FIG 5 .

CPL activity is required for normal replication. (A) CPL-deficient parasites have a replication defect. Parasites were cultured in the monolayer HFF cells, and samples were collected at 17 and 26 h postinfection, fixed, stained with DAPI (4′,6-diamidino-2-phenylindole) and rabbit anti-SAG1 antibody, and quantified by fluorescence microscopy. At least 100 vacuoles were counted from 6 different fields of view. The percentages of different replication stages in the population for each strain were plotted. Results represent means ± SD from n = 3 experiments. Statistical significance by unpaired Student’s t test: *, P < 0.05; **, P < 0.01. (B) GRA2 does not significantly influence replication. Parasites were inoculated into HFF cells in chamber slides and allowed to replicate for the indicated times. Monolayers were fixed, stained with crystal violet, and enumerated by light microscopy. Parasitophorous vacuoles containing 1 parasite were not included in the data for 28 and 36 h to avoid skewing and to allow assessment of the logarithmic growth phase. Results represent means ± SD from n = 2 independent experiments, each with duplicate samples. Statistical significance by unpaired Student’s t test: *, P < 0.05. (C) Replication of PruΔku80LUC and PruΔku80LUCΔcpl assessed by luciferase activity. Results represent the mean ± standard error of the mean (SEM) fold change normalized to the respective 0-h value set at 1. n = 3 experiments. Statistical significance by paired Student’s t test: *, P < 0.05; **, P < 0.01.