scholarly journals Control of Archetype BK Polyomavirus MicroRNA Expression

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.

scholarly journals Control of archetype BK polyomavirus miRNA expression

2020 ◽  
Author(s):  
Wei Zou ◽  
Gau Shoua Vue ◽  
Benedetta Assetta ◽  
Heather Manza ◽  
Walter J. Atwood ◽  
...  
Keyword(s):  
Hemorrhagic Cystitis ◽  
Antigen Expression ◽  
Marrow Transplant ◽  
T Antigen ◽  
Replication Capacity ◽  
Late Gene Expression ◽  
Cis Acting ◽  
Transplant Patients ◽  
Bk Polyomavirus ◽  
Dna Replication Origin

AbstractBK polyomavirus (BKPyV) is a ubiquitous human pathogen, with over 80% of adults worldwide 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 non-coding 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 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 the 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 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 the intron of these greater-than-genome size primary transcripts.

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

2021 ◽  
Author(s):  
Zongsong Wu ◽  
Fabrice E Graf ◽  
Hans H. Hirsch
Keyword(s):  
Retinoic Acid ◽  
Protein Expression ◽  
Hemorrhagic Cystitis ◽  
Vero Cells ◽  
T Antigen ◽  
Large T Antigen ◽  
Urothelial Cells ◽  
Sv40 Large T Antigen ◽  
Treatment And Prevention ◽  
Bk Polyomavirus

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.

scholarly journals BK Polyomavirus Genomic Integration and Large T Antigen Expression: Evolving Paradigms in Human Oncogenesis

2017 ◽  
Vol 17 (6) ◽  
pp. 1674-1680 ◽  
Author(s):  
D. J. Kenan ◽  
P. A. Mieczkowski ◽  
E. Latulippe ◽  
I. Côté ◽  
H. K. Singh ◽  
...  
Keyword(s):  
Antigen Expression ◽  
T Antigen ◽  
Large T Antigen ◽  
Genomic Integration ◽  
Bk Polyomavirus

scholarly journals BK polyomavirus (BKPyV) is a risk factor for bladder cancer through induction of APOBEC3-mediated genomic damage

2021 ◽  
Author(s):  
Simon Charles Baker ◽  
Andrew S Mason ◽  
Raphael G Slip ◽  
Katie T Skinner ◽  
Andrew Macdonald ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bladder Cancer ◽  
Risk Factor ◽  
The Elderly ◽  
T Antigen ◽  
Transplant Patients ◽  
Adult Kidney ◽  
Bk Polyomavirus ◽  
Vitro Model

Limited understanding of bladder cancer aetiopathology hampers progress in reducing incidence. BK polyomavirus (BKPyV) is a common childhood infection that can be reactivated in the adult kidney leading to viruria. Here we used a mitotically-quiescent, differentiated, normal human urothelial in vitro model to study BKPyV infection. BKPyV infection led to significantly elevated APOBEC3A and APOBEC3B protein, increased deaminase activity and greater numbers of apurinic/apyrimidinic sites in the host urothelial genome. BKPyV Large T antigen (LT-Ag) stimulated re-entry into the cell cycle via inhibition of Retinoblastoma protein and activation of EZH2, E2F1 and FOXM1, which combined to push urothelial cells from G0 into an arrested G2 cell cycle state. The single-stranded DNA displacement loops formed during BKPyV-infection, provide a substrate for APOBEC3 enzymes where they interacted with LT-Ag. These results support reactivated BKPyV infections in adults as a risk factor for bladder cancer in immune-insufficient populations, including transplant patients and the elderly.

scholarly journals A pilot study of alternative TrkAIII splicing in Merkel cell carcinoma: a potential oncogenic mechanism and novel therapeutic target

2019 ◽  
Vol 38 (1) ◽  
Author(s):  
Lucia Cappabianca ◽  
Stefano Guadagni ◽  
Rita Maccarone ◽  
Michela Sebastiano ◽  
Alessandro Chiominto ◽  
...  
Keyword(s):  
Pilot Study ◽  
Therapeutic Target ◽  
Normal Skin ◽  
Stage Iv ◽  
Antigen Expression ◽  
T Antigen ◽  
Activation Mechanism ◽  
Merkel Cell ◽  
Large T Antigen ◽  
Sv40 Large T Antigen

