Acitretin and Retinoic Acid Derivatives Inhibit BK Polyomavirus Replication in Primary Human Proximal Renal Tubular Epithelial and Urothelial Cells

Small-molecule drugs inhibiting BK polyomavirus (BKPyV) represent a significant unmet clinical need in view of polyomavirus-associated nephropathy or hemorrhagic cystitis which complicate 5% to 25% of kidney and hematopoietic cell transplantations. We characterized the inhibitory activity of acitretin on BKPyV-replication in primary human renal proximal tubular epithelial cells (RPTECs). Effective inhibitory concentration 50% (EC50) and 90% (EC90) were determined in dilution series measuring BKPyV loads, transcripts and protein expression, using cell proliferation, metabolic activity, and viability to estimate cytotoxic concentrations and selectivity indices (SI). Acitretin EC50 and EC90 in RPTECs were 0.64 (SI50 250) and 3.25 μM (SI90 49.2), respectively. Acitretin effectively inhibited BKPyV-replication until 72 h post-infection when added 24 h before until 12 h after infection, but decreased to <50% at later timepoints. Acitretin did not interfere with nuclear delivery of BKPyV genomes, but decreased large T-antigen transcription and protein expression. Acitretin did not inhibit the initial round of BKPyV-replication following transfection of full-length viral genomes, but affected subsequent rounds of re-infection. Acitretin also inhibited BKPyV-replication in human urothelial cells and in Vero cells, but not in COS-7 cells constitutively expressing SV40-large T-antigen. Retinoic acid-agonists (all-trans-retinoic acid, 9-cis-RA, 13-cis-RA, bexarotene, tamibarotene) and the RAR/RXR-antagonist RO41-5253 also inhibited BKPyV-replication, pointing to as yet undefined mechanism. Importance Acitretin selectively inhibits BKPyV-replication in primary human cell culture models of nephropathy and hemorrhagic cystitis. Since acitretin is an approved drug in clinical use reaching BKPyV-inhibiting concentrations in systemically treated patients, further studies are warranted to provide data for clinical repurposing of retinoids for treatment and prevention of replicative BKPyV-diseases.

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Protein expression of the RB-related gene family and SV40 large T antigen in mesothelioma and lung cancer

Oncogene ◽

10.1038/sj.onc.1203815 ◽

2000 ◽

Vol 19 (40) ◽

pp. 4632-4639 ◽

Cited By ~ 25

Author(s):

Sanjay Modi ◽

Akihito Kubo ◽

Herbert Oie ◽

Amy B Coxon ◽

Ahad Rehmatulla ◽

...

Keyword(s):

Lung Cancer ◽

Protein Expression ◽

Gene Family ◽

T Antigen ◽

Large T Antigen ◽

Related Gene ◽

Sv40 Large T Antigen

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Vectors bicistronically linking a gene of interest to the SV40 large T antigen in combination with the SV40 origin of replication enhance transient protein expression and luciferase reporter activity

BioTechniques ◽

10.2144/000113720 ◽

2011 ◽

Cited By ~ 3

Author(s):

Matthew Mahon

Keyword(s):

Protein Expression ◽

T Antigen ◽

Luciferase Reporter ◽

Origin Of Replication ◽

Large T Antigen ◽

Sv40 Large T Antigen ◽

Reporter Activity ◽

Transient Protein Expression ◽

Sv40 Origin

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Control of Archetype BK Polyomavirus MicroRNA Expression

Journal of Virology ◽

10.1128/jvi.01589-20 ◽

2020 ◽

Vol 95 (2) ◽

pp. e01589-20

Author(s):

Wei Zou ◽

Gau Shoua Vue ◽

Benedetta Assetta ◽

Heather Manza ◽

Walter J. Atwood ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Viral Genome ◽

Hemorrhagic Cystitis ◽

Antigen Expression ◽

T Antigen ◽

Replication Capacity ◽

Large T Antigen ◽

Transplant Patients ◽

Bk Polyomavirus ◽

Splice Junctions

ABSTRACTBK polyomavirus (BKPyV) is a ubiquitous human pathogen, with over 80% of adults worldwide being persistently infected. BKPyV infection is usually asymptomatic in healthy people; however, it causes polyomavirus-associated nephropathy in renal transplant patients and hemorrhagic cystitis in bone marrow transplant patients. BKPyV has a circular, double-stranded DNA genome that is divided genetically into three parts: an early region, a late region, and a noncoding control region (NCCR). The NCCR contains the viral DNA replication origin and cis-acting elements regulating viral early and late gene expression. It was previously shown that a BKPyV microRNA (miRNA) expressed from the late strand regulates viral large-T-antigen expression and limits the replication capacity of archetype BKPyV. A major unanswered question in the field is how expression of the viral miRNA is regulated. Typically, miRNA is expressed from introns in cellular genes, but there is no intron readily apparent in BKPyV from which the miRNA could derive. Here, we provide evidence for primary RNA transcripts that circle the genome more than once and include the NCCR. We identified splice junctions resulting from splicing of primary transcripts circling the genome more than once, and Sanger sequencing of reverse transcription-PCR (RT-PCR) products indicates that there are viral transcripts that circle the genome up to four times. Our data suggest that the miRNA is expressed from an intron spliced out of these greater-than-genome-size primary transcripts.IMPORTANCE The BK polyomavirus (BKPyV) miRNA plays an important role in regulating viral large-T-antigen expression and limiting the replication of archetype BKPyV, suggesting that the miRNA regulates BKPyV persistence. However, how miRNA expression is regulated is poorly understood. Here, we present evidence that the miRNA is expressed from an intron that is generated by RNA polymerase II transcribing the circular viral genome more than once. We identified splice junctions that could be generated only from primary transcripts that contain tandemly repeated copies of the viral genome. The results indicate another way in which viruses optimize expression of their genes using limited coding capacity.

