FIG. 4.

Kinetics of CMX001 at 0.31 μM treatment of BKV-infected RPTECs. (a) Extracellular BKV load. Cells were infected for 2 h, and CMX001 was added for 22, 46, 70, or 94 h, respectively. At the indicated times, supernatant was removed, cells were washed, and new medium added. At 96 hpi all supernatants were harvested and qPCR was performed. Data are presented as BKV load in log Geq/ml. Determinations were in triplicate. The mean values are shown, and the error bars represent standard deviations. (b) Infection and indirect immunofluorescence. The supernatant collected 96 hpi from the cells described above where diluted 1:10 and seeded on new RPTEC cells. At 72 hpi cells were methanol fixed, and immunofluorescence staining with polyclonal rabbit anti-agno serum (green) and the SV40 LT-ag monoclonal Pab416 was performed (red). Cell nuclei (blue) were stained with Drac 5. The pictures were taken with the 10× objective. (c) Extracellular BKV load. CMX001 was added 24 h before infection was indicated. Before infection, all cells were washed once with complete medium. BKV was added for 2 h and then removed, and cells were washed once with medium. Where indicated, previously untreated cells now were treated with CMX001 for 24 h. After 24 h, all supernatants were removed and cells were washed once more before complete medium was given for 48 h. Supernatants were harvested 72 hpi, and BKV loads were measured by qPCR. Determinations were in triplicate. The mean values are shown, and the error bars represent standard deviations. (d) Indirect immunofluorescence of BKV-infected RPTECs treated as outlined above. The cells were methanol fixed and stained using the primary antibodies polyclonal rabbit anti-agno serum (green) for the visualization of the viral late gene protein agno and the SV40 LT-ag monoclonal Pab416 for the visualization of viral early protein LT-ag (red). Cell nuclei (blue) were stained with Drac 5. The pictures were taken with a 20× objective.