Novel Human Polyomavirus Noncoding Control Regions Differ in Bidirectional Gene Expression according to Host Cell, Large T-Antigen Expression, and Clinically Occurring Rearrangements.

Human polyomavirus (HPyV) DNA genomes contain three regions denoted the early viral gene region (EVGR), encoding the regulatory T-antigens and one microRNA, the late viral gene region (LVGR), encoding the structural Vp capsid proteins, and the noncoding control region (NCCR). The NCCR harbors the origin of viral genome replication and bidirectional promoter/enhancer functions governing EVGR and LVGR expression on opposite DNA strands. Despite principal similarities, HPyV NCCRs differ in length, sequence, and architecture. To functionally compare HPyV NCCRs, sequences from human isolates were inserted into a bidirectional reporter vector using dsRed2 for EVGR expression and green fluorescent protein (GFP) for LVGR expression. Transfecting HPyV NCCR reporter vectors into human embryonic kidney 293 (HEK293) cells and flow cytometry normalized to archetype BKPyV NCCR revealed a hierarchy of EVGR expression levels with MCPyV, HPyV12, and STLPyV NCCRs conferring stronger levels and HPyV6, HPyV9, and HPyV10 NCCRs weaker levels, while LVGR expression was less variable and showed comparable activity levels. Transfection of HEK293T cells expressing simian virus 40 (SV40) large T antigen (LTag) increased EVGR expression for most HPyV NCCRs, which correlated with the number of LTag-binding sites (Spearman's r, 0.625; P < 0.05) and decreased following SV40 LTag small interfering RNA (siRNA) knockdown. LTag-dependent activation was specifically confirmed for two different MCPyV NCCRs in 293MCT cells expressing the cognate MCPyV LTag. HPyV NCCR expression in different cell lines derived from skin (A375), cervix (HeLaNT), lung (A549), brain (Hs683), and colon (SW480) demonstrated that host cell properties significantly modulate the baseline HPyV NCCR activity, which partly synergized with SV40 LTag expression. Clinically occurring NCCR sequence rearrangements of HPyV7 PITT-1 and -2 and HPyV9 UF1 were found to increase EVGR expression compared to the respective HPyV archetype, but this was partly host cell type specific.IMPORTANCE HPyV NCCRs integrate essential viral functions with respect to host cell specificity, persistence, viral replication, and disease. Here, we show that HPyV NCCRs not only differ in sequence length, number, and position of LTag- and common transcription factor-binding sites but also confer differences in bidirectional viral gene expression. Importantly, EVGR reporter expression was significantly modulated by LTag expression and by host cell properties. Clinical sequence variants of HPyV7 and HPyV9 NCCRs containing deletions and insertions were associated with increased EVGR expression, similar to BKPyV and JCPyV rearrangements, emphasizing that HPyV NCCR sequences are major determinants not only of host cell tropism but also of pathogenicity. These results will help to define secondary HPyV cell tropism beyond HPyV surface receptors, to identify key viral and host factors shaping the viral life cycle, and to develop preclinical models of HPyV persistence and replication and suitable antiviral targets.

Donor-derived, metastatic urothelial cancer after kidney transplantation associated with a potentially oncogenic BK polyomavirus.

BK polyomavirus has been linked to urothelial carcinoma in immunosuppressed patients. Here, we performed comprehensive genomic analysis of a BK polyomavirus-associated, metachronous, multifocal and metastatic micropapillary urothelial cancer in a kidney transplant recipient. Dissecting cancer heterogeneity by sorting technologies prior to array-comparative genomic hybridization followed by short tandem repeat analysis revealed that the metastatic urothelial cancer was of donor origin (4-year-old male). The top 50 cancer-associated genes showed no key driver mutations as assessed by next-generation sequencing. Whole genome sequencing and BK polyomavirus-specific amplification provided evidence for episomal and subgenomic chromosomally integrated BK polyomavirus genomes, which carried the same unique 17-bp deletion signature in the viral non-coding control region (NCCR). Whereas no role in oncogenesis could be attributed to the host gene integration in chromosome 1, the 17-bp deletion in the NCCR increased early viral gene expression, but decreased viral replication capacity. Consequently, urothelial cells were exposed to high levels of the transforming BK polyomavirus early proteins large tumour antigen and small tumour antigen from episomal and integrated gene expression. Surgery combined with discontinuation of immunosuppression resulted in complete remission, but sacrificed the renal transplant. Thus, this report links, for the first time, BK polyomavirus NCCR rearrangements with oncogenic transformation in urothelial cancer in immunosuppressed patients. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.