FIG. 1.

vGPCR and LAT genes increase ROS production in a Rac1-dependent manner. (A) Superoxide production was measured by DHE staining in mECK36 cells transfected with a vGPCR-expressing plasmid and treated with siRac1 (Rac1 siRNA) or siCTL (control siRNA). LPS/TPA-stimulated macrophages were used as positive control. KSHV-null mECK36 cells, which do not express vGPCR, served as negative control. Groα is a vGPCR agonist. (B) Superoxide production of vGPCR-expressing mECK36 cells induced with Groα was measured by DHE staining using DPI or FCCP inhibitors. (C) ROS production measured by enhanced luminol assay in iSLK.219 cells upon stimulation with 0.5 μg/ml doxycyclin to induce lytic gene expression. (D) qRT-PCR analysis of KSHV lytic gene expression (RTA, vGPCR) in iSLK.219 cells upon stimulation with 0.5 μg/ml doxycyclin to induce lytic gene expression. Data indicate fold increase of mRNA as measured by duplicates in two independent experiments. (E) DCF staining of 293 cells either transfected with LAT vector expressing the major LAT encoding KSHV LANA, v-cyclin and vFLIP and control vector. ^†p<0.05. DCF, 2′,7′-dichlorofluorescin; DHE, dihydroethidium; DPI, diphenylene iodonium; FCCP, p-(tri-fluromethoxy) phenyl-hydrazone; KSHV, Kaposi's sarcoma herpesvirus; LAT, latent transcript; LPS, lipopolysaccharide; qRT-PCR, quantitative real-time polymerase chain reaction; ROS, reactive oxygen species; RTA, replication transcriptional activator; TPA, 12-O-tetradecanoylphorbol-13-acetate; vGPCR, viral gene G protein-coupled receptor. (To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars.)