Conserved long-range interactions are required for stable folding of orthoflaviviral genomic RNA

Figures

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  Figure 1.. Orthoflaviviral RNA can fold into alternative secondary structures. (A) Organization of orthoflavivirus vRNA. The vRNA polyprotein ORF is subdivided into the protein products produced by proteolytic cleavage during infection. Conserved terminal structures are shown as cartoons, structural proteins as blue boxes, and nonstructural proteins as purple boxes. Numbering corresponds to a model orthoflavivirus, DENV2 strain 16681. Regions shown in panels (B) and (C) are boxed. (B) Proposed secondary structure of the terminal regions of DENV2 strain 16681, in the extended conformation. A schematic of vRNA organization is shown at the top, with conserved terminal structures shown as cartoons, structural protein genes as blue boxes, and nonstructural protein genes as purple boxes. Start codons for the viral ORF are highlighted in bold green font, and conserved complementary sequences are annotated with purple, yellow, and maroon lines. Named secondary structure elements are labeled below each stem. (C) Proposed secondary structure of the cyclized conformation of DENV2 strain 16681. Annotations are the same as in panel (B).

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  Figure 2.. The extended and cyclized conformations of DENV vRNA are evolutionarily conserved. (A) Overview of pan-serotype bioinformatic analysis of DENV sequence and structure conservation. (B) Sequence-structure conservation diagram of the proposed extended conformation of DENV vRNA. Sequence conservation and base pair covariation for 1418 sequences were analyzed and diagram prepared with R2R [46]. Base pairs supported by phylogenetic analysis are shown with a solid line, and base pairs that are only supported by secondary structure prediction are indicated with a dashed line. Evolutionarily supported base pairs were annotated as covarying and highlighted in green if both nucleotides changed identities in one or more strains (e.g. G-C to A-U), and were annotated as compatible and highlighted in blue if just one residue changed (e.g. G-C to G-U). Predicted base pairs between universally conserved nucleotides are highlighted with a red box. The font color of individual residues indicates primary sequence conservation. R and Y stand for positions that must be purines or pyrimidines, respectively. A filled red circle indicates a residue that is absent (gap in the MSA) in ≤3% of DENV sequences…

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  Figure 3.. Orthoflaviviral RNA and mRNA conformations are heterogeneous. (A) Schematic of the 5′-3′ smFRET experiment using two-color TIRF microscopy. (B) Overview of approach to probe RNA conformational ensembles using smFRET. Single illumination experiments identify different conformational states distinguishable by 5′-3′ end distance and measure the kinetics of exchange between these states. Dual illumination experiments count the number of doubly labeled molecules that either display 5′-3′ FRET or remain in EFRET = 0 states for the duration of the observation window. (C) Cartoon schematic of minigenome and mRNA constructs analyzed. Positions of conserved complementary sequences are highlighted with the UAR in purple, DAR in yellow, and CS in maroon. The ORF of β-globin mRNA is colored in light blue. (D) Representative 5′-3′ smFRET traces collected with 100 ms exposure time for the DENV2 5′-3′ UTR and β-globin mRNA. The single-step drops in magenta and green fluorescence intensity for DENV2 5′-3′ UTR are due to photobleaching of Cy5 and Cy3, respectively. The solid lines are fluorescence counts smoothed with a sliding window over n = 5 frames. Raw data are displayed in light coloring.…

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  Figure 4.. Stable 5′-3′ FRET states depend on long-range interactions. (A) Overview of perturbations used to probe the functions of conserved complementary sequences. Examples shown (clockwise from top left) are stabilization of the cyclized state with additional base pairs, disruption and reconstitution of 5′-3′ CS base pairing, and inhibition of the 5′-3′ UAR interaction with a DNA oligonucleotide. Binding of the blocking oligonucleotides to the predicted positions in the 3′ UTR was validated by digesting the RNA–DNA duplex with RNase H and observing the resulting RNA fragments by denaturing gel electrophoresis (Supplementary Fig. S4B and C). (B) The high FRET state is stabilized by long-range base pairing. FRET efficiency distributions of representative DENV2 5′-3′ UTR mutants are plotted with open circles. The solid lines are the Gaussian fit to the observed data. Number of single illumination and dual illumination traces analyzed for each construct, respectively, were n = 114 and n = 309 for WT DENV2 5′-3′ UTR, n = 99 and n = 359 for CS mutant 1 5′-3′ UTR, n = 104 and n = 314 for CS rescue 1 5′-3′ UTR, and n = 196 and n = 263 for Cyc + 5′-3′ UTR. (C) Representative 5′-3′ smFRET trace…

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  Figure 5.. Cyclization of full-length DENV2 vRNA. (A) Full-length and truncated DENV2 strain 16681 vRNA constructs analyzed by 5′-3′ smFRET. ORFs are shown as boxes proportional to the length of the sequence. The vRNA polyprotein ORF is subdivided into the protein products produced by proteolytic cleavage during infection. Terminal regions are shown as cartoon structures, not to scale with the ORF. (B) FRET efficiency distribution of doubly-labeled constructs shown in panel (A). n = 90 single illumination and n = 423 dual illumination traces were analyzed for full-length vRNA and n = 23 single illumination and n = 350 dual illumination traces were analyzed for truncated vRNA. (C) Representative 5′-3′ smFRET trace for the full-length DENV2 vRNA, showing a single stable high FRET state that persists until photobleaching of Cy5. The solid lines are fluorescence counts smoothed with a sliding window over n = 5 frames. Raw data are displayed in light coloring.

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  Figure 6.. Conformational remodeling of DENV2 vRNA by eIF4 proteins. (A) Schematic of eIF4 delivery experiments on the custom RS instrument. The final concentrations of protein factors after delivery were 1 μM eIF4A, 220 nM eIF4B, 130 nM eIF4G, and 160 nM eIF4E. (B) Representative 5′-3′ smFRET traces for the full-length DENV2 vRNA in the presence and absence of eIF4 proteins. Buffer or factors were delivered att = 0 s. The solid lines are fluorescence counts smoothed with a sliding window over n = 5 frames. Raw data are displayed in light coloring. (C) Initial FRET lifetime observed after delivery of either buffer or eIF4 proteins in the presence of 1 mM ATP. n = 108 traces were analyzed for the vRNA only condition, and n = 78 traces were analyzed for eIF4F + ATP delivery. The data were fit to single exponential equations to extract rate constants. The error reported represents the 95% CI of the fit. (D) Density maps of normalized doubly-labeled vRNA FRET efficiency after buffer or factor delivery for traces quantified in panel (C).

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  Figure 7.. Model for orthoflaviviral conformations. (A) Proposed secondary structure conformations of distinct FRET states observed in this work. (B) Model for the essential role of host and viral RBPs in mediating the conformational switch between extended and cyclized states of vRNA. The slow exchange rate observed between extended and cyclized conformations may require active remodeling to enable cis-regulatory functions of the conserved complementary sequences during infection. RBPs that bind the terminal regions could modulate long-range base pairing or other tertiary contacts to disrupt the high stability of the extended and cyclized conformations observed with RNA alone in this study.