Fig. 2.

Dissection of stability and replication using a dual-luciferase replicon.a Illustration of the Bi-Luc-SG construct. miR-122 binding to UTR1 acts on RNA stability and replication (as measured by Nano luciferase activity), translation of non-structural proteins is driven by UTR2 (as measured by firefly luciferase activity). b Northern blot analysis of HCV RNA stability over 72 h. Stability of different Bi-Luc construct RNAs was analyzed in the presence of miR^mut or miR^WT. Band #1: full-length RNA, #2: truncated product. U:A mutations in SLI of UTR2 (red dots) prevented the formation of truncated RNA. c Illustration of miR^WT (green) and miR^mut (black) binding upon introduction of a matching point mutation in the target (underlined). d, e Translation assay of point mutants cloned into the MBR of UTR1 or 2. The activity of Nano (d) and firefly (e) luciferase was measured in the presence of miR^mut or miR^WT. f Assessment of the full intracellular replication cycle of the Bi-Luc-SG constructs after electroporation with miR^mut or miR^WT. Replication was monitored with the nano luciferase signal. Mean values (±SD), n = 3, duplicates. RLU relative light units, ΔGDD replication deficient mutant. For translation, statistical significance was determined for miR^WT against miR^mut, for replication miR^WT and miR^mut conditions were tested against ΔGDD. *P<0.05, **P<0.01, ***P<0.001