Structural and functional analysis of the roles of the HCV 5′ NCR miR122-dependent long-range association and SLVI in genome translation and replication

Figure 1. Schematics of RNA structures and templates used. (A) The predicted ‘open’ and ‘closed’ conformations of the HCV 5′UTR from SL I-VI with the addition, or loss, of miR122 as shown. S1 and S2 highlight the known binding sites of miR122. Black arrows indicate position and directionality of SHAPE primers. The red box shows an expanded view of nucleotides 1–42 of JFH-1 with miR122 (lowercase) bound to sites S1 and S2, with the blue box showing expanded view of nucleotides 428–507 of JFH-1 encompassing SLVI. Nucleotides in red are those predicted to be directly involved in formation of the LRA and mutations are indicated by faint black arrows indicating the substitutions made. Figure recreated and adapted from (Díaz-Toledano et al., 2009). (B) The JFH-1 bicistronic translation reporter (JFH1-luc-trans:ΔNS5B), the core-extended translation reporter (JFH1-CEluc-trans), and replicon (JFH1-CEluc-rep). (C) The miR122 binding sites and SLVI with 5′ stem (L), 3′ stem (R) and S1 mutations displayed. The predicted blocking of miR122 binding, or SLVI formation, of each pair of mutations is shown. Black arrows represent the relative likely conformation, ‘open’ or ‘closed’, predicted to be favoured…

Figure 2. RNA-RNA electrophoretic mobility gel shift assays of miR122 binding to JFH-1 5′UTR. A synthetic RNA of nts 1–50 of JFH-1 (JFH11–50) was complexed with increasing molar ratios of (A) wild type or (B) antisense synthetic miR122 and separated by non-denaturing PAGE. JFH11–50 mutated at the S1 binding site was similarly complexed with (C) wild type or (D) wild type plus an S1-mutated miR122 and separated by non-denaturing PAGE. miR122 binding was denoted by 1 (+ miR122) or 2 (++ miR122) reductions in RNA mobility compared to JFH11–50 control with no miR122 present (lane 1, A–D).

Figure 3. SHAPE analysis of parental template plus/minus miR122. SHAPE reactivities are shown for (A) JFH1-CEtrans plus miR122, (B) JFH1-CEtrans minus miR122, and (C) JFH1-CEtrans plus LNA J22, with predicted (P) and observed (O) conformations given top right below template name. Black bars show normalised SHAPE reactivities of nucleotides 427–447 and 487–507, encompassing the 5′ and 3′ basal stems of SLVI, respectively. Nucleotides with a reactivity of <0.4 are considered unreactive and therefore base-paired. Shaded regions highlight nucleotides of importance in determining ‘open’ or ‘closed’ conformations: specifically the 5′ G434G435 motif and 3′ nucleotides 494–507. The superimposed red line indicates the exposure of JFH1-CEtrans plus miR122, and is included on all plots for comparison of reactivities between a demonstrated ‘open’ conformation and the observed reactivity of additional templates. A maximum negative reactivity was set at −0.1. Unless otherwise stated, error bars represent the SD of a minimum of two independent gel analyses for two replicate RNA–NMIA folding reactions. Figure 3C was derived from only one replicate folding reaction.

Figure 4. SHAPE analysis of S1 and SLVI mutants with predicted ‘open’ conformation. SHAPE reactivities are shown for (A) JFH1-CEtrans-S1, (B) JFH1-CEtrans-L, (C) JFH1-CEtrans-L/R and (D) JFH1-CEtrans-S1/R, with predicted (P) and observed (O) conformations given top right below template name. Data presentation as described in Fig. 3.

Figure 5. SHAPE analysis of S1 and SLVI mutants with predicted ‘closed’ conformation. SHAPE reactivities are shown for (A) JFH1-CEtrans-S1/L, (B) JFH1-CEtrans-R and (C) JFH1-CEtrans-S1/L/R, with predicted (P) and observed (O) conformations given top right below template name. Data presentation as described in Fig. 3.

Figure 6. Phenotypic characterisation of JFH-1 reporter bearing S1 and SLVI mutations. Translation levels were determined by luciferase assay for (A) JFH1-luc-trans:ΔNS5B and JFH1-CEtrans, (B) JFH1-CEtrans, S1 and SLVI mutants and (C) JFH1-luc-trans:ΔNS5B and JFH1-CEtrans in HeLa cells. Cell lysates were harvested at 4 h and luciferase readings calculated as a ratio of Firefly luciferase to a co-transfected Renilla luciferase control RNA. Replication kinetics of (D) JFH1-CErep, S1 and SLVI mutants and (E) JFH1-CErep-S1 supplemented with S1-miR122, were determined by luciferase assays at 4, 21, 28 and 45 h post-transfection. Luciferase readings are expressed as a percentage of the 4 h reading to normalise against translation of input RNA. A polymerase active site mutant, GDD to GNN, was included as replication control (Pol −ve). For all assays error bars represent SD of three replicate transfections from triplicate experiments, with statistical significance calculated by unpaired t-test analysis using GraphPad Prism V7.