FIG 4.

Silencing of enzymes involved in IMP synthesis cascade facilitates HEV replication. (A) Huh7 cells were transduced with lentiviral shRNAs to stably silence the corresponding genes for PPAT, GART, and ATIC (a set of independent shRNA clones targeting each gene was used). Huh7 cells transduced with lentiviral shRNA targeting GFP (shCTR) were used as a control. The efficiency of gene knockdown was analyzed by qRT-PCR. (B) Silencing of PPAT, GART, and ATIC resulted in significant elevation of viral RNA upon inoculation of HEV. HEV RNA levels were determined 72 h after inoculation. (C) Silencing of PPAT, GART, and ATIC abrogated the pro-HEV effects of 6-TG, lometrexol, and MTX. Data were normalized to that for cells without treatment with the three compounds (green bar; set as 1). All data were normalized to two housekeeping genes and are presented relative to the control (CTR) (set as 1) (means ± SEM from four to eight experiments). *, P < 0.05; **, P < 0.01; ***, P < 0.001.