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

Sztuba-Solinska J, Teramoto T, Rausch JW, Shapiro BA, Padmanabhan R, Le Grice SF - Nucleic Acids Res. (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: RT Biochemistry Section, HIV Drug Resistance Program, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.

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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Related in: MedlinePlus

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).
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gkt203-F5: 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).

Mentions: The NMIA sensitivity profile of PK1Flip mutant indicated extensive alternations within the 5′- and 3′-UTRs (Figure 5 and Supplementary Figures S7 and S8). The 5′ UAR no longer formed a double-stranded region, but instead, it became a part of the novel SLB hairpin (G70–C105) with a reactive apical loop (G86–G89) and internal loops (U73–A77 and G99–U102). The 5′ DAR region, previously double-stranded in DENV-MINI, rearranged into a reactive single-stranded loop (C105–G110). In addition, the PK1 ‘flipping’ mutation abolished base pairing between 5′ and 3′ CS, disrupting the previous double-stranded 5′–3′ CS. The 5′ CS (C135–G144) now formed a reactive single-stranded region downstream of cHP, whereas the 3′ CS (C613–U622) and the ‘flipped’ PK1 region (C613–A617) comprised a reactive loop downstream of the 3′ DB. In addition, the two hairpins previously formed within the sequence of the 5′-end of the capsid protein gene were fused into an extended hairpin (G153–C197) with a weakly reactive internal loop (A158–G163 and A187–A192) and an A–C mismatch.Figure 5.


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

Sztuba-Solinska J, Teramoto T, Rausch JW, Shapiro BA, Padmanabhan R, Le Grice SF - Nucleic Acids Res. (2013)

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).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643606&req=5

gkt203-F5: 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).
Mentions: The NMIA sensitivity profile of PK1Flip mutant indicated extensive alternations within the 5′- and 3′-UTRs (Figure 5 and Supplementary Figures S7 and S8). The 5′ UAR no longer formed a double-stranded region, but instead, it became a part of the novel SLB hairpin (G70–C105) with a reactive apical loop (G86–G89) and internal loops (U73–A77 and G99–U102). The 5′ DAR region, previously double-stranded in DENV-MINI, rearranged into a reactive single-stranded loop (C105–G110). In addition, the PK1 ‘flipping’ mutation abolished base pairing between 5′ and 3′ CS, disrupting the previous double-stranded 5′–3′ CS. The 5′ CS (C135–G144) now formed a reactive single-stranded region downstream of cHP, whereas the 3′ CS (C613–U622) and the ‘flipped’ PK1 region (C613–A617) comprised a reactive loop downstream of the 3′ DB. In addition, the two hairpins previously formed within the sequence of the 5′-end of the capsid protein gene were fused into an extended hairpin (G153–C197) with a weakly reactive internal loop (A158–G163 and A187–A192) and an A–C mismatch.Figure 5.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: RT Biochemistry Section, HIV Drug Resistance Program, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.

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.

Show MeSH
Related in: MedlinePlus