BK Polyomavirus Replication in Renal Tubular Epithelial Cells Is Inhibited by Sirolimus, but Activated by Tacrolimus Through a Pathway Involving FKBP-12

Abstract

BK polyomavirus (BKPyV) replication causes nephropathy and premature kidney transplant failure. Insufficient BKPyV-specific T cell control is regarded as a key mechanism, but direct effects of immunosuppressive drugs on BKPyV replication might play an additional role. We compared the effects of mammalian target of rapamycin (mTOR)- and calcineurin-inhibitors on BKPyV replication in primary human renal tubular epithelial cells. Sirolimus impaired BKPyV replication with a 90% inhibitory concentration of 4 ng/mL by interfering with mTOR-SP6-kinase activation. Sirolimus inhibition was rapid and effective up to 24 h postinfection during viral early gene expression, but not thereafter, during viral late gene expression. The mTORC-1 kinase inhibitor torin-1 showed a similar inhibition profile, supporting the notion that early steps of BKPyV replication depend on mTOR activity. Cyclosporine A also inhibited BKPyV replication, while tacrolimus activated BKPyV replication and reversed sirolimus inhibition. FK binding protein 12kda (FKBP-12) siRNA knockdown abrogated sirolimus inhibition and increased BKPyV replication similar to adding tacrolimus. Thus, sirolimus and tacrolimus exert opposite effects on BKPyV replication in renal tubular epithelial cells by a mechanism involving FKBP-12 as common target. Immunosuppressive drugs may therefore contribute directly to the risk of BKPyV replication and nephropathy besides suppressing T cell functions. The data provide rationales for clinical trials aiming at reducing the risk of BKPyV replication and disease in kidney transplantation.

Keywords: calcineurin inhibitor (CNI); calcineurin inhibitor: tacrolimus; immunosuppressant; infection and infectious agents; mechanistic target of rapamycin: sirolimus; viral: BK / JC / polyoma, kidney biology.

Figure 1

SIR inhibition of BKPyV replication. (A) SIR inhibition of supernatant BKPyV loads. RPTECs infected with BKPyV(DUN), mock (0), or SIR were added at 2 hpi at the indicated concentrations, and BKPyV load in supernatants was measured after 72 hpi. (B) SIR inhibition of BKPyV protein expression. RPTECS were infected with BKPyV(DUN) and at 2 hpi treated with SIR (4 ng/mL) or mock, and immunofluorescence staining was performed at 24, 48, and 72 hpi (red: large T‐antigen, LTag; green: agnoprotein, Agno; cyan, capsid protein VP1; blue, DNA, and images were taken at 100× magnification). (C) Quantification of total cell count DAPI‐positive cells; (D) LTag‐positive cells; (E) VP1‐positive cells. Cells were counted by Image J64 and normalized to the value of the mock‐treated cells at the time points 48 and 72 hpi. BKPyV, BK polyomavirus; DAPI, 4′,6‐diamidino‐2‐phenylindole; hpi, hours postinfection; LTag, large T‐antigen; RPTECs, renal proximal tubule epithelial cells; SIR, sirolimus.

Figure 2

SIR inhibition of intracellular BKPyV load and time course. (A) SIR inhibits intracellular BKPyV replication. Infected RPTECs were treated with 4 ng/mL SIR at 2 hpi and lysed at 24, 36, and 48 hpi. Intracellular viral DNA was extracted and analyzed by qPCR for BKPyV load. The calculated values represented the GEq/150 000 cells and normalized to infected mock‐treated cells at 24 hpi. (B) SIR inhibition is time dependent. Infected RPTECs were treated with 4 ng/mL SIR at the indicated time points postinfection. The supernatants were measured at 72 hpi and expressed as percent of mock treatment. BKPyV, BK polyomavirus; hpi, hours postinfection; qPCR, quantitative polymerase chain reaction; RPTECs, renal proximal tubule epithelial cells; SIR, sirolimus.

