PMC

Determination of the CMX001 EC₅₀/EC₉₀ and CC₅₀/CC₉₀ values by curve fitting. The effect of increasing CMX001 on JCV supernatant loads and on BrdU incorporation was analyzed by curve fitting using the XLfit program for EC₅₀ and EC₉₀ as well as CC₅₀ and CC₉₀. The data display means for two independent experiments, determined in triplicate. Error bars indicate the means ± SD. (A) JCV load in PDA cell culture supernatants at 7 dpi; (B) BrdU incorporation of PDA cells at 7 dpi; (C) JCV load in COS-7 cell culture supernatants at 5 dpi; (D) BrdU incorporation of COS-7 cells at 5 dpi.

CMX001 inhibition of JCV-infected cells. (A) JCV (Mad-4)-infected PDA cells stained for large T antigen (LTag) at 7 dpi. (B) JCV (Mad-4)-infected COS-7 cells stained for the JCV capsid protein VP1 at 5 dpi. Indirect immunofluorescence of the respective cells, treated with indicated concentrations of CMX001, fixed, and stained with the cross-reacting mouse anti-SV40 LTag or the rabbit anti-JCV VP1 serum (red, left panel) and with Hoechst-33342 dye to stain DNA (blue, middle panel). Magnification, 100-fold. Ratio: number of LTag- or VP1-positive cells per 1,000 cells. Mean for 3 10× microscopic fields.

CMX001 inhibition of JCV infectious progeny in PDA cell supernatants. Supernatants were recovered from JCV (Mad-4)-infected PDA cells that had been treated with the indicated concentrations of CMX001, diluted 5-fold, and seeded on COS-7 cells. The COS-7 cells were fixed at 5 dpi and stained with rabbit anti-JCV VP1 serum (red; left panel) for visualization of the late JCV capsid protein VP1 and with Hoechst-33342 dye to stain nuclear DNA (blue; middle panel). The merged images are shown in the right panel. Images were recorded with 100-fold magnification (insets, 400-fold). Ratio: number of VP1-positive cells per 1,000 cells. Mean for 3 10× microscopic fields is given.

Influence of CMX001 on metabolic activity and total host cell DNA replication. The metabolic activity (dark-gray bars) was determined by measuring WST-1 cleavage. The cellular DNA replication (light-gray bars) was examined with a cell proliferation enzyme-linked immunosorbent assay (ELISA) monitoring BrdU incorporation (see Materials and Methods for details). Absorbance for unifected and untreated cells was set as 100% (black bars). The data display a mean for two independent experiments, determined in triplicate. Error bars indicate the means ± SD. (A) PDA cells at 7 days post-CMX001 treatment; (B) COS-7 cells at 5 days post-CMX001 treatment.

CMX001 inhibition of supernatant JCV DNA load. The indicated cells were infected with JCV (Mad-4), CMX001 was added at 2 h postinfection, and cell culture supernatants were harvested at the indicated days postinfection (dpi) and quantified by real-time PCR as described in Materials and Methods. Replication of JCV is shown as log₁₀ genome equivalents per ml (geq/ml) and percentages of untreated cells. The data display a mean of data for two independent experiments, determined in triplicate. Error bars indicate the means ± SD. (A) JCV loads in PDA cell supernatants. (B) Percent inhibition by CMX001 at 7 dpi. (C) JCV loads in COS-7 cell supernatants. (D) Percent inhibition by CMX001 at 5 dpi.