Characterization of BK viral responses to the dual-PI3K/MTOR inhibitor dactolisib (NVP BEZ-235) in a renal cell culture model
Lerner, Gabriel B.
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BK virus (BKV) is a ubiquitous polyomavirus known to asymptomatically reside in the renal tissues of up to 90% of the human population. BK virions reactivate during periods of intense immunosuppression and can cause disease in renal transplant recipients, such as BKV-associated nephropathy (BKVAN). BKVAN can lead to loss of the transplanted renal grafts. For this reason, the study of BKV biology is of importance to the transplant community. Previous studies have shown that BKV upregulates the pro-growth mTOR pathway in host cells, thereby increasing BKV replicative efficiency. Downstream effectors of the mTOR pathway, particularly p70S6 kinase, control the basal rate of protein translation, in part through regulation of ribosomal biogenesis. It was hypothesized that viral upregulation of the mTOR pathway is beneficial for viral replication due to an increase in the number of ribosomes available to translate viral proteins. Therefore, inhibition of the mTOR pathway could reduce viral replication. This study investigated whether host cell mTOR inhibition could reduce BK viral replication in an in vitro model. We utilized the dual PI3K/mTOR inhibitor NVP BEZ-235 (Novartis Pharmaceuticals), which potently downregulates expression of both upstream (PI3K) and central (mTOR) effectors of the mTOR pathway. Immortalized renal epithelial cells were exposed to varying concentrations of BEZ-235 for a period of 48 hours, infected with BK virus for three hours, and allowed to grow for a further 48 hours. Cell populations were then assayed via quantitative PCR (qPCR), Western blotting and fluorescent immunohistochemical staining to determine the effect of BEZ-235 on BK viral replication. Western blot experiments confirmed the effectiveness of BEZ-235's inhibition of the mTOR pathway in a renal epithelial cell culture model, as well as downregulation of the mTOR pathway during BK viral infection. Western blotting for the key BK replicative protein Large T antigen showed a dose-dependent decrease in expression, with increasing concentrations of BEZ-235. Fluorescent immunohistochemical staining showed a dose-dependent decrease in expression of Large T antigen staining in host cell nuclei. qPCR results were inconclusive, in that no clear pattern in the number of BKV genomes per cell population could be observed across the range of BEZ-235 concentrations tested. While results from our study indicate that BEZ-235 can reduce BKV replication in vitro, further in vitro experimentation, including repetition of approaches already carried out as well as novel approaches, will be needed to definitively confirm inhibition of the mTOR pathway as a viable antiviral strategy.