change in LC3 localization (magnification 1000×). The number in the top right corner is the average number of punctae scored for 80 cells. (H) The average number of LC3-GFP+ punctae per cell for 80 cells was scored. The treatments were as follows: 100 nM rapamycin, DMSO, and 5 uM spautin-1 for 24 h, and serum free media and 5 mM 3-MA for 3 h. Graphs represent data from three or more experiments. Error bars on bar graphs represent the SEM. ⁎p-value=0.02–0.05 and ⁎⁎p-value<0.02, or p-value was included.
Fig. 3
Fig. 3
Autophagy Inhibitors decrease BKPyV infection early in the viral life cycle. Cells were treated for the indicated times before or after BKPyV infection with 5 mM 3-MA, 5 uM Spautin-1, 100 nM bafilomycin-A, and the media- or DMSO-control. Cells were fixed at 72 h post infection, and infection was determined by scoring the number of VP1 expressing cells. The effect of the drugs on infection levels was calculated by comparing the average number of infected cells in control-treated conditions to the average number of infected cells in inhibitor-treated cells. A positive value indicates the autophagy-inhibitor reduced BKPyV infection. Each bar graph represent the average of three experiments ⁎p-value<0.02. Error bars on bar graphs represent the SEM.
Fig. 4
Fig. 4
Knockdown of autophagy proteins, Beclin-1 and ATG7, reduced in BKPYV infection. (A) HeLa cells were transfected with 70 nM siRNA. After 48 h, cells were harvested, protein lysates were resolved by SDS-PAGE gel, and immunoblot analysis for LC3, Beclin-1, ATG7, and tubulin was performed. Tubulin expression was used as a loading control. Numeric values represent the ratio of immunoblot band intensity of indicated gene/tubulin. (B) HeLa cells were transfected with 70 nM siRNA, which contained a mixture of 3–5 siRNA molecules directed to different regions of a gene, and incubated for 48 h. Cells were then infected with BKPyV and fixed at 72 h post infection. Infected cells were by scored by VP1 expression. The graph represents the percentage of infected cells from three or more experiments, each which have been normalized to scrambled siRNA control sequence (Scr). ⁎p-value of siRNA treatment compared to Scr control is <0.02. Error bars on bar graphs represent the SEM.
Fig. 5
Fig. 5
BKPyV localizes to the autophagosome. (A–D) Vero cells were plated on coverslips and transfected with 0.5 µg LC3-GFP plasmid and incubated for 24 h. Cells were then infected with BKPyV-AF633 at 37 °C for 3 h, fixed and mounted onto slides using DAPI mounting media. Cells were analyzed for appearance of LC3-positive autophagosomes (green) and infection of labeled BKPyV (red) use a Zeiss 710 confocal microscope and Zen imaging software. Images analyzed using ImageJ image analysis software. Four representative cells show that BKPyV-Af633 resides within the LC3-GFP+ autophagosome and with smaller LC3-GFP+ punctae (magnification 630X). (A and B) (ii) An orthogonal Z slice of (iii) (along yellow x-axis). (iii) A magnified image of BKPyV and LC3-GFP+ as shown in (i). (iv) An orthogonal Z slice of (iii) (along yellow y-axis). (E) LC3-GFP transfected cells were scored for the pattern of LC3-GFP distribution (n=60, left). BKPyV-633 infected/LC3-GFP transfected cells were scored for LC3-GFP distribution (n=60, middle). BKPyV-633 infected that showed colocalization with LC3-GFO were scored for pattern of LC3-GFP distribution (n=60, right).
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