FIG 2.

RNAi screening to identify specific DUBs involved in HCV propagation. (A) Schematic representation of the experimental procedure for our RNAi screening. Huh7.5.1 cells were infected with retroviruses expressing shRNA targeting DUBs (shDUBs). To validate the knockdown of each DUB gene in Huh7.5.1 cell lines expressing shDUBs, qRT-PCR was performed by using the primer sets shown in Table 1. shDUB Huh7.5.1 cell lines that exhibited a >40% reduction in the expression of their specific DUB were selected for further screening. DUB knockdown Huh7.5.1 cells were infected with HCV at an MOI of 0.5, and intracellular HCV RNA levels were quantified after 4 days. (B) The levels of intracellular HCV RNAs at 4 days postinfection were determined by qRT-PCR as a relative value against the GAPDH mRNA level in cells. Data are presented as relative values compared to those in Huh7.5.1 cells expressing shRNA against the LacZ gene. (C) The expression level of USP15 in Huh7.5.1 cells and those expressing shRNA targeting either LacZ (shLacZ) or USP15 (shUSP15) was quantified by qRT-PCR. (D) The intracellular HCV RNA levels in Huh7.5.1 cells and those expressing either shLacZ or shUSP15 upon infection with HCV at an MOI of 0.5 were determined by qRT-PCR at the indicated time points. dpi, days postinfection.