Sp1 Sites in the Noncoding Control Region of BK Polyomavirus Are Key Regulators of Bidirectional Viral Early and Late Gene Expression

Abstract

In kidney transplant patients with BK polyomavirus (BKPyV) nephropathy, viral variants arise bearing rearranged noncoding control regions (rr-NCCRs) that increase viral early gene expression, replicative fitness, and cytopathology. rr-NCCRs result from various deletions and duplications of archetype NCCR (ww-NCCR) sequences, which alter transcription factor binding sites (TFBS). However, the role of specific TFBS is unclear. We inactivated 28 TFBS in the archetype NCCR by selective point mutations and examined viral gene expression in bidirectional reporter constructs. Compared to the archetype, group 1 mutations increased viral early gene expression similar to rr-NCCR and resulted from inactivating one Sp1 or one Ets1 TFBS near the late transcription start site (TSS). Group 2 mutations conferred intermediate early gene activation and affected NF1, YY1, and p53 sites between early and late TSS. BK polyomavirus (BKPyV) is one of now more than 12 human PyVs (1) and is known to infect more than 90% of the human population during early childhood (2-6). Following primary infection, BKPyV persists latently in the renourinary tract, with intermittent periods of asymptomatic shedding into urine (5, 7). BKPyV disease typically occurs in individuals with altered immune functions, to which viral determinants are thought to contribute (3, 8). Thus, BKPyV causes nephropathy in 1 to 14% of kidney transplant patients and hemorrhagic cystitis in 5 to 20% of allogeneic hematopoietic stem cell transplant recipients (2, 9).BKPyV strains excreted in urine of healthy immunocompetent individuals typically bear a noncoding control region (NCCR) of linear archetype (ww) architecture (ww-NCCR) (5, 10, 11) and grow slowly in primary human urothelial and renal tubular epithelial cells (8,12). In immunosuppressed kidney transplant patients with early stages of BKPyV-associated nephropathy, the virus contains mostly archetype ww-NCCR (8, 13). However, when immunosuppression is not readily reduced to permit specific T cells to resume control over BKPyV replication (14-17), viral variants with rearranged NCCRs (rr-NCCRs) emerge that are associated with higher plasma viral loads and more severe renal allograft pathology (8). Similar rr-NCCRs of JC polyomavirus have been linked with the emergence of progressive multifocal leukoencephalopathy, a debilitating, often fatal brain disease in immunocompromised patients with HIV/AIDS or receiving transplantation or therapies for cancer and immune diseases (18)(19)(20)(21)(22). The archetype NCCR of polyomaviruses is approximately 400 bp in length and harbors the origin of replication as well as bidirectional promoter/ enhancer functions that, in concert with the host cell signals, control the timing and sequential activation of early viral gene region (EVGR) expression, viral DNA genome replication, and late viral gene region (LVGR) expression. Notably, EVGR and LVGR are encoded and transcribed in opposite directions from the NCCR (23). The ϳ2.3-kb EVGR encodes the small T antigen, the large ...

Imperfect Symmetry of Sp1 and Core Promoter Sequences Regulates Early and Late Virus Gene Expression of the Bidirectional BK Polyomavirus Noncoding Control Region

“…The bidirectional reporter construct used to measure expression of the early (red fluorescent protein [RFP]) and late (green fluorescent protein [GFP]) virus gene regions has been described before (31). Furthermore, we described a fluorescenceactivated cell sorter (FACS)-based quantification method for this reporter construct (32). While the use of flow cytometry for the reporter assay enables a convenient means of visualization of expression, at the same time, accurate and reproducible quantification is achieved.…”

“…Group 2 TFBS mutations showed an intermediate phenotype with respect to EVGR expression and replication rates, whereas group 3 TFBS mutations reduced both EVGR and LVGR expression and permitted little or no viral replication (32).…”

“…In contrast, rearranged NCCRs (rr-NCCRs) associated with high blood viral loads and severe renal allograft pathology carry partial duplications and/or deletions of the archetype ww-NCCR sequence and give rise to virus strains with elevated levels of EVGR expression, faster viral replication, and more pronounced cytopathic effects in cell culture (31,32,40).…”

“…However, immu-nosuppressive drug-specific effects (20,30) and viral determinants (31,32) may also contribute to the evolution, kinetics, and severity of disease. At the core of what distinguishes archetype BKPyV strains with a low replicative capacity from the highly replicative strains found in kidney transplant patients are changes in the noncoding control region (NCCR) of the BKPyV genome (33).…”

“…These consist of an unaltered EVGR-proximal region covering the O 143 block with the origin of replication and the central P 68 block. Insertion strains carry complete or partial duplications of the P 68 block, while deletion strains are characterized by the absence of the LVGR-proximal Q 39 and R 63 blocks and, rarely, the S 63 block (31,32,40,41).…”