Johannes Korth1, Olympia E Anastasiou2, Jens Verheyen3, Julia Dickow3, Helene Sertznig3, Nicola Frericks3, Barbara Bleekmann3, Andreas Kribben4, Alexandra Brinkhoff4, Benjamin Wilde4, Kathrin Sutter3, Ulf Dittmer3, Sandra Ciesek3, Oliver Witzke5, Marek Widera6. 1. Department of Nephrology, University of Duisburg-Essen, University Hospital Essen, Hufelandstr. 55, 45147, Essen, Germany; Institute for Virology, University of Duisburg-Essen, University Hospital Essen, Virchowstr. 179, 45147, Essen, Germany. 2. Institute for Virology, University of Duisburg-Essen, University Hospital Essen, Virchowstr. 179, 45147, Essen, Germany; Department of Gastroenterology, University of Duisburg-Essen, University Hospital Essen, Hufelandstr. 55, 45147, Essen, Germany. 3. Institute for Virology, University of Duisburg-Essen, University Hospital Essen, Virchowstr. 179, 45147, Essen, Germany. 4. Department of Nephrology, University of Duisburg-Essen, University Hospital Essen, Hufelandstr. 55, 45147, Essen, Germany. 5. Department of Infectious Diseases, University of Duisburg-Essen, University Hospital Essen, Hufelandstr. 55, 45147, Essen, Germany. 6. Institute for Virology, University of Duisburg-Essen, University Hospital Essen, Virchowstr. 179, 45147, Essen, Germany. Electronic address: marek.widera@uni-due.de.
Abstract
BACKGROUND: Reactivation of the BK-Polyomavirus (BKPyV) can cause a polyomavirus associated nephropathy in approx. 10% of kidney transplant recipients. In these cases, current therapy is based on the reduction of immunosuppression. Since BKPyV-transcription is driven by the Non-Coding-Control-Region (NCCR) we were interested whether NCCR-activity is affected by immunosuppressive agents. METHODS: Plasma samples from 45 BKPyV-positive patients after renal transplantation were subjected to PCR-analysis. NCCR-amplicons were cloned into a plasmid that allows the quantification of early and late NCCR-activity by tdTomato and eGFP expression, respectively. HEK293T-cells were transfected with the reporter-plasmids, treated with immunosuppressive agents, and subjected to FACS-analysis. In addition, H727-cells were infected with patient derived BKPyV, treated with mTOR-inhibitors, and NCCR activity was analysed using qRT-PCR. RESULTS: While tacrolimus and cyclosporine-A did not affect NCCR-promoter-activity, treatment with mTOR1-inhibitor rapamycin resulted in the reduction of early, but not late-NCCR-promoter-activity. Treatment with dual mTOR1/2 inhibitors (INK128 or pp242) led to significant inhibition of early, however, concomitantly enhanced late-promoter-activity. In BKPyV infected cells both rapamycin and INK128 reduced early expression, however, INK128 resulted in higher late-mRNA levels when compared to rapamycin treatment. CONCLUSIONS: Our results demonstrate that mTOR1-inhibitors are able to reduce early-expression of wildtype and rearranged NCCRs, which might contribute to previously described inhibition of BKPyV-replication. Dual mTOR1/2-inhibitors, however, additionally might shift viral early into late-expression promoting synthesis of viral structural proteins and particle production.
BACKGROUND: Reactivation of the BK-Polyomavirus (BKPyV) can cause a polyomavirus associated nephropathy in approx. 10% of kidney transplant recipients. In these cases, current therapy is based on the reduction of immunosuppression. Since BKPyV-transcription is driven by the Non-Coding-Control-Region (NCCR) we were interested whether NCCR-activity is affected by immunosuppressive agents. METHODS: Plasma samples from 45 BKPyV-positive patients after renal transplantation were subjected to PCR-analysis. NCCR-amplicons were cloned into a plasmid that allows the quantification of early and late NCCR-activity by tdTomato and eGFP expression, respectively. HEK293T-cells were transfected with the reporter-plasmids, treated with immunosuppressive agents, and subjected to FACS-analysis. In addition, H727-cells were infected with patient derived BKPyV, treated with mTOR-inhibitors, and NCCR activity was analysed using qRT-PCR. RESULTS: While tacrolimus and cyclosporine-A did not affect NCCR-promoter-activity, treatment with mTOR1-inhibitor rapamycin resulted in the reduction of early, but not late-NCCR-promoter-activity. Treatment with dual mTOR1/2 inhibitors (INK128 or pp242) led to significant inhibition of early, however, concomitantly enhanced late-promoter-activity. In BKPyV infected cells both rapamycin and INK128 reduced early expression, however, INK128 resulted in higher late-mRNA levels when compared to rapamycin treatment. CONCLUSIONS: Our results demonstrate that mTOR1-inhibitors are able to reduce early-expression of wildtype and rearranged NCCRs, which might contribute to previously described inhibition of BKPyV-replication. Dual mTOR1/2-inhibitors, however, additionally might shift viral early into late-expression promoting synthesis of viral structural proteins and particle production.
Authors: Julia Dickow; Sandra Francois; Rouven-Luca Kaiserling; Anna Malyshkina; Ingo Drexler; Astrid Maria Westendorf; Karl Sebastian Lang; Mario L Santiago; Ulf Dittmer; Kathrin Sutter Journal: Front Immunol Date: 2019-09-24 Impact factor: 7.561
Authors: Hana Rohn; Rafael Tomoya Michita; Sabine Schramm; Sebastian Dolff; Anja Gäckler; Johannes Korth; Falko M Heinemann; Benjamin Wilde; Mirko Trilling; Peter A Horn; Andreas Kribben; Oliver Witzke; Vera Rebmann Journal: Cells Date: 2019-08-07 Impact factor: 6.600
Authors: Maxim Cherneha; Johannes Korth; Meike Kaulfuß; Mirko Trilling; Marek Widera; Hana Rohn; Sebastian Dolff; Nina Babel; André Hoerning; Andreas Kribben; Oliver Witzke Journal: Viruses Date: 2021-03-06 Impact factor: 5.048
Authors: Jennifer Alvarez Orellana; Hyun Jin Kwun; Sara Artusi; Yuan Chang; Patrick S Moore Journal: J Infect Dis Date: 2021-10-13 Impact factor: 5.226