Transforming growth factor-beta-mediated regulation of BK virus gene expression

Abstract

The increasing prevalence of BK virus (BKV)-associated diseases in immunosuppressed patients has prompted an investigation of the immune response to BKV, especially the role of cytokines in regulating viral replication. We examined the effect of TGF-beta, a cytokine that is stimulated by certain immunosuppressive therapies, on BKV gene expression during lytic infection of renal proximal tubule epithelial cells. Viral gene expression, and specifically the activity of the BKV early promoter, is regulated by TGF-beta in a strain-dependent manner. Promoter activity is upregulated in the presence of TGF-beta for the TU strain of BKV, and not for the Dik, Dunlop, or Proto-2 strains. Using site-directed mutagenesis, we have identified a small segment of the TU promoter that is required for stimulation in response to TGF-beta. These results demonstrate that BKV strains can respond differently to cytokine treatment and suggest that TGF-beta may play a role in the reactivation of BKV.

FIGURE 1. TGF-β upregulates BKV TU gene expression during infection

A) RPTE cells were infected with the indicated strains of BKV at an MOI of 0.5 IU/cell and treated with 10 ng/ml TGF-β at three to four hpi. Total cell protein was harvested at three dpi and analyzed by Western blot, probing for TAg and GAPDH. Mock, mock-infected samples with no TGF-β treatment. B) RPTE cells were infected with the TU strain of BKV at an MOI of 0.5 IU/cell in the presence or absence of 10 ng/ml TGF-β, and total cell RNA was prepared at 24, 36, 48, 72, and 96 hpi. Relative TAg transcript levels were determined using real time RT-PCR, normalizing to levels of GAPDH transcripts in each sample. One representative experiment is shown; triplicate samples were analyzed in the same assay. Fold expression of TAg at 24 hpi, untreated was arbitrarily set to one. Mock, mock-infected samples with no TGF-β treatment.

FIGURE 2. BKV early promoter activity in the presence of TGF-β

A) RPTE cells were cotransfected with BKV early promoter-firefly luciferase constructs and a promoterless control Renilla luciferase plasmid. TGF-β was added at three to four hpt and total cell lysates were harvested at 48 hpt. Luciferase assays were performed on triplicate samples and data are represented as relative light units (RLU) of firefly luciferase activity, normalized to RLU of Renilla luciferase activity. Data shown represent results obtained from three independent experiments. B) HT-1080 cells were cotransfected with BKV early promoter-firefly luciferase constructs and a promoterless control Renilla luciferase plasmid and assayed as described in (A). Data shown represent results obtained from three independent experiments.

FIGURE 3. Alignment of BKV NCCRs

The segments of the NCCRs (TU, Dik, Dunlop, Proto-2) from the nucleotide before the start codon of agnoprotein through the P block directly adjacent to the O block are shown. The O block is not shown because it is highly homologous between strains. Bold type indicates nucleotides that are identical in at least three of the strains. Underlined regions indicate the predicted Smad3 and ZEB-1 binding sites. Dots indicate nucleotides not found within a sequence. The starred nucleotides indicate the region of the TU NCCR that does not align anywhere in the Dik, Dunlop, or Proto-2 NCCRs. The early promoter is read from left to right (top to bottom), in the direction of the early coding region. The late promoter is read from right to left (bottom to top), in the direction of the late coding region.

FIGURE 4. Regions of the TU promoter required for TGF-β-mediated regulation of BKV

A) Schematic representation of promoter constructs. TU-Smt has a single base change in the core of the predicted Smad3 binding site (hatched). Dik+6TU has a 6 bp insertion that creates the predicted ZEB-1 binding site. The early promoter is read from left to right, with the start codon for the firefly luciferase gene following the right end of the promoter. B) RPTE cells were cotransfected with wildtype or mutant BKV early promoter-firefly luciferase constructs and a promoterless control Renilla luciferase plasmid. TGF-β was added at three to four hpt and total cell lysates were harvested at 48 hpt. Luciferase assays were performed on triplicate samples and data are represented as relative light units (RLU) of firefly luciferase activity, normalized to RLU of Renilla luciferase activity. Data shown represent results obtained from three independent experiments.