Figure 3

SIR treatment reduces BKPyV protein expression and blocks mTOR pathway activated by BKPyV infection. Cell lysates were prepared at the different times postinfection in the presence and absence of SIR (4 ng/mL) as indicated and analyzed with Western blotting for the indicated viral and host cell proteins. (A) SIR delays and reduces BKPyV protein expression. The EVGR protein small T‐antigen (sTag) and LVGR proteins VP1 and Agno are analyzed by Western blot as indicated, and tubulin was used as a loading control. (B) BKPyV infection activates the mTOR‐p70S6Kinase pathway. Cell lysates were prepared at the indicated times after BKPyV infection in the presence or absence of SIR (4 ng/mL) and analyzed by Western blotting on 10% SDS‐PAGE for 70S6kinase phosphorylation (P‐p70SK1) and total p70S6Kinase (p70S6K1). (C) BKPyV virus‐like particles (BKVLP) activate Akt‐mTOR pathway. RPTECs were starved for 48 h and then exposed to BKVLPs (hemaglutination titer of 1:3200) or mock treatment before preparing cell lysates for Western blot analysis on 10% Tris‐Tricine gel for the indicated targets P‐Akt, total Akt, P‐p70 S6K1, and total p70 S6K1. (D) BKPyV infection activates the Akt‐mTOR pathway. RPTECs were starved for 48 h and then exposed BKPyV(DUN) virions before preparing cell lysates for Western blot analysis for the indicated targets P‐Akt, P‐mTOR, 4E‐BP, and tubulin. (E) BKPyV infection activates p38 and JNK kinase pathway. RPTECs were starved for 48 h and then exposed BKPyV(DUN) virions before preparing cell lysates at the indicated time points for Western blot analysis for the indicated targets P‐p38, P‐Erk1/2, P‐JNK, and tubulin. Agno, agnoprotein; BKPyV, BK polyomavirus; 4E‐BP, translation inhibitory factor 4E binding protein; EVGR, early viral gene region; hpi, hours postinfection; LVGR, late viral gene region; mTOR, mammalian target of rapamycin; P‐Akt, phosphorylated serine‐threonine kinase‐Akt; SDS‐PAGE, sodium dodecylsulfate–polyacrylamide gel electrophoresis; SIR, sirolimus.

Figure 4

Torin1 inhibits BKPyV replication in RPTECs. (A) Dose‐dependent inhibition of supernatant BKPyV loads. RPTECs were infected with BKPyV(DUN) for 2 h, and treated with the indicated final concentrations of Torin1. At 72 hpi, the BKPyV load in culture supernatants was determined by qPCR. (B) Dose‐dependent inhibition of p70S6kinase phosphorylation. RPTECs were infected with BKPyV(DUN) for 2 h, and treated with the indicated final concentrations of Torin1. Cell lysates were prepared at 6 hpi and analyzed by Western blotting for P‐p70‐S6K1 and total p70 S6K1. (C) Time‐dependent inhibition of BKPyV replication. RPTECs were infected with BKPyV(DUN) for 2 h, and treated at the indicated time points postinfection with 100 nM of Torin1. At 2 hpi, 100 nM Torin1 was added. At 72 hpi, the BKPyV load in culture supernatants was determined by qPCR. BKPyV, BK polyomavirus; hpi, hours postinfection; p70‐S6K1, total p70S6Kinase; P‐p70‐S6K1, phosphorylated S6‐kinase of 70kD; qPCR, quantitative polymerase chain reaction; RPTECs, renal proximal tubule epithelial cells.

