Figure 5

ORF2 protein inhibits the expression of MHC-I heavy chain. (A) EGFP, ORF2 or Δ35 ORF2 expressing cells were treated with LPS for 45 minutes after 48 hours of transfection. Lysates were immunoprecipitated and immunoblotted with anti-MHC-I heavy chain antibody (upper panel). An aliquot of the lysate was immunoblotted with anti-calnexin antibody (lower panel). (B) To test p65 NF-κB binding to the MHC-I heavy chain and IL-8 promoters ChIP assay was performed. Huh7 Cells were transfected with EGFP, ORF2 or Δ35 ORF2 expression plasmids and treated with LPS for 45 minutes prior to harvesting. PCR was performed using primers flanking the proximal promoter region of MHC-I heavy chain and IL-8 genes. Lane 7 shows ChIP from EGFP expressing cells immunoprecipitated with rabbit pre-immune serum. Lane 8 shows ChIP from mock immuonprecipitation (without cell lysate) using p65 antibody. 10% of the initial amount of chromatin used for one immuonprecipitation assay was used as input in each PCR. (C) SP1 binding to the MHC-I heavy chain ptomoter was again assayed by ChIP. Huh7 Cells were transfected with EGFP or full-length ORF2 expression plasmids and treated with LPS for 45 minutes prior to harvesting. PCR for MHC-I promoter regions was performed using primers mentioned above. Lane 5 shows ChIP from EGFP expressing cells immunoprecipitated with rabbit pre-immune serum. Lane 6 shows ChIP from mock immunoprecipitation using SP1 antibody. 10% of the initial amount of chromatin used for one immuonprecipitation assay was used as input in each PCR. (D) Quantitative RT-PCR analysis of IL-6 and IL-8 RNA level in Huh7 cells, treated as indicated. Values were normalized with respect to that of HPRT and represented as mean ± SEM.