FIGURE 2.

Herpes simplex virus 1 UL2 inhibits NF-κB–driven cytokine expression. (A) HEK293T cells were cotransfected with Flag vector or diverse concentrations (100, 250, and 500 ng) of UL2-Flag expression plasmid along with reporter plasmids pXP2-pIL-8-Luc and pRL-TK. Twenty-four hours posttransfection, cells were treated with TNF-α (10 ng/mL) for 6 h, and luciferase activity was measured as described in Figure 1. (B) HEK293T cells were transfected with 1 μg of HA control vector or UL2-HA expression plasmid; 24 h posttransfection, cells were treated with TNF-α (10 ng/mL) for 6 h, and then RT-qPCR analysis was performed to analyze the relative expression level of IL-8 mRNA. Glyceraldehyde-3-phosphate dehydrogenase was used as the housekeeping gene. (C) was carried out as (B), except that HeLa cells were used for transfection. The expression of UL2 was analyzed by WB using anti-Flag mAb or anti-HA mAb, and β-actin was used to verify equal loading of protein in each lane. (D) HEK293T cells were mock-infected or infected with WT, UL2 Del, or UL2 Rev HSV-1 BAC GFP Luc virus at an MOI of 1 for 16 h. Flow cytometry analysis was then carried out to detect the GFP fluorescence. (E) HEK293T cells were infected with WT, UL2 Del, or UL2 Rev HSV-1 BAC GFP Luc virus at an MOI of 1. Sixteen hours postinfection, cells were treated with TNF-α (10 ng/mL) for 6 h. Then, RT-qPCR analysis was performed to detect the relative expression level of IL-8 mRNA. Data were expressed as means ± SD from three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.