Transactivation of the human polyomavirus BK early promoter by cellular and viral factors.

摘要

The human polyomavirus BK (BKV) infects up to 90% of the human population. Immunosuppressed individuals, such as bone marrow transplant recipients, renal transplant recipients, and AIDS patients have an increased risk of developing a clinically manifest reactivation of BKV.; In renal transplant recipients, BKV reactivation is associated with ureteric stenosis and a severe interstitial nephritis termed BKV allograft nephropathy, or BKVAN. While it is well established that BKV is the polyomavirus responsible for BKVAN, to date the mechanism of reactivation of BKV from latency remains unknown. It has been suggested that specific changes in the allograft must be present to promote BKVAN, since only the allograft is affected. Previous episodes of acute rejection may render renal tubular cells permissive for BKV reactivation, since mouse kidney cells become permissive to acute polyomavirus infection in response to cellular damage. We hypothesized that one possible mechanism for the reactivation of BKV may be the induction of transcription factor binding to the promoter thereby leading to activation of BKV transcription. Computer analysis identified binding sites for the inducible transcription factors NFkappaB and CCAAT/enhancer binding protein beta (C/EBPbeta) within the BKV promoter. Transient transfection experiments showed that the BKV early promoter is activated synergistically by overexpressed p65 and C/EBPbeta in the green monkey kidney cell line CV-1. Electrophoretic mobility shift assays (EMSA) verified the binding of NFkappaB and C/EBPbeta to the sites identified by computer analysis. We next demonstrated via glutathione-S-transferase affinity chromatography a direct physical interaction between p65 and C/EBPbeta. Site-directed mutagenesis of the NFkappaB and C/EBPbeta binding sites in the BKV early promoter demonstrated that synergism between p65 and C/EBPbeta requires only the NFkappaB site. Subsequent experiments showed that only the c-terminal bZIP domain of C/EBPbeta is required for synergistic activation of BKV transcription by p65 and C/EBPbeta.; Though BKV is best known for its role as the etiologic agent of BKVAN, in recent years reactivation of BKV has been shown to have the potential to cause several complications ranging from nephritis, atypical retinitis, and hemorrhagic cystitis to a disseminated infection involving multiple organs in patients with AIDS. We investigated the potential effect of the HIV-1-encoded proteins Tat and Vpr on BKV transcriptional activity and found that Tat dramatically stimulates the BKV early promoter that is responsible for producing the viral early protein T-antigen. We employed site-directed mutagenesis analysis of potential Tat-responsive transcriptional motifs complemented by EMSA and found that Tat activates BKV-early transcription by inducing the binding of the p65 subunit of NFkappaB to the kappaB motif in the BKV promoter. We also identified an RNA binding site within the 5' untranslated region of BKV-early transcripts BKV-early-TAR with a sequence identical to the bulge of the HIV-1 transactivation responsive RNA to which Tat binds. RNA EMSA complemented by deletion of the BKV-early-TAR sequence demonstrated that Tat binds to BKV-early-TAR and BKV-early-TAR is required for transactivation of BKV-early transcription by Tat. We therefore conclude that Tat positively affects BKV-early transcription and this supports the idea that BKV reactivation-induced pathologies should be considered in the differential diagnosis of complications in HIV-1 infected individuals. Furthermore, our findings add the BKV promoter to the growing list of heterologous promoters through which Tat acts to enhance AIDS-related pathologies.