Subpopulations of non-coding control region variants within a cell culture-passaged stock of BK virus: sequence comparisons and biological characteristics

Abstract

In the circular DNA genome of the human polyomavirus BK an approximately 400 bp non-coding control region (NCCR) separates the early and late genes. The NCCR contains the origin of replication as well as the promoter/enhancer with a mosaic of cis-acting elements involved in the regulation of both early and late transcription. The NCCR has been shown to be very heterogeneous between different BK virus (BKV) strains. This may affect the host cell permissivity and oncogenic potential of a given BKV strain. Our previous studies with BKT-1B, a continuous cell line established from a BKV (Gardner) -induced hamster fibrosarcoma, revealed that the BKV DNA is integrated in the host genome in multiple copies. The sequence of the integrated BKV NCCR was substantially different from (and even contained sequences not found in) that of the BKV (Gardner) strain supposedly used to establish the BKT-1B cell line. PCR amplification, cloning and subsequent sequencing revealed that the original BKV (Gardner) stock contained at least seven different subpopulations of viral genomes. None of them had a control region 'anatomy' which was identical to either the BKV (Gardner) strain, the variant found integrated in BKT-1B cells or any previously published NCCR. In order to study the biological significance of these new BKV NCCR variants we developed a simple cassette model allowing the NCCRs of the new variants to be cloned in an identical genomic background of BKV protein-coding sequences and performed transfection studies with the recombinant genomes in non-permissive rodent cells and in semi-permissive monkey cells. The results demonstrated that the NCCR variants conferred striking differences, in both transforming capacity and host cell permissivity, to the recombinant BKV genomes. Sequence comparisons suggested genetic explanations for these differences, as well as evolutionary relationships between BKV NCCRs.

Early and late promoters of BK polyomavirus, Merkel cell polyomavirus, Trichodysplasia spinulosa-associated polyomavirus and human polyomavirus 12 are among the strongest of all known human polyomaviruses in 10 different cell lines

“…Rearrangements in the NCCR of the BKPyV and JCPyV have been shown to affect virus propagation in vitro (Ault, 1997;Broekema et al, 2010;Gosert et al, 2008Gosert et al, , 2010Johnsen et al, 1995). The NCCR structure in novel HPyV isolates has been studied less and the effect of mutations on viral gene expression and replication has not been investigated.…”

“…BKPyV and JCPyV clinical isolates with rearranged NCCR have been isolated and the architecture of the NCCR has been shown to be a major determinant of viral gene expression and replication in vitro (Ault, 1997;Broekema et al, 2010;Gosert et al, 2008Gosert et al, , 2010Johnsen et al, 1995). Less is known about the NCCR of the novel HPyV that circulate in the human population.…”

Replication of Oral BK Virus in Human Salivary Gland Cells

“…The BKPyV NCCR is the main determinant of in vitro replication (9), and rearrangements confer significant differences in rep- lication capacity, transforming potential, host cell permissivity, and tropism (12,37,39,40,(51)(52)(53). NCCR rearrangements are defined as changes in the NCCR block sequences compared to the archetype.…”

“…The NCCR is the major determinant of in vitro replication (9) and is arbitrarily divided into five block sequences: O (142 bp), P (68 bp), Q (39 bp), R (63 bp), and S (63 bp). The blocks contain the origin of DNA replication and an array of transcription factor binding sites (TFBS) (10)(11)(12)(13). The late region encodes the agnoprotein and the structural proteins VP1, VP2 and VP3 (4,14).…”

The Presumed Polyomavirus Viroporin VP4 of Simian Virus 40 or Human BK Polyomavirus Is Not Required for Viral Progeny Release

“…All SV40 experiments were performed with strain 776 and virus with start codon mutations of VP4 (Table 1). All mutants were created by long PCR of pBKV (34-2) (ATCC 45025) (18), pBKV WWT (19), or pWTSV40 JL (20) using overlapping primers that targeted the ATG start codon (Table 1) and the high-fidelity Phusion polymerase (M0530S; New England BioLabs) according to the manufacturer's instructions as previously described (21). Sanger sequencing confirmed successful mutagenesis.…”