A conserved arginine in NS5 binds genomic 3′ stem–loop RNA for primer-independent initiation of flavivirus RNA replication

Analysis of DENV2 NS5 binding to 3′SL RNA and 3′SL RNA with top-loop or side-loop deletion. (A) Secondary structures of 3′SL RNA and 3′SL with deleted top lop (3′SL-TL^del) or side-loop (3′SL-SL^del) were predicted by mfold web server with folding free energy ΔG at 37°C shown below. Corresponding RNA samples produced by in vitro transcription followed by purification were analyzed by agarose gel electrophoresis. (B,C) RNA electrophoretic mobility shift assay (REMSA) for NS5 WT (B) or R888A mutant (C) with 3′ SL, 3′SL-TL^del, or 3′SL-SL^del analyzed on 1.2% agarose gel with lane number indicated at the bottom. Lanes 2, 8, and 15 in both gels represented 3′SL, 3′SL-TL^del, 3′SL-SL^del RNA alone in binding buffer. Lanes 3–7, 9–13, and 15–19 represented binding reactions of NS5 with RNA at molar ratios of 0:1, 1:1, 2:1, 4:1, 8:1, and 16:1 as indicated on top of each lane. The REMSA experiments were performed three times with similar results. (D) Binding affinity of WT NS5 or R888A mutant with 3′ SL, 3′SL-TL^del, or 3′SL-SL^del reflected by apparent dissociation constant (K_d) was calculated from three independent repeat experiments represented as average ± SD. The statistical significance of difference between the two groups was evaluated by Student's t-test and a P-value <0.05 was considered significant (≤0.0001, ≤0.001, ≤0.01, ≤0.05 were represented by [****], [***], [**], [*], respectively).