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. 2013 May;41(9):5075-89.
doi: 10.1093/nar/gkt203. Epub 2013 Mar 26.

Structural complexity of Dengue virus untranslated regions: cis-acting RNA motifs and pseudoknot interactions modulating functionality of the viral genome

Joanna Sztuba-Solinska  1 , Tadahisa Teramoto, Jason W Rausch, Bruce A Shapiro, Radhakrishnan Padmanabhan, Stuart F J Le Grice
Affiliations

Structural complexity of Dengue virus untranslated regions: cis-acting RNA motifs and pseudoknot interactions modulating functionality of the viral genome

Joanna Sztuba-Solinska et al. Nucleic Acids Res. 2013 May.

Abstract

The Dengue virus (DENV) genome contains multiple cis-acting elements required for translation and replication. Previous studies indicated that a 719-nt subgenomic minigenome (DENV-MINI) is an efficient template for translation and (-) strand RNA synthesis in vitro. We performed a detailed structural analysis of DENV-MINI RNA, combining chemical acylation techniques, Pb(2+) ion-induced hydrolysis and site-directed mutagenesis. Our results highlight protein-independent 5'-3' terminal interactions involving hybridization between recognized cis-acting motifs. Probing analyses identified tandem dumbbell structures (DBs) within the 3' terminus spaced by single-stranded regions, internal loops and hairpins with embedded GNRA-like motifs. Analysis of conserved motifs and top loops (TLs) of these dumbbells, and their proposed interactions with downstream pseudoknot (PK) regions, predicted an H-type pseudoknot involving TL1 of the 5' DB and the complementary region, PK2. As disrupting the TL1/PK2 interaction, via 'flipping' mutations of PK2, previously attenuated DENV replication, this pseudoknot may participate in regulation of RNA synthesis. Computer modeling implied that this motif might function as autonomous structural/regulatory element. In addition, our studies targeting elements of the 3' DB and its complementary region PK1 indicated that communication between 5'-3' terminal regions strongly depends on structure and sequence composition of the 5' cyclization region.

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Figures

Figure 1.
Figure 1.
Secondary structure of the nucleotides 1–719 of DENV-MINI RNA. (A) Processed SHAPE reactivities of DENV-MINI RNA are presented as a function of nucleotide position. Red and orange notations are expected to fall in single-stranded regions, whereas bases indicated in black and green correspond predominantly to either double-stranded regions or putative tertiary interactions. (B) Three distinct DENV RNA domains have been annotated, namely, 5′–3′-UTRs, DBs and VR. Nucleotide positions are numbered every 20 nt. The abbreviations correspond to the following cis-acting RNA elements: UTR, untranslated region; SLA, stem–loop A; dsUAR, double-stranded upstream of AUG region; cHP, capsid hairpin; CS, cyclization sequences; DB, dumbbell; TL, top loop; SL, stem–loop. The pattern of Pb2+-induced hydrolysis obtained for interacting regions is indicated in the upper left insert.
Figure 2.
Figure 2.
TL1/PK2 pseudoknot interaction in DENV-MINI RNA. (A) SHAPE-predicted hairpin-type pseudoknot interaction comprises two helical regions, including base paring between PK2 (G526–C530) and TL1 residues (G470–U474), as well as the stem of the leftmost hairpin (C462–G466 and C478–G482), and three single-stranded loops: A468–G470, U474–C476 and C516–C525. (B) The 2D map of TL1/PK2 interaction forming H-type pseudoknot generated by PseudoViewer3 (32).
Figure 3.
Figure 3.
Structural responses of the TL1/PK2 pseudoknot motif to antisense oligomers. (A) aiSHAPE principal. Hybridization of an interfering oligonucleotide (green) to one partner of the proposed RNA duplex increases acylation sensitivity of its base-paired counterpart. (B) Influence of LNA/DNA oligonucleotide (orange) hybridized to PK2 on chemical reactivity of TL1 residues. (C) The effect of LNA 1B hybridization to TL1 loop on acylation of PK2 nucleotides. The gray residues represent the formation of an extensive stop during reverse transcription at the place of LNA hybridization. The native traces (red) are compared with antisense-interfered DENV-MINI RNA (violet). Nucleotide positions exhibiting increased NMIA reactivity in the presence of given LNA are distinguished by orange brackets.
Figure 4.
Figure 4.
Chemical probing of PK2Flip mutant RNA. The description of the scheme follows the convention of the Figure 1 legend. The flipping mutation within PK2 is marked by a blue rectangle. The insert in the left top corner represents increased Pb2+-induced hydrolysis within PK2 and TL1 regions.
Figure 5.
Figure 5.
Chemical probing of PK1Flip mutant RNA. The description of the scheme follows the convention of Figure 1 legend. The flipping mutation within PK1 region is marked by blue rectangle. The insert in the left top corner represents increased Pb2+-induced hydrolysis within ‘flipped’ PK1 residues and the novel motif, stem–loop B (SLB).
Figure 6.
Figure 6.
Chemical probing of TL2FlipPK1Flip-3mutant RNA. The description of the scheme follows the convention of Figure 1 legend. The 3-nt compensatory mutations introduced within 5′ CS, the ‘flipping’ mutations within PK1 and TL2 region are marked by blue rectangles and trapezoid. The insert in the left top corner represents the pattern of increased Pb2+-induced hydrolysis within ‘flipped’ PK1 and compensatory residues.
Figure 7.
Figure 7.
Proposed 3D model of truncated DENV-MINI RNA. The internally deleted DENV-MINI RNA is depicted (A) in its entirety or (B) highlighting the proximal 5′- and 3′-UTRs. Specific RNA domains are color-coded as shown in the key. The cis-acting motifs are labeled as follows: SLA TL, stem–loop A top loop; SLA SSL, stem–loop A side stem–loop; 3′ SL, 3′ stem–loop; dsUAR, double-stranded upstream of AUG region; PK, pseudoknot; TL, top loop. For clarity, the remainder of the RNA molecule is colored in gray.
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