1. Effects of immunosuppressive drugs on
BK polyomavirus (BKPyV) replication and host cell in vitro
Ksenia Yakhontova
Transplantation & Clinical Virology
Department Biomedicine (Haus Petersplatz)
University of Basel, Switzerland
Master Thesis defense
Haus Petersplatz, Basel
10:00, 11.03.2014

2.  Family of Polyomaviridae
 Non-enveloped DNA virus
 Circular double-stranded DNA of 5.2 kb
 Early proteins large and small T antigens
• Activate the host and late viral genes
 Late proteins VP1, VP2, VP3 and agnoprotein
• Capsid formation
• Agno, small regulatory protein
 BKPyV can be reactivated in kidney transplant recipients under
immunosuppressie conditions.
 In some cases the patients can develop BKPyV-associated
nephropathy (PyVAN) which might lead to graft loss.
BK Polyomavirus (BKPyV)
2

3.  No antiviral treatment available, only down regulation of
immunosuppression
 Role of immunosuppressive drugs in kidney transplantation
• Tacrolimus (TAC) causes higher rate of BKPyV associated diseases
• Sirolimus (SIR) associated with less frequent PyVAN manifestation
Immunosuppression
 Hirsch et al. (2013) American Journal of Transplantation
 Sanches Fructuoso et al. (2011) Transplant Infectious Disease
Allograft
rejection
BKPyV
viremia
IMMUNOSUPPRESSION

4. T-cell signaling: Tacrolimus (TAC) , Sirolimus (SIR)
Vicari-Christensen
et al. 2009
S6K1
Tacrolimus
Sirolimus
FKBP12
FKBP12
4E-BP

5. Aim of the Thesis
In vitro characterization of the effect of TAC and
SIR on the BKPyV replication and its host cell.
Possibly optimize choice of
immunosuppressive drugs in renal transplant
recipients with BKPyV replication

6. Experiment organization
The characterization of immunosuppressive drug-effect on the viral
replication and host cells was performed by analyzing:
Viral protein expression in the host cell
Host cell DNA synthesis
Activity of mTORC1 on its downstream substrate S6K1
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7. Influence of SIR and TAC on the expression of
BKPyV(DUN)-proteins in RPTECs
SIR-treatment leads to a reduction in BKPyV-protein expression.
TAC-treatment has a stimulating effect on BKPyV-protein
expression.
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8. Influence of SIR and TAC on the DNA synthesis
in RPTECs
4ng/mL of SIR on the other hand interferes with DNA synthesis.
Treatment with 20ng/mL TAC stimulates cell proliferation and
therefore might favor the replication of BKPyV.
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9. Influence of SIR and TAC on the mTORC1 activity
in kidney cells
The addition of 2ng/mL SIR on the other hand inhibits the ability of
mTORC1 to phosphorylate S6K1 at position T389.
TAC increases the mTORC1-mediated phosphorylation of S6K1.
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10. Conclusion
SIR-treatment at the clinically relevant concentration of 4ng/mL,
leads to a downregulation of multiple cellular processes including
translation, DNA replication and metabolism.
 Reduction of intracellular viral replication!
Treatment with 20ng/mL TAC can upregulate all of these
processes.
 Stimulation of intracellular viral replication!
One has now a better understanding of the effect of
immunosuppressive drugs TAC and SIR on the replication of
BKPyV and its host cells.
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11. Outlook for further experiments
Next step: Immunoprecipitation of BKPyV(DUN) proteins with HA-
FKBP12-coupled beads using Flip-In HEK293 cells transfected
with a plasmid encoding the BKPyV genome.
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Does one of the BKPyV proteins interact with FKBP12?

12. Acknowledgments
 Prof. Dr. med. Hans H. Hirsch
 Julia Manzetti
 Marion Wernli
 Min Ji-Lu
 Elvis Ajuh
 Dr. Tobias Bethge
 Dr. Rainer Gosert
 Piotr Kardas
 Dr. Céline Leboeuf
 Flavio Lombardo
 Dr. Gunhild Unterstab
 Fabian Weissbach
 Dr. Sabrina Wilk
Thank you for your attention
 Prof. Dr. Petr Broz
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13. Influence of SIR and TAC on the DNA synthesis
in RPTEC and RPTEC-TERT cells
Treatment with 20ng/mL of TAC stimulates cell proliferation and
therefore might favor the replication of BKPyV.
4ng/mL of SIR on the other hand interferes with DNA synthesis.
The drugs had the same effect on RPTECs and RPTEC-TERT cells.
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14. Influence of SIR and TAC on the metabolism
in kidney cells
SIR-treatment of RPTECs and RPTEC-TERT cells leads to a
decrease in cellular metabolism, while TAC has the opposite effect.
TAC increases the mTORC1-mediated phosphorylation of S6K1.
The addition of 2ng/mL SIR on the other hand inhibits the ability of
mTORC1 to phosphorylate S6K1. 14