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BK polyomavirus with archetypal and rearranged non-coding control regions is present in cerebrospinal fluids from patients with neurological complications

Journal of General Virology ◽

10.1099/vir.0.042143-0 ◽

2012 ◽

Vol 93 (8) ◽

pp. 1780-1794 ◽

Cited By ~ 15

Author(s):

Ana Bárcena-Panero ◽

Juan E. Echevarría ◽

Marijke Van Ghelue ◽

Giovanni Fedele ◽

Enrique Royuela ◽

...

Keyword(s):

Opportunistic Pathogen ◽

Vero Cells ◽

Neurological Complications ◽

T Antigen ◽

Cns Infection ◽

Large T Antigen ◽

Serum Samples ◽

Bk Polyomavirus ◽

Neurological Patients ◽

Coding Control

BK polyomavirus (BKPyV) has recently been postulated as an emerging opportunistic pathogen of the human central nervous system (CNS), but it is not known whether specific strains are associated with the neurotropic character of BKPyV. The presence of BKPyV large T-antigen DNA was examined in 2406 cerebrospinal fluid (CSF) samples from neurological patients with suspected JC polyomavirus infection. Twenty patients had a large T-antigen DNA-positive specimen. The non-coding control region (NCCR) of the BKPyV strains amplified from CSF from these 20 patients, strains circulating in renal and bone marrow transplant recipients and from healthy pregnant women was sequenced. The archetypal conformation was the most prevalent in all groups and 14 of the neurological patients harboured archetypal strains, while the remaining six patients possessed BKPyV with rearranged NCCR similar to previously reported variants from non-neurological patients. Transfection studies in Vero cells revealed that five of six early and four of six late rearranged promoters of these CSF isolates showed significantly higher activity than the corresponding archetypal promoter. From seven of the neurological patients with BKPyV DNA-positive CSF, paired serum samples were available. Five of them were negative for BKPyV DNA, while serum from the remaining two patients harboured BKPyV strains with archetypal NCCR that differed from those present in their CSF. Our results suggest that NCCR rearrangements are not a hallmark for BKPyV neurotropism and the dissemination of a rearranged NCCR from the blood may not be the origin of BKPyV CNS infection.

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Comparison of Immunohistochemical Staining for Large T Antigen and Capsid Protein VP1 in BK Polyomavirus-Associated Nephropathy

Nephron ◽

10.1159/000510967 ◽

2020 ◽

pp. 1-9

Author(s):

Kosuke Masutani ◽

Yuta Matsukuma ◽

Akihiro Tsuchimoto ◽

Yasuhiro Okabe ◽

Atsushi Doi ◽

...

Keyword(s):

Serum Creatinine ◽

Capsid Protein ◽

Immunohistochemical Staining ◽

T Antigen ◽

Tubular Damage ◽

Large T Antigen ◽

Sv40 Large T Antigen ◽

Negative Results ◽

Bk Polyomavirus ◽

Capsid Protein Vp1

Aim: Most transplant centres use SV40 large T antigen (TAg) staining for the diagnosis and assessment of BK polyomavirus-associated nephropathy (BKPyVAN). This study was performed to evaluate the significance of capsid protein VP1 expression in BKPyVAN. Methods: We performed immunohistochemical staining using anti-SV40 TAg and anti-BKPyV VP1 antibodies in 16 index biopsies and 12 re-biopsies of BKPyVAN and compared the patterns of positivity and the percentage of positive tubules by counting whole specimens. We investigated the correlation between serum creatinine increase from baseline and the percentage of positive tubules for both markers in 16 index biopsies. Results: In VP1 staining, positive findings were observed not only in the nuclei of tubular epithelial cells but also in the cytoplasm, cells shedding into the lumen, intra-tubular casts, and in the interstitium. Two of 28 biopsies (7.1%) showed TAg-positive and VP1-negative results, in which TAg-positive cells were detected only in a single tubule. The median (interquartile range) percentage of positive tubules was 2.8% (0.7–9.8%) for TAg and 1.4% (0.5–3.9%) for VP1 staining (p = 0.2). In 16 index biopsies, serum creatinine increases significantly correlated with the percentage of VP1-positive tubules (r = 0.49, p = 0.02), while this correlation revealed borderline significance with TAg-positive tubules. Conclusions: VP1 expression showed various patterns, but was detected in half as many tubules as TAg staining, which might lead to false negatives in the samples with minimal viral replication. However, increased VP1-positive tubules indicate advanced tubular damage and possible association with graft dysfunction.

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