HAV antigenome-encoded hav-miR-N1-3p is a biologically functional miRNA. (A) Construction of sensor and sensor-mut plasmids. Sensor construct with two tandem repeats of artificial target sites perfectly complementary to the miRNA sequence were inserted into the 3' UTR of the luciferase gene in pmirGLO vector (sensor), and mutant construct with two tandem repeats of mutated nucleotides in the artificial target sites (sensor-mut). (B) Silencing activity of exogenous viral hav-miR-N1-3p in HEK293T cells transfected with viral miRNA mimics. (C) Silencing activity of endogenous viral hav-miR-N1-3p in HAV-infected and mock -infected cells. Luciferase activity was assessed 48 hours after transfection. A cellular miR-154 served as a positive control and the mutated plasmid was used as a negative control. For all treatments, relative luciferase activity was expressed as ratio of firefly to Renilla luciferase activity. Error bars indicate standard deviation obtained in three replicates, and significant differences are indicated with *at P < 0.05 and **at P < 0.01.