FIG. 5.

Effects of endocytic inhibitors on KSHV entry into HUVEC cells. (A) Kinetics of KSHV entry into HUVEC cells. HUVEC cells were infected with KSHV (100 DNA copies per cell), and amounts of internalized viral DNA at different time points were estimated as described in the Fig. 2D and E legend. Each reaction was done in duplicate, and each bar represents the mean ± standard deviation of the results of three experiments. (B) Kinetics of KSHV DNA delivery into infected-cell nuclei. Nuclear fractions from HUVEC cells infected with KSHV at 100 DNA copies per cell for the indicated times (', min) were isolated, and total DNA extracted, normalized to 100 ng/5 μl, and analyzed by real-time DNA PCR with KSHV ORF73 primers. Each reaction was done in duplicate, and each bar represents the mean ± standard deviation of the results of three experiments. (C) Kinetics of KSHV latent gene (ORF73) and lytic gene (ORF50, K8, and gpK8.1) expression in HUVEC cells. For RNA isolation, PCR primers, and methodology, refer to Materials and Methods. Each sample was measured in triplicate, and data were analyzed by the threshold cycle method for comparing relative expression results. For each experiment, PCR amplifications without cDNA were performed as negative controls. (D) Effect of endocytic inhibitors on KSHV gene expression in HUVEC cells. HUVEC cells grown in six-well plates were either left untreated or preincubated with various nontoxic concentrations of agents for 1 h at 37°C, washed, and incubated with KSHV for 2 h, and total RNA was isolated at 8 h and 48 h p.i. and subjected to real-time RNA PCR. Histogram depicts the percent inhibition in KSHV ORF73 and ORF50 gene RNA copy numbers in the presence of indicated drugs, obtained by comparison with copy numbers in cells incubated with virus alone. Each reaction was done in duplicate, and each bar represents the mean ± standard deviation of the results of three experiments. (E) Effect of endocytic inhibitors on KSHV internalization in HUVEC cells. HUVEC cells grown in six-well plates were either left untreated or preincubated with various nontoxic concentrations of agents at 37°C for 1 h and infected with KSHV for 2 h, and the amount of internalized viral DNA at different time points was estimated as described in the Fig. 2D and E legend. The data are represented as the percent inhibition of KSHV DNA internalization in comparison with that in cells incubated with virus alone. Each reaction was done in duplicate, and each bar represents the mean ± standard deviation of the results of three experiments. Chlor, chlorpromazine; Rot, rottlerin; CytoD, cytochalasin D; CTB, cholera toxin B; nys, nystatin.