The oncogenic potential of BK-polyomavirus is linked to viral integration into the human genome

Abstract

It has been suggested that BK-polyomavirus is linked to oncogenesis via high expression levels of large T-antigen in some urothelial neoplasms arising following kidney transplantation. However, a causal association between BK-polyomavirus, large T-antigen expression and oncogenesis has never been demonstrated in humans. Here we describe an investigation using high-throughput sequencing of tumour DNA obtained from an urothelial carcinoma arising in a renal allograft. We show that a novel BK-polyomavirus strain, named CH-1, is integrated into exon 26 of the myosin-binding protein C1 gene (MYBPC1) on chromosome 12 in tumour cells but not in normal renal cells. Integration of the BK-polyomavirus results in a number of discrete alterations in viral gene expression, including: (a) disruption of VP1 protein expression and robust expression of large T-antigen; (b) preclusion of viral replication; and (c) deletions in the non-coding control region (NCCR), with presumed alterations in promoter feedback loops. Viral integration disrupts one MYBPC1 gene copy and likely alters its expression. Circular episomal BK-polyomavirus gene sequences are not found, and the renal allograft shows no productive polyomavirus infection or polyomavirus nephropathy. These findings support the hypothesis that integration of polyomaviruses is essential to tumourigenesis. It is likely that dysregulation of large T-antigen, with persistent over-expression in non-lytic cells, promotes cell growth, genetic instability and neoplastic transformation.

Keywords: BK virus; carcinoma; cell transformation, neoplastic; kidney transplantation; large T Antigen; oncogenesis; polyomavirus; polyomavirus nephropathy; virus integration.

Figure 1

Allograft nephrectomy specimen, bivalved. The invasive tumour (*) infiltrates the renal medulla and extends into the inner cortex; the pelvis shows hydronephrosis; a nephrostomy drain is seen in situ; the ureter and a portion of the urinary bladder extend downwards.

Figure 2

Histology of renal allograft tumour (FFPE tissue). (A) Top of image displays an in situ component and the bottom displays an invasive component; H&E stain; magnification = ×100. (B) The invasive carcinoma shows glandular differentiation with central foci of debris and moderate nuclear atypia; H&E stain; magnification = ×200. (C) Immunohistochemistry with an antibody against SV40 large T‐antigen shows strong intranuclear staining; magnification = ×200. (D) Immunohistochemistry with an antibody against polyomavirus capsid protein VP1 shows no expression of VP1 in the tumour; magnification = ×200.

Figure 3

Urothelial carcinoma cell nucleus with so‐called 'nuclear granules' (arrows; N, tumour cell nucleus). There is no evidence of viral particles. EM magnification = ×25 000.

Figure 4

Radial phylogenetic tree of VP1 capsid protein DNA sequences using ClustalW2 and FigTree v 1.4.2, comparing the Chapel Hill tumour‐associated polyomavirus‐1 (termed CH‐1) sequence to 14 polyomavirus sequences representing the major genotype groups of BK and JC‐polyomaviruses, as indicated.

Figure 5

Schematic of the BKV integration site and molecular details of the virus–host junctions. Human chromosomal integration of the circular BK‐polyomavirus genome results in linearization of the virus, with a breakpoint between codons 309 and 310 of the VP1 gene, and integration into exon 26 of the MYBVC1 gene at position 102 069 082 on chromosome 12. The coding sequences for capsid protein VP1 are indicated by a light red arrow in the circular genome. Linearization and integration disrupts the VP1 gene, with the C‐terminus encoded at the left end of the integrated fusion (indicated by # in the small red arrow) and the N‐terminus encoded at the right end of the integrated fusion (larger red rectangle). Integration further results in the generation of a putative fusion protein between the N‐terminus of VP1 and a short six‐amino acid segment within out‐of‐frame MYBPC1 exonic sequences before a stop codon is encountered (indicated by * in the right green MYBPC1 exon). The blue NCCR (non‐coding control region) in the virus is located towards the middle of the integrated virus, where both the early and late promoters (P‐early and P‐late, respectively) are expected to remain intact. Likewise, large T‐antigen and small t‐antigen sequences remain intact (black arrows), as well as the late gene product agnoprotein (dark red arrow). Transcription of the MYBPC1 gene proceeds from left to right, and the direction of transcription of the early and late BK‐polyomavirus gene products is indicated by the direction of the arrows representing these genes.

Figure 6

Proposed mechanism of oncogenesis by integration of BK‐polyomavirus. Viral infection leads to either productive infection, resulting in cell death, or latency, resulting in cell survival but with minimal viral replication. Either mechanism may lead to sporadic viral integration through unknown mechanisms. Once integrated, the virus is no longer replication‐competent and cannot produce new virions. Integration with linearization may occur before or after rearrangement of the NCCR, which can influence BK‐polyomavirus gene expression and transcription of the large T‐antigen gene sequence. Large T‐antigen or an active fragment thereof is typically expressed and up‐regulated via alteration of the NCCR region and attenuation of negative feedback loops from late gene products. Dysregulated large T‐antigen expression is known to inhibit cellular p53 and Rb function and can thereby promote oncogenesis.