FIG. 1.

Intact microtubules (MTs) are required for target cell infection by HHV-8. (A) MT depolymerization inhibits GFP-HHV-8 infectivity. HFF cell monolayers in eight-well chamber slides were incubated with Dulbecco's modified Eagle's medium (DMEM) containing different nontoxic concentrations of MT-depolymerizing nocodazole (Nocod) or MF-depolymerizing cyto D or lat A at 37°C for 1 h before being infected with GFP-HHV-8. The cells were incubated with virus for 2.5 h at 37°C in the presence of inhibitors, washed, and incubated with growth medium for 3 days at 37°C. Green fluorescent cells indicative of GFP-HHV-8 entry and infection were counted. In control wells, approximately 300 GFP-expressing cells were detected per well. Each reaction was done in triplicate, and each bar represents the mean ± standard deviation (SD) for three experiments. (B) Nocodazole inhibits HHV-8 ORF73 mRNA expression. HFF cells or HFF cells preincubated with nontoxic doses of nocodazole, cyto D, or lat A were infected with GFP-HHV-8 at a multiplicity of infection (MOI) of 5 DNA copies/cell for 2.5 h, washed, and incubated in fresh medium for 8 h. Total cellular RNAs were analyzed by real-time RT-PCRs using gene-specific Taqman probes to quantitate the HHV-8 ORF73 mRNA. Known concentrations of gene-specific transcripts from an ORF73 clone were used as standards. All samples were used in separate real-time PCRs without RT to confirm the absence of contaminating DNA. The relative copy numbers of the transcripts were calculated and normalized to GAPDH control reaction values. Data are presented as percentages of the inhibition of ORF73 mRNA copy numbers obtained when the cells were incubated with virus alone. Each reaction was done in duplicate, and each point represents the mean ± SD for three experiments.