Molecular interplay between T-Antigen and splicing factor, arginine/serine-rich 1 (SRSF1) controls JC virus gene expression in glial cells

In order to investigate the role of JCV regulatory proteins T-antigen and agnoprotein in SRSF1-mediated suppression of JCV gene expression, primary human fetal astrocytes (PHFA) were transiently transfected with a luciferase reporter construct in both early and late orientations (pLuc.JCV-Early or pLuc.JCV-Late). In addition to transfection with the luciferase reporter construct, PHFA cells were co-transfected with plasmids encoding SRSF1 (pCGT7-SRSF1), T-antigen (pCDNA3.1-T-ag), and agnoprotein (pCGT7-agno) in varying combinations. At 48 h post transfections, JCV early and late transcription was analyzed by luciferase reporter assay as described in materials and methods. Luciferase assay of JCV-early gene transcription showed a strong suppression with SRSF1, which was rescued by co-expression of T-antigen, as well as a significant increase in transcription with the expression of T-antigen alone (Fig. 1a). However, there was no observed effect of agnoprotein on transcriptional suppression by SRSF1. Similarly, luciferase assay of JCV-late gene transcription yielded strong suppression of transcription with SRSF1, and a significant increase in transcription with T-antigen. When compared with the early gene transcription, SRSF1 possessed a much stronger suppression of late gene transcription (compare panel A, lane 3 with panel C, lane 3). Consistent with early gene transcription, T-antigen was also able to rescue the SRSF1-mediated transcriptional suppression of late promoter (Fig. 1c). Western blot analysis of the whole cell protein extracts were completed, demonstrating overexpression of SRSF1 and the expression of the viral proteins that were transfected in parallel to luciferase assays (Fig. 1b, d). These results suggest a novel role of viral T-antigen in suppression of cellular SRSF1 in glial cells.

To determine possible impact of T-antigen on cellular expression of SRSF1, both primary human fetal astrocytes (PHFA) and a human glioblastoma cell line (T98G) were transiently transfected with an expression vector encoding T-antigen in increasing concentrations. After 48 h post-transfection, cells were harvested and the whole cell protein extracts were analyzed by Western blotting for the detection of SRSF1 expression. As shown in Fig. 2a and b, expression of T-antigen caused a significant reduction in the basal expression levels of SRSF1 in PHFA cells in a dose dependent manner. On the other hand, consistent with primary glial cultures, expression of T-antigen also caused a significant reduction in SRSF1 levels in T98G cells (Fig. 2c and d). The early gene product small t antigen was also expressed by alternative splicing of T-antigen pre-mRNA and detected in the same immunoblots. These results suggest that JCV T-antigen can target and downregulate the expression of cellular SRSF1.

In order investigate the molecular mechanism of T-antigen-mediated suppression of SRSF1 expression in glial cells, a luciferase assay reporting the transcriptional activity of the SRSF1 promoter was utilized. T98G cells were transiently transfected with a luciferase reporter construct consist of −1000 to +48 bp promoter region of SRSF1. Along with luciferase reporter construct, cells were co-transfected with plasmids encoding T-antigen or agnoprotein to determine the impact of these viral proteins on SRSF1 transcription. Cells were also transfected with an expression plasmid encoding green fluorescein protein (GFP) to access the transfection efficiencies. After 48 h, cells were harvested and processed for luciferase assay. It was observed that T-antigen, but not agnoprotein, suppressed the transcriptional activity of SRSF1 promoter (Fig 3a and b). Similar to our previous experiments (Fig. 2), it was found that higher levels of T-antigen correspond to a greater decrease in the transcriptional activity of SRSF1, which accounts for the decreased expression of SRSF1 in T-antigen expressing cells. To better understand the events associated with T-antigen suppression of SRSF1 promoter activity, we designed a series of experiments to assess the ability of T-antigen to bind to the SRSF1 promoter sequences. T98G cells were transfected with expression plasmid encoding T-antigen, cross-linked and possible interaction of T-antigen with SRSF1 promoter was analyzed by ChIP assay using antibodies to T-antigen as described in Materials and Methods. ChIP analysis of the cells demonstrated the association of T-antigen with SRSF1 promoter sequences (Fig. 3b).

The human glioblastoma multiforme cell line, T98G, was obtained from American Type Culture Collection (ATCC) and grown in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 5 % heat-inactivated fetal bovine serum (FBS) and penicillin/streptomycin (100 ug/ml). T98G cells were maintained at 37 °C in a humidified environment with 5 % CO₂. Primary human fetal astrocytes were cultured from the fetal human brain of a donor and provided by the Comprehensive NeuroAIDS Core facility at Temple University and cultured in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F-12) containing 10 % heat-inactivated fetal bovine serum (FBS) penicillin/streptomycin (100 μg/ml), GlutaMax (100 μg/mL) and insulin (100 μg/mL). Cultured PHFA cells were maintained at 37 °C in a humidified atmosphere with 5 % CO₂.

