Pervasive tertiary structure in the dengue virus RNA genome

Abstract

RNA virus genomes are efficient and compact carriers of biological information, encoding information required for replication both in their primary sequences and in higher-order RNA structures. However, the ubiquity of RNA elements with higher-order folds-in which helices pack together to form complex 3D structures-and the extent to which these elements affect viral fitness are largely unknown. Here we used single-molecule correlated chemical probing to define secondary and tertiary structures across the RNA genome of dengue virus serotype 2 (DENV2). Higher-order RNA structures are pervasive and involve more than one-third of nucleotides in the DENV2 genomic RNA. These 3D structures promote a compact overall architecture and contribute to viral fitness. Disrupting RNA regions with higher-order structures leads to stable, nonreverting mutants and could guide the development of vaccines based on attenuated RNA viruses. The existence of extensive regions of functional RNA elements with tertiary folds in viral RNAs, and likely many other messenger and noncoding RNAs, means that there are significant regions with pocket-containing surfaces that may serve as novel RNA-directed drug targets.

Keywords: RING-MaP; RNA tertiary structure; genome circularization; viral packaging.

Fig. 1.

Well-determined secondary structure elements in the DENV2 RNA genome. The first half of the genome is shown (the entire genome is shown in SI Appendix, Fig. S1, panel 1). Median ex virion 1M7 SHAPE reactivities (black) and Shannon entropies (dark blue) are plotted over centered 55-nt windows. Regions with both low SHAPE and low Shannon entropy are highlighted by dark-gray shading, with light-gray shading extended to encompass entire intersecting helices. The first 12 elements (out of 24 total elements) with well-determined structures are numbered. Base pair probability arcs are colored by probability (see scale), with green arcs indicating the most probable base pairs; black arcs indicate plausible pseudoknots (PK). The minimum free-energy secondary structure (inverted black arcs) was obtained using both 1M7 and differential SHAPE reactivities as constraints (1, 12, 13). Secondary structures of elements 1–12 are colored by SHAPE reactivity; high-resolution structures are provided in SI Appendix, Fig. S1, panel 2.

Fig. 2.

Tertiary interactions and structural conservation in the DENV2 RNA genome. (A and B) The entire genome is split into two panels. (A) Characterization of DENV2 RNA secondary and tertiary structures. Base pairs common to both ex virion and refolded RNA are depicted by black arcs; base pairs unique to refolded RNA are in gray. RINGs are depicted by inverted arcs. Secondary structure RINGs are in black. Tertiary RINGs (colored by correlation coefficient; see key) were summed over 101-nt windows, and positions with a value greater than the median plus 1 SD were assigned as having a significant degree of tertiary structure (yellow shading). (B) Evolutionary and RING support for the 24 elements with low SHAPE and low entropy (gray boxes; lowSS). Filled boxes indicate element-supporting data: minor allele frequency tests (allele; green), synonymous substitution rates (dS; blue), coevolution (coev; purple), and RINGs (black). For RING data, open boxes indicate regions that fold differently between the ex virion and refolded RNA, and the lack of a black line indicates regions with very few RINGS. (C) 5′-UTR and 3′-UTR RINGs detected using gene-specific primers. Positions shown: 5′-UTR, 1–275; 3′-UTR, 10,247–10,660.

Fig. 3.

Encapsidated DENV2 RNA is in its circular form. Nucleotides protected in virion (blue boxes) are shown on both linear and circular genome structures. (A) Secondary structure of the proposed linear form of the DENV2 genomic RNA 5′-UTR, capsid-coding region, and 3′-UTR with nucleotides colored by ex virion SHAPE reactivity. Pseudoknots DCS-PK and 3′DB-PK are shown. The 3′DB-PK does not form in the ex virion RNA, as indicated by a dashed gray line. Positions with no data are in gray. (B) Secondary structure of the proposed circular form of the DENV2 genomic RNA color-coded by in virion SHAPE reactivity.

Fig. 4.

Hydrodynamic radii of WT and mutant DENV2 RNAs. (A) Dynamic light scattering curves from individual experiments, representative of multiple replicate experiments. The mean radius values for WT, Env^mut, and NS2A^mut are 54, 81, and 75 nm, respectively. (B) RNA radii of WT and mutant RNAs. Values are mean and SD calculated from four experiments. Control NS5^mut is denoted by an open symbol.

Fig. 5.

Replication fitness of DENV2 tertiary structure mutants and control. (A) DENV2-positive BHK-21 cells after transfection of WT or mutant in vitro transcribed DENV2 RNAs. Virus was visualized by immunostaining of DENV2 envelope protein and nuclei (n ≥3, with ≥1,000 cells counted per condition). (B) Representative fields showing DENV2 RNA-transfected cells (green) and nuclei (DAPI, blue) at 72 h posttransfection. (C) Viral titer relative to WT following transfection of DENV2 RNA at 72 h (n ≥3). (D) Percent DENV2 RNA relative to WT at 72 h posttransfection as quantified by RT-qPCR (n ≥3). Values plotted are mean ± SEM of at least three independent experiments. Pol⁻, a lethal mutation in the DENV2 NS5 RNA-dependent RNA polymerase; n.s., not significant. *P < 0.05; **P < 0.01; ***P < 0.001, two-way ANOVA with Bonferroni correction in A, Student’s t test in C and D.

Fig. 6.

Higher-order structures throughout the DENV2 genome. (A) Secondary structure of the NS2A element. RINGs used as tertiary structure constraints in the DMD simulation are depicted by gray lines. (B) Medoid of DMD models of the NS2A element. (C) The 10 lowest aligned free-energy DMD models of the NS2A element. (D) From top to bottom, the DENV2 RNA genome, the secondary structure (black arcs) and tertiary RINGS (inverted colored arcs) of elements with tertiary structures, and tertiary structure models. RINGs reporting tertiary interactions are color-coded by correlation coefficient as in Fig. 2, and 3D folds are color-coded by secondary structure (SI Appendix, Figs. S11–S14). Previously identified elements in the 5′- and 3′-UTRs (–9) are labeled. For each element, the 10 lowest free-energy models from the largest cluster are shown.