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Brincidofovir (CMX001) inhibits BK polyomavirus replication in primary human urothelial cells.

ABSTRACT: BK polyomavirus (BKPyV)-associated hemorrhagic cystitis (PyVHC) complicates 5 to 15% of allogeneic hematopoietic stem cell transplantations. Targeted antivirals are still unavailable. Brincidofovir (BCV; previously CMX001) has shown inhibitory activity against diverse viruses, including BKPyV in a primary human renal tubule cell culture model of polyomavirus-associated nephropathy. We investigated the effects of BCV in BKPyV-infected and uninfected primary human urothelial cells (HUCs), the target cells of BKPyV in PyVHC. The BCV concentrations causing 50 and 90% reductions (EC50 and EC90) in the number of intracellular BKPyV genome equivalents per cell (icBKPyV) were 0.27 ?M and 0.59 ?M, respectively. At 0.63 ?M, BCV reduced viral late gene expression by 90% and halted progeny release. Preinfection treatment for only 24 h reduced icBKPyV similarly to treatment from 2 to 72 h postinfection, while combined pre- and postinfection treatment suppressed icBKPyV completely. After investigating BCV's effects on HUC viability, mean selectivity indices at 50 and 90% inhibition (SI50 and SI90) calculated for cellular DNA replication were 2.7 and 2.9, respectively, those for mitochondrial activity were 8.9 and 10.4, those for total ATP were 8.6 and 8.2, and those for membrane integrity were 25.9 and 16.7. The antiviral and cytostatic effects, but less so the cytotoxic effects, were inversely related to cell density. The cytotoxic effects at concentrations of ?10 ?M were rapid and likely related to BCV's lipid moiety. After carefully defining the antiviral, cytostatic, and cytotoxic properties of BCV in HUCs, we conclude that a preemptive or prophylactic approach in PyVHC is likely to give the best results.

SUBMITTER: Tylden GD

PROVIDER: S-EPMC4432119 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Brincidofovir (CMX001) inhibits BK polyomavirus replication in primary human urothelial cells.

Tylden Garth D GD Hirsch Hans H HH Rinaldo Christine Hanssen CH

Antimicrobial agents and chemotherapy 20150323 6

BK polyomavirus (BKPyV)-associated hemorrhagic cystitis (PyVHC) complicates 5 to 15% of allogeneic hematopoietic stem cell transplantations. Targeted antivirals are still unavailable. Brincidofovir (BCV; previously CMX001) has shown inhibitory activity against diverse viruses, including BKPyV in a primary human renal tubule cell culture model of polyomavirus-associated nephropathy. We investigated the effects of BCV in BKPyV-infected and uninfected primary human urothelial cells (HUCs), the targ ...[more]

PMID: 25801568

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Project description:UNLABELLED:BK polyomavirus (BKPyV) reactivation is associated with severe human disease in kidney and bone marrow transplant patients. The interplay between viral and host factors that regulates the productive infection process remains poorly understood. We have previously reported that the cellular DNA damage response (DDR) is activated upon lytic BKPyV infection and that its activation is required for optimal viral replication in primary kidney epithelial cells. In this report, we set out to determine what viral components are responsible for activating the two major phosphatidylinositol 3-kinase-like kinases (PI3KKs) involved in the DDR: ataxia telangiectasia mutated (ATM) kinase and ATM and Rad3-related (ATR) kinase. Using a combination of UV treatment, lentivirus transduction, and mutant virus infection experiments, our results demonstrate that neither the input virus nor the expression of large T antigen (TAg) alone is sufficient to trigger the activation of ATM or ATR in our primary culture model. Instead, our data suggest that the activation of both the ATM- and ATR-mediated DDR pathways is linked to viral DNA replication. Intriguingly, a TAg mutant virus that is unable to activate the DDR causes substantial host DNA damage. Our study provides insight into how DDRs are activated by polyomaviruses in primary cells with intact cell cycle checkpoints and how the activation might be linked to the maintenance of host genome stability. IMPORTANCE:Polyomaviruses are opportunistic pathogens that are associated with several human diseases under immunosuppressed conditions. BK polyomavirus (BKPyV) affects mostly kidney and bone marrow transplant patients. The detailed replication mechanism of these viruses remains to be determined. We have previously reported that BKPyV activates the host DNA damage response (DDR), a response normally used by the host cell to combat genotoxic stress, to aid its own replication. In this study, we identified that the trigger for DDR activation is viral replication. Furthermore, we show that the virus is able to cause host DNA damage in the absence of viral replication and DDR activation. These results suggest an intricate relationship between viral replication, DDR activation, and host genome instability.

Project description:Most humans have a lifelong imperceptible BK Polyomavirus (BKPyV) infection in epithelial cells lining the reno-urinary tract. In kidney transplant recipients, unrestricted high-level replication of donor-derived BKPyV in the allograft underlies polyomavirus-associated nephropathy, a condition with massive epithelial cell loss and inflammation causing premature allograft failure. There is limited understanding on how BKPyV disseminates throughout the reno-urinary tract and sometimes causes kidney damage. Tubule epithelial cells are tightly connected and have unique apical and basolateral membrane domains with highly specialized functions but all in vitro BKPyV studies have been performed in non-polarized cells. We therefore generated a polarized cell model of primary renal proximal tubule epithelial cells (RPTECs) and characterized BKPyV entry and release. After 8 days on permeable inserts, RPTECs demonstrated apico-basal polarity. BKPyV entry was most efficient via the apical membrane, that in vivo faces the tubular lumen, and depended on sialic acids. Progeny release started between 48 and 58 hours post-infection (hpi), and was exclusively detected in the apical compartment. From 72 hpi, cell lysis and detachment gradually increased but cells were mainly shed by extrusion and the barrier function was therefore maintained. The decoy-like cells were BKPyV infected and could transmit BKPyV to uninfected cells. By 120 hpi, the epithelial barrier was disrupted by severe cytopathic effects, and BKPyV entered the basolateral compartment mimicking the interstitial space. Addition of BKPyV-specific neutralizing antibodies to this compartment inhibited new infections. Taken together, we propose that during in vivo low-level BKPyV replication, BKPyV disseminates inside the tubular system, thereby causing minimal damage and delaying immune detection. However, in kidney transplant recipients lacking a well-functioning immune system, replication in the allograft will progress and eventually cause denudation of the basement membrane, leading to an increased number of decoy cells, high-level BKPyV-DNAuria and DNAemia, the latter a marker of allograft damage.

Dataset Information

Brincidofovir (CMX001) inhibits BK polyomavirus replication in primary human urothelial cells.

Publications

Brincidofovir (CMX001) inhibits BK polyomavirus replication in primary human urothelial cells.

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