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Functional RNA elements in the dengue virus genome - PubMed

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Functional RNA elements in the dengue virus genome

Leopoldo G Gebhard et al. Viruses. 2011 Sep.

Abstract

Dengue virus (DENV) genome amplification is a process that involves the viral RNA, cellular and viral proteins, and a complex architecture of cellular membranes. The viral RNA is not a passive template during this process; it plays an active role providing RNA signals that act as promoters, enhancers and/or silencers of the replication process. RNA elements that modulate RNA replication were found at the 5' and 3' UTRs and within the viral coding sequence. The promoter for DENV RNA synthesis is a large stem loop structure located at the 5' end of the genome. This structure specifically interacts with the viral polymerase NS5 and promotes RNA synthesis at the 3' end of a circularized genome. The circular conformation of the viral genome is mediated by long range RNA-RNA interactions that span thousands of nucleotides. Recent studies have provided new information about the requirement of alternative, mutually exclusive, structures in the viral RNA, highlighting the idea that the viral genome is flexible and exists in different conformations. In this article, we describe elements in the promoter SLA and other RNA signals involved in NS5 polymerase binding and activity, and provide new ideas of how dynamic secondary and tertiary structures of the viral RNA participate in the viral life cycle.

Keywords: NS5 protein; RNA structures; dengue virus; flavivirus RNA; genome cyclization; viral RNA replication; viral RdRp.

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Figures

Figure 1.
Figure 1.

Schematic representation of the Dengue virus (DENV) genome. (a) The viral 5′ and 3′ untranslated regions (UTRs) and the open reading frame indicating structural proteins (C-prM-E) and non-structural proteins (NS1-NS2AB-NS3-NS4AB-NS5). The location of the complementary sequences 5′-3′CS and 5′-3′UAR are also indicated by solid and dashed lines, respectively. (b) Predicted secondary structure of the 5′ terminal region of the DENV genome. Structural elements located at the 5′ end: stem loop A (SLA), stem loop B (SLB), oligo(U) track spacer, translation initiator AUG, capsid region hairpin (cHP), and the 5′CS element. (c) Schematic representation of predicted RNA elements at the 3′UTR of the DENV genome. The predicted secondary structures of the three defined domains are indicated: domain I (variable region, VR), domain II (dumbbell structures, DB1 and DB2), and domain III (conserved sequence CS1 and 3′SL). In addition, the location and sequence of each of the conserved elements corresponding to RCS2, CS2, 3′CS, and 3′UAR are shown.

Figure 2.
Figure 2.

Schematic representation of predicted conformational changes in the transition between the linear and circular conformation of the DENV genome. Predicted changes of conserved RNA structures upon 5′-3′ end hybridization are indicated: a) the SLB and the sHP of the 3′SL observed in the linear conformation of the RNA open to form an extended duplex in the circular form, b) the large stem of the 3′SL observed in the linear form opens, releasing the last nucleotides of the genome in the circular form, and c) the complementary sequences 5′ and 3′ CS interact in the circular form to generate a double stranded region.

Figure 3.
Figure 3.

Representation of the in vitro trans initiation assay for RNA synthesis. (a) Formation of an RNA-RNA complex between the first 160 nucleotides of the viral genome (5′UTR-5′CS) and the viral 3′UTR allows the RNA-dependent RNA polymerase (RdRp) to initiate RNA synthesis at the 3′ end of both molecules, as indicated schematically in the figure. On the right, a representative native polyacrylamide gel shows the radiolabeled RNA products obtained after incubation of the viral RdRp with the templates described at the top of the figure. (b) Schematic representation of the role of a spacer sequence for RNA synthesis. The oligo(U) track spacer located between the SLA and the hybridized 5′-3′ UAR sequences allows accommodation of the RdRp to initiate RNA synthesis at the 3′UTR. In contrast, an RNA molecule carrying an intact SLA but a deletion of the oligo(U) spacer is unable to promote RNA synthesis in trans.

Figure 4.
Figure 4.

Representation of a model showing the requirement of a balance between different conformations of the DENV genome. Viruses carrying mutations that increase the stability of the circular or the linear form of the RNA, by increasing 5′-3′UAR complementarity (Mut Cyc+) or by stabilizing the sHP (Mut sHP+) respectively, evolve in culture incorporating spontaneous mutations that restore the relative stability of the two competing structures.

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