Figure 12. Knockdown of the Rho guanine exchange factor (GEF) p115 prevents KSHV-induced disruption of p-bodies in latently infected endothelial cells.

A–B) HUVECs were transduced with recombinant GFP-expressing lentiviruses that express short hairpin RNAs (shRNAs) against the Rho guanine exchange factors (GEFs; p115 [numbered −3, −4, and −9] and H1 [numbered −1, −2]) or the non-specific (NS) shRNA control. Positive transductants were selected by puromycin treatment and positive GFP-expression. After seeding cells on coverslips for 24 hours, transduced HUVECs were either not infected or infected with KSHV for 48 hours before being fixed with 4% paraformaldehyde. Cells were permeabilized in 0.1% Triton X-100 (in PBS), and blocked in 1% human AB serum before staining with the following primary antibodies: anti-hedls (p-bodies, false-colored blue) and anti-LANA (to mark infected cells, red). Cells expressing the shRNA are GFP-positive (green). Representative IF images are shown in A. To quantify the effect of latent infection on p-bodies, the number of LANA-expressing cells that displayed p-bodies of normal size (>300 nm in diameter) was counted and compared to uninfected cells. Three independent experiments were performed and results are shown in B. Scale bar  = 10 µm.