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 ...