Figure 4.

gD facilitates IRF7-mediated IFN-β promoter activity through enhancing the interaction of viperin with IRAK1 and increasing K63-linked polyubiquitination of IRAK1. (A–C) HEK293T cells were co-transfected with IFN-β-Luc reporter, pRL-TK and gD-EYFP or pViperin-Flag or plasmids combination of gD-EYFP and pViperin-Flag, with or without IRF3/5D (A) or IRF7/6D (B,C) expression plasmid. Twenty-four hours post-transfection, luciferase activity was analyzed. (D) HEK293T cells were co-transfected with the indicated plasmids as described in (C), except for the reporter plasmids. Twenty-four hours post-transfection, RT-qPCR was performed to analyze the relative mRNA expression level of IFN-β. Data were expressed as means ± SD from three independent experiments. (E,F) HEK293T cells co-transfected with expression plasmids pViperin-Flag, IRAK1-HA and gD-EYFP, or EYFP control construct were harvested and immunoprecipitated with mouse anti-Flag mAb or non-specific mouse IgG, and IB analysis was probed with the indicated antibodies. Densitometry of the IRAK1 and viperin interaction bands were normalized to the loading control β-actin. (G,H) HA-tagged Ub (WT), Ub (K48), or Ub (K63) expression plasmid was co-transfected with plasmids combination of pCMV-Flag-IRAK1 and pViperin-Flag into HEK293T cells, with or without the presence of gD-EYFP. Twenty-four hours post-transfection, cells were collected, followed by Co-IP with mouse anti-Flag mAb and IB analysis with mouse anti-HA mAb. Densitometry of IRAK1 polyubiquitination bands were normalized to the loading control β-actin. Data were expressed as means ± SD from three independent experiments. Statistical analyses were performed using one-way ANOVA, except (F) using student t test. *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001.