Abstract Background Merkel cell carcinomas (MCCs) are rare, aggressive, therapeutically-challenging skin tumours that are increasing in incidence and have poor survival rates. The majority are caused by genomic Merkel cell polyomavirus (MCPyV) integration and MCPyV T-antigen expression. Recently, a potential oncogenic role for the tropomyosin-related tyrosine kinase A receptor (TrkA) has been proposed in MCC. Alternative TrkAIII splicing is a TrkA oncogenic activation mechanism that can be promoted by SV40 large T-antigen, an analogue of MCPyV large T-antigen. In this pilot study, therefore, we have evaluated TrkAIII splicing as a novel potential oncogenic mechanism and therapeutic target in MCPyV positive MCC. Methods Formalin-fixed paraffin-embedded MCC tissues, consisting of 10 stage IV, 1 stage IIIB, 1 stage IIB, 4 stage IIA and 2 stage I tumours, from patients diagnosed and treated from September 2006 to March, 2019, at the University of L’Aquila, L’Aquila, Italy, were compared to 3 primary basal cell carcinomas (BCCs), 3 primary squamous cell carcinomas (SCCs) and 2 normal skin samples by RT-PCR for MCPyV large T-antigen, small T-antigen, VP-1 expression and alternative TrkAIII splicing and by indirect IF for evidence of intracellular TrkA isoform expression and activation. Results 9 of 10 Recurrent stage IV MCCs were from patients (P.1–3) treated with surgery plus loco-regional Melphalan chemotherapy and remaining MMCs, including 1 stage IV tumour, were from patients treated with surgery alone (P. 4–11). All MCPyV positive MCCs exhibiting MCPyV large T-antigen expression (17 of 18MCCs, 90%) exhibited alternative TrkAIII mRNA splicing (100%), which was exclusive in a significant number and predominant (> 50%) in all stage IV MCCs and the majority of stage 1-III MCCs. MCCs with higher TrkAIII to 18S rRNA expression ratios also exhibited strong or intermediate immunoreactivity to anti-TrkA antibodies, consistent with cytoplasmic TrkAIII expression and activation. In contrast, the MCPyV negative MCC, BCCs, SCCs and normal skin tissues all exhibited exclusive fully-spliced TrkA mRNA expression, associated with variable immunoreactivity for non-phosphorylated but not phosphorylated TrkA. Conclusions MCPyV positive MCCs but not MCPyV negative MCC, BCCs and SCCs exhibit predominant alternative TrkAIII splicing, with evidence of intracellular TrkAIII activation. This establishes a new potential MCC subset, unveils a novel potential MCPyV oncogenic mechanism and identifies TrkAIII as a novel potential therapeutic target in MCPyV positive MCC.

scholarly journals JC virus promoter/enhancers contain TATA box-associated Spi-B-binding sites that support early viral gene expression in primary astrocytes

2012 ◽  
Vol 93 (3) ◽  
pp. 651-661 ◽  
Author(s):  
Leslie J. Marshall ◽  
Lisa D. Moore ◽  
Matthew M. Mirsky ◽  
Eugene O. Major
Keyword(s):  
Gene Expression ◽  
Binding Sites ◽  
Jc Virus ◽  
Viral Gene Expression ◽  
Antigen Expression ◽  
T Antigen ◽  
Viral Gene ◽  
Large T Antigen ◽  
Viral Promoter ◽  
The Brain

JC virus (JCV) is the aetiological agent of the demyelinating disease progressive multifocal leukoencephalopathy, an AIDS defining illness and serious complication of mAb therapies. Initial infection probably occurs in childhood. In the working model of dissemination, virus persists in the kidney and lymphoid tissues until immune suppression/modulation causes reactivation and trafficking to the brain where JCV replicates in oligodendrocytes. JCV infection is regulated through binding of host factors such as Spi-B to, and sequence variation in the non-coding control region (NCCR). Although NCCR sequences differ between sites of persistence and pathogenesis, evidence suggests that the virus that initiates infection in the brain disseminates via B-cells derived from latently infected haematopoietic precursors in the bone marrow. Spi-B binds adjacent to TATA boxes in the promoter/enhancer of the PML-associated JCV Mad-1 and Mad-4 viruses but not the non-pathogenic, kidney-associated archetype. The Spi-B-binding site of Mad-1/Mad-4 differs from that of archetype by a single nucleotide, AAAAGGGAAGGGA to AAAAGGGAAGGTA. Point mutation of the Mad-1 Spi-B site reduced early viral protein large T-antigen expression by up to fourfold. Strikingly, the reverse mutation in the archetype NCCR increased large T-antigen expression by 10-fold. Interestingly, Spi-B protein binds the NCCR sequence flanking the viral promoter/enhancer, but these sites are not essential for early viral gene expression. The effect of mutating Spi-B-binding sites within the JCV promoter/enhancer on early viral gene expression strongly suggests a role for Spi-B binding to the viral promoter/enhancer in the activation of early viral gene expression.

Selection against large T-antigen expression in cells transformed by lymphotropic papova virus

Virology ◽  
1992 ◽  
Vol 190 (1) ◽  
pp. 155-167 ◽  
Author(s):  
Volker von Hoyningen-Huene ◽  
Marion Kurth ◽  
Wolfgang Deppert
Keyword(s):  
Antigen Expression ◽  
T Antigen ◽  
Large T Antigen ◽  
Papova Virus ◽  
In Cells

Extended lifespan of normal human B lymphocytes experimentally infected by SV40 or transfected by SV40 large T antigen expression vector

2013 ◽  
Vol 37 (6) ◽  
pp. 681-689 ◽  
Author(s):  
Franca Nneka Alaribe ◽  
Elisa Mazzoni ◽  
Gian Matteo Rigolin ◽  
Lara Rizzotto ◽  
Stefania Maniero ◽  
...  
Keyword(s):  
B Lymphocytes ◽  
Expression Vector ◽  
Antigen Expression ◽  
T Antigen ◽  
Large T Antigen ◽  
Sv40 Large T Antigen ◽  
Extended Lifespan ◽  
Normal Human
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