FIG. 2.

Effect of endocytic inhibitors on KSHV gene expression, binding, and entry in HMVEC-d and HFF cells. (A and B) Cells were left untreated or pretreated with endocytic inhibitors for 1 h at 37°C, washed, and infected with KSHV at an MOI of 10. Total RNA was isolated at 2 h and 24 h p.i., and 50 ng of DNase-treated RNA/μl was subjected to real-time RT-PCR with ORF73 and ORF50 gene-specific primers and TaqMan probes. Known concentrations of DNase-treated, in vitro-transcribed ORF50 and ORF73 transcripts were used in real-time RT-PCR to construct a standard graph from which the relative copy numbers of viral transcripts were calculated and normalized to the amount of GAPDH. Histograms depict KSHV ORF73 and ORF50 gene RNA copy numbers in untreated cells (KSHV) or cells in the presence of indicated nontoxic concentrations of chlorpromazine (Chlor), EIPA, rottlerin (Rot), cytochalasin D (CytoD), filipin, cholera toxin B (CTB), MβCD, or nystatin (nys) in HMVEC-d (A) and HFF (B) cells. Each reaction was done in duplicate, and each bar represents the mean ± standard deviation of the results of three independent experiments. (C, D, and E) Effect of endocytic inhibitors on KSHV binding (C) and internalization (D) in HMVEC-d cells and on internalization (E) in HFF cells. (C) HMVEC-d cells grown in 24-well plates were either left untreated or pretreated with various nontoxic concentrations of agents for 1 h at 37°C and incubated with a fixed concentration of [³H]thymidine-labeled virus for 1 h at 4°C. As a control, labeled KSHV was preincubated with 100 μg of heparin/ml for 1 h at 37°C before being added to the cells. After incubation, cells were washed, lysed, and precipitated with trichloroacetic acid and the cell-associated-virus radioactivity (in cpm) was counted. The cell-associated cpm in the presence of drugs compared to virus binding to untreated cells was calculated as the percent inhibition of virus binding. Each reaction was done in triplicate, and each bar represents the average ± standard deviation of the results of three independent experiments. (D and E) Cells grown in six-well plates were either left untreated or preincubated with drugs at 37°C for 1 h. Cells were incubated with KSHV for 2 h, washed to remove unbound virus, treated with trypsin-EDTA for 5 min at 37°C to remove unbound, noninternalized virus, and washed, and the total DNA was isolated. KSHV preincubated with 100 μg of heparin/ml for 1 h at 37°C before being added to the cells was used as a control. KSHV ORF73 DNA copy numbers were estimated by real-time DNA PCR (21). Each reaction was done in duplicate, and each bar represents the mean ± standard deviation of the results of three experiments.