Review Functional RNA Elements in the Dengue Virus Genome

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Structural and Functional Studies of the Promoter Element for Dengue Virus RNA Replication ▿

The 5′ untranslated region (5′UTR) of the dengue virus (DENV) genome contains two defined elements essential for viral replication. At the 5′ end, a large stem-loop (SLA) structure functions as the promoter for viral polymerase activity. Next to the SLA, there is a short stem-loop that contains a cyclization sequence known as the 5′ upstream AUG region (5′UAR). Here, we analyzed the secondary structure of the SLA in solution and the structural requirements of this element for viral replication. Using infectious DENV clones, viral replicons, and in