The T-antigen gene was cloned into the eukaryotic expression vector pcDNA3.1 (+) at the EcoRI restriction enzyme site and designated as pcDNA3.1-T-antigen previously [25]. pCGT7-SF2/ASF (SRSF1) expression plasmid was kindly provided by Javier F. Cáceres (Medical Research Council Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, Scotland, United Kingdom) and was described previously [26]. The pCGT7-agno plasmid was described previously [27]. Briefly, agnoprotein coding sequence was cloned into the eukaryotic expression plasmid pCGT7 at Xba1/BamH1 restriction enzyme sites and designated as pCGT7-agno. The luciferase reporter construct pLuc-JCV-Early and pLuc-JCV-Late were made by blunt end cloning of the full-length Mad-1 NCCR into the SmaI site immediately upstream of the luciferase gene in the plasmid pGL3 (Promega, Madison WI) and described previously [28]. The luciferase reporter plasmid pLuc-SRSF1 was made by cloning the −1000 to +49 promoter region of SRSF1 gene into pGL3 vector at BamH1 site.

T98G cells were plated in 6-well tissue culture dishes and transiently transfected with pLuc-JCV-Early, pLuc-JCV-Late, or pLuc-SRSF1-1000 bp reporter plasmids in the presence or absence of expression plasmids for T-antigen and agnoprotein or pCDNA3.1 as control. At 48 h post-transfection, cells were extracted and lysed using reporter lysis buffer for the luciferase reporter system provided by the manufacturer Promega. After cell lysis, luciferase activity of samples was determined through the use of luciferase assay reagent (LAR). The luciferase activities were then corrected for protein concentrations and normalized to the basal levels of transcription, allowing for determination of the fold changes.

T98G cells were transfected with pCDNA3.1-T-antigen. At 48 h post-transfection, proteins were cross-linked to DNA using formaldehyde at a final concentration of 1 %. After cross-linking, cells were lysed and subject to sonication to fragment the chromatin. After sonication, cells were incubated overnight with Protein G beads and anti-T antigen antibody (pAb-416, Calbiochem) for immunoprecipiation. After immunoprecipiation, the beads were washed and bound proteins were eluted using ChIP elution buffer (1 % SDS, 100 mM NaHCO₃). After elution, cross-linking was reversed by using 5 M NaCl followed by Proteinase and RNase treatment. The sample was then purified for DNA using phenol/chloroform extraction followed by ethanol precipitation. Obtained DNA fragments were analyzed by PCR using primers; SRSF1-Promoter-Forward (−1000 to +47): 5’-ACCTTCCAAAGCTTTCCAGATTTCAG-3’ and SRSF1-Promoter-Reverse (+47 to +27): 5’-ACCTTCCACTCGAGGAAGGAAACAGC-3’. The PCR conditions were as follows: denaturing at 95° for 30 s, annealing at 58 °C for 40 s, and extension at 72 °C for 65 s. The PCR products were then resolved on a 1 % agarose gel.

Whole cell protein extracts were washed with PBS and lysed with TNN lysis buffer with protease inhibitors. Purified protein extracts were then heated to 95 °C for 5 min and resolved through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After resolution, the gel was transferred to a nitrocellulose membrane (Whatman, Germany) for three hours at 250 mÅ at 4 °C in transfer buffer (25 mM Tris pH 7.4, 200 mM glycine, 20 % methanol). After transfer, membranes were blocked for thirty minutes at room temperature with 5 % non-fat dry milk in 1X phosphate-buffered saline containing 0.1 % Tween-20 (PBST). After blocking, membranes were washed and incubated with primary antibodies at a 1:1000 dilution overnight at 4 °C. After primary antibody incubation, membranes were washed three times in PBST and incubated with secondary antibodies at a 1:5000 dilution at room temperature. Following secondary antibody incubation, membranes were visualized with the Odyssey CLx Imaging System (LI-COR). Primary antibodies used were anti-T-antigen (pAb-416, Calbiochem), anti-SRSF1 (ab12910, Abcam), anti-agnoprotein (Pab7903, house raised), anti-β-Tubulin (LI-COR), and anti-GAPDH (Cell Signaling Technology). Secondary antibodies used were IRDye 800CW goat anti-mouse (LI-COR) and IRDye 680RD goat anti-rabbit (LI-COR).