Disruption of actin dynamics reduces the entry and trafficking of KSHV particles to the perinuclear region.

(A, E and I) HUVEC were treated with chemicals to disrupt actin dynamics at the indicated doses for 1 h. Cells were inoculated with KSHV in the presence of inhibitors, fixed at 4 hpi, and stained for Orf65+ viral particles (red) and nuclei (blue). (B, F and J) The total number of nuclei bearing at least one Orf65+ viral particle was determined and calculated as Orf65+ nuclei (%). In parallel experiments, cells were subjected to the same treatments and evaluated for viability by PI staining. (C, G and K) The total number of Orf65+ viral particles docked at each nucleus was determined. (D, H and L) Disruption of the actin cytoskeleton by cytochalasin (D), latrunculin A (H) and jasplakinolide (L). HUVEC were treated with the inhibitors as described in A, E and I, and stained for the actin cytoskeleton with AlexaFluor 488-phalloidin except for cells treated with jasplakinolide, which were stained with an antibody to β-actin. (M) Disruption of actin dynamics reduces the internalization of AlexaFluor 488-transferrin. HUVEC were pretreated with chemicals to disrupt actin dynamics for 1 h prior to exposure to AlexaFluor 488-transferrin. The reduced internalization and perinuclear accumulation of transferrin (green) compared to untreated control cells demonstrates the effective inhibition of clathrin-mediated endocytosis when actin dynamics are disrupted.