FIG 2.

ISG15 is not required for HEV replication. Huh7-S10-3 liver cells were cotransfected with 20 nM ISG15-targeted siRNA (siISG15) or control siRNA (siCnt) along with HEV P6GLuc replicon RNA (A and C) or HEV P6 infectious clone viral genomic RNA (B and D). The GLuc activity levels in culture supernatant (A) and the intracellular viral RNA levels (B) were determined by GLuc assay and qPCR, respectively, at 5 dpt. The efficiency of knockdown of ISG15 by siISG15 was determined by measuring ISG15 protein levels in siRNA and HEV P6GLuc replicon RNA cotransfected samples at 5 dpt (C) and ISG15 mRNA levels in siRNA and HEV P6 viral RNA cotransfected samples at 5 dpt (D); fold change compared to untransfected control cells was calculated using the 2^−ΔΔCT method, and the RPS18 gene was used as the housekeeping gene. The data represent means ± SEM of results from two independent transfection experiments.