Fig. 2: Ago2 shields the HCV 5’ end from enzymatic attack.

a 3D structures (left) and schematic representation (right) of the miR-122:Site-1 three-way junction, composed of the seed duplex, SL1, and the supplementary duplex. b Cartoon representation of Ago2 with major HCV RNA contacts highlighted (determined by PDB PISA⁵⁰). c Multiple sequence alignment of HCV genotype 1 subtype sequences (top) and genotype 1-8 subtype sequences (bottom). The total height of the letters at each position corresponds to the conservation at that position, while the height of the letters within the stack corresponds to their relative frequency. Supplementary contacts, SL1, t9, and seed contacts are indicated. d Surface representation showing closeup of the HCV RNA (blue) 5’ end bound to miR-122 (red) and positioned against the N-terminal domain residues E64, K65, and C66 (orange). e Ago2:miR-122 off-rate experiment using Site-1 WT and Site-1 with supplementary-pairing region mutated (GCCA > GAAA). Data shown as means of 3 independent replicates with error bars representing SEM. f XRN1 mediated degradation of Site-1 WT and with mutated supplementary-pairing region RNAs in the presence of Ago2:miR-122. Data shown as means of 3 independent replicates with error bars representing SEM. g XRN1 mediated degradation of WT Site-1 in presence of Ago2 loaded with indicated miR-122 3’ isomiRs, or no Ago2. Data shown as means of 3 independent replicates with error bars representing SEM. h Polyacrylamide gels showing XRN1 mediated degradation of Site-1 RNA alone (top), Site-1 with Ago2:miR-122 (middle), and Site-1 containing a 5’ U₁₀ extension with Ago2:miR-122. Images are representative gels of triplicate experiments. Ago2:miR-122:Site-1 structure with U₁₀ extension modelled (right) to illustrate potential 5’ end exposure.