Common Mechanisms of Virus-Mediated Oncogenesis in Bladder Cancers Arising In Solid Organ Transplant Recipients

A small percentage of bladder cancers in the general population have been found to harbor polyomaviruses and papillomaviruses. In contrast, up to 25% of tumors of solid organ transplant recipients, who are at an increased risk of developing bladder cancer, have been reported to harbor BK polyomavirus (BKPyV). To better understand the biology of the tumors and the mechanisms of carcinogenesis from any potential oncovirus, we performed whole genome sequencing and transcriptomic sequencing on bladder cancer specimens from 43 transplant patients. Roughly a third of the tumors from this patient population contained viral sequences, among which the most common were from BKPyV (N=9, 21%), JC polyomavirus (N=7, 16%), carcinogenic human papillomaviruses (N=3, 7%), and torque teno viruses (N=5, 12%). BKPyV presence was validated by Large T antigen staining and revealed heterogeneous staining patterns between cases ranging from less than one percent to 100% of tumor tissue staining positive. In most cases of BKPyV-positive tumors, the viral genome was integrated into the host chromosome consistent with microhomology-mediated end joining and, in some cases, resulting in focal amplifications of the tumor genome. In BKPyV-positive tumors, significant changes in the expression of E2F- and DREAM-regulated genes amongst others were also observed consistent with the depletion of RB-family members by BKPyV Large T antigen in the tumors. We identified four mutation signatures in our cases with APOBEC3 and SBS5 mutations being the most abundant. We also identified a widespread mutation signature most similar to that caused by the antiviral, ganciclovir, and several cases harbored a high mutation burden associated with aristolochic acid, a nephrotoxic compound found in some herbal medicines. The results indicate that viruses may play a causal role in the development of bladder cancer among immunosuppressed individuals.

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.

Evidence of BK Polyomavirus Infection in Urothelial but not Renal Tumors from a Single Center Cohort of Kidney Transplant Recipients

Emerging evidence indicates that reactivation of BK polyomavirus (BKPyV) in the kidney and urothelial tract of kidney transplant recipients (KTRs) may be associated with cancer in these sites. In this retrospective study of a single center cohort of KTRs (n = 1307), 10 clear cell renal cell carcinomas and 5 urinary bladder carcinomas were analyzed from 15 KTRs for the presence of BKPyV infection through immunohistochemistry and fluorescent in situ hybridization (FISH). Three of these patients had already exhibited biopsy-proven polyomavirus-associated nephropathies (PyVAN). Although the presence of BKPyV large-T antigen was evident in the urothelium from a kidney removed soon after PyVAN diagnosis, it was undetectable in all the formalin-fixed and paraffin-embedded (FFPE) blocks obtained from the 10 kidney tumors. By contrast, large-T antigen (LT) labeling of tumor cells was detected in two out of five bladder carcinomas. Lastly, the proportion of BKPyV DNA-FISH-positive bladder carcinoma nuclei was much lower than that of LT-positive cells. Taken together, our findings further strengthen the association between BKPyV reactivation and cancer development in KTRs, especially bladder carcinoma.

Human Polyomavirus-Encoded Circular RNAs

ABSTRACTCircular RNAs (circRNAs) are a conserved class of RNAs with diverse functions. A subset of circRNAs are translated into peptides. Here we describe circular RNAs encoded by human polyomaviruses (HPyVs), including circular forms of RNAs encoding variants of the previously described alternative large T antigen open reading frame (ALTO) gene. Circular ALTO RNAs (circALTOs) can be detected in virus positive Merkel cell carcinoma (VP-MCC) cell lines and tumor samples. CircALTOs are stable, predominantly located in the cytoplasm, and N6-methyladenosine (m6A) modified. MCPyV circALTOs produce ALTO protein in cultured cells. MCPyV ALTO promotes the transcription of co-transfected reporter genes. MCPyV circALTOs are enriched in exosomes derived from VP-MCC lines and circALTO-transfected 293T cells, and purified exosomes can mediate ALTO expression and transcriptional activation. The related trichodysplasia spinulosa polyomavirus (TSPyV) also expresses a circALTO that can be detected in infected tissues and produces ALTO protein in cultured cells. Thus, human polyomavirus circRNAs are expressed in human tumors and tissues, encode for proteins, and may contribute to the infectious and tumorigenic properties of these viruses.

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

<b><i>Aim:</i></b> 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. <b><i>Methods:</i></b> 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. <b><i>Results:</i></b> 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 (<i>p</i> = 0.2). In 16 index biopsies, serum creatinine increases significantly correlated with the percentage of VP1-positive tubules (<i>r</i> = 0.49, <i>p</i> = 0.02), while this correlation revealed borderline significance with TAg-positive tubules. <b><i>Conclusions:</i></b> 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.

Control of Archetype BK Polyomavirus MicroRNA Expression

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.

Merkel Cell Polyomavirus Large T Antigen is Dispensable in G2 and M-Phase to Promote Proliferation of Merkel Cell Carcinoma Cells

Merkel cell carcinoma (MCC) is an aggressive skin cancer frequently caused by the Merkel cell polyomavirus (MCPyV), and proliferation of MCPyV-positive MCC tumor cells depends on the expression of a virus-encoded truncated Large T antigen (LT) oncoprotein. Here, we asked in which phases of the cell cycle LT activity is required for MCC cell proliferation. Hence, we generated fusion-proteins of MCPyV-LT and parts of geminin (GMMN) or chromatin licensing and DNA replication factor1 (CDT1). This allowed us to ectopically express an LT, which is degraded either in the G1 or G2 phase of the cell cycle, respectively, in MCC cells with inducible T antigen knockdown. We demonstrate that LT expressed only in G1 is capable of rescuing LT knockdown-induced growth suppression while LT expressed in S and G2/M phases fails to support proliferation of MCC cells. These results suggest that the crucial function of LT, which has been demonstrated to be inactivation of the cellular Retinoblastoma protein 1 (RB1) is only required to initiate S phase entry.