FIG 9.

ORF21 and ORF50 strongly inhibit TLR2 and TLR4 signaling. (A, B) HEK293T cells were cotransfected with pNF-κB-Luc, pTK-RL, Flag-TLR2, and empty pcDNA vector or increasing concentrations (1×, 2×, or 4×) of the indicated KSHV ORF-Myc. At 18 h posttransfection, cells were unstimulated or stimulated for 6 h with the TLR2 agonist FSL-1 and then lysed in passive lysis buffer. (C, D) HEK293 TLR4-MD2-CD14 cells were cotransfected with pNF-κB-Luc, pTK-RL, and empty pcDNA vector or increasing concentrations (1×, 2×, or 4×) of the indicated KSHV ORF-Myc. At 18 h posttransfection, cells were unstimulated or stimulated for 9 h with the TLR4 agonist LPS and then lysed in passive lysis buffer for measurement of luciferase activity. (A, C) NF-κB response in unstimulated cells, normalized to renilla and scaled to empty vector. (B, D) Fold induction of NF-κB in response to FSL-1 (B) or LPS (D), determined as follows: firefly normalized to renilla and then stimulated divided by unstimulated. Data shown are averages with standard deviations from 3 or 4 replicates total in two independent experiments (A, B) or duplicates from each of 3 independent experiments (C, D). Transformed results were analyzed by one-way ANOVA followed by Dunnett's multiple comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (E) Flag and Myc immunoblotting was used to verify Flag-TLR2 and ORF-Myc expression levels in whole-cell RIPA lysates. Actin was used as a loading control.