Figure 5

Comparing SIR treatment on BKPyV replication in RPTECs after FKBP‐12 knockdown. (A) RPTECs were seeded into a T25 flask and FKBP‐12 was transfected with siRNA tFKBP‐12 targeting siRNA (+) or scrambled control siRNA (–) as described in Materials and Methods. On the next day, the respective RPTECs were seeded (2.5*10⁵ cells/well; six‐well plate) and left to adhere overnight. The cells were infected with BKPyV and treated at 2 hpi with the indicated final concentrations of SIR. At 48 hpi, cell lysates were prepared and analyzed by a 15% Tris‐Tricine SDS‐PAGE and Western blotting for FKBP‐12, BKPyV VP1, Agno, and tubulin. (B) RPTECs transfected with siRNA‐FKBP‐12 and siRNA control were infected with BKPyV and treated with SIR (4 ng/mL) at 2 hpi. At 72 hpi, the BKPyV load in culture supernatants was determined by qPCR. Agno, agnoprotein; BKPyV, BK polyomavirus; FKBP, FK binding protein; hpi, hours postinfection; qPCR, quantitative polymerase chain reaction; RPTECs, renal proximal tubule epithelial cells; SDS‐PAGE, sodium dodecylsulfate–polyacrylamide gel electrophoresis; SIR, sirolimus; VP1, viral capsid protein 1.

Figure 6

TAC reverses SIR inhibition of BKPyV replication in RPTECs. (A) BKPyV supernatant loads. RPTECs were infected with BKPyV for 2 h, and treated with the indicated final concentrations of SIR and TAC as described in Materials and Methods. At 72 hpi, the BKPyV load in culture supernatants was determined by qPCR. (B) p70S6 Kinase phosphorylation. RPTECs were infected with BKPyV for 2 h, and treated with the indicated final concentrations of SIR and TAC as described in Materials and Methods. At 6 hpi, cell lysates were analyzed by Western blotting for P‐p70 S6K1 and p70 S6K1. (C) Effect of TAC on BKPyV replication in RPTECs after FKBP‐12 knockdown. RPTECs transfected with siRNA FKBP‐12 or scrambled control were infected with BKPyV for 2 h, and then treated with the indicated final concentrations of TAC. At 72 hpi, the BKPyV load in culture supernatants was determined by qPCR. BKPyV, BK polyomavirus; FKBP, FK binding protein; hpi, hours postinfection; P‐p70 S6K1, phosphorylated S6‐kinase of 70kD; qPCR, quantitative polymerase chain reaction; RPTECs, renal proximal tubule epithelial cells; SIR, sirolimus; TAC, tacrolimus.

Figure 7

The effect on SIR and TAC on the DNA synthesis in RPTECs. RPTECs were seeded onto coverslips overnight, and mock‐treated, or treated with 4 ng/mL SIR, or 20 ng/mL TAC for 22 h. EdU labeling was performed for 2 h and then the cells were fixed and analyzed with immunofluorescence. Cellular DNA (blue) and the newly labeled cellular DNA (pink). Image J64 was used to quantify and normalize the Hoechst‐positive and EdU‐positive cells. RPTECs, renal proximal tubule epithelial cells; SIR, sirolimus; TAC, tacrolimus.

Figure 8

TAC stimulates and CsA inhibits BKPyV replication in RPTECs. RPTECs were infected with BKPyV for 2 h, and treated with the indicated final concentrations of TAC and CsA as described in Materials and Methods. At 72 hpi, the BKPyV load in culture supernatants was determined by qPCR. BKPyV, BK polyomavirus; CsA, cyclosporine A; hpi, hours postinfection; qPCR, quantitative polymerase chain reaction; RPTECs, renal proximal tubule epithelial cells; TAC, tacrolimus.

Figure 9

Effects of calcineurin inhibitors and mTOR inhibitors on BKPyV replication. Akt, plasma membrane located, inositol‐activated serine‐threonine kinase; BKPyV, BK polyomavirus; CsA, cyclosporine A; FKBP, FK binding protein; mTOR, mammalian target of rapamycin; mTORC1, mammalian target of rapamycin complex 1; SIR, sirolimus; S6K, S6 kinase; TAC, tacrolimus; TSC, tuberous sclerosis factor; 4E‐BP, translation inhibitor 4E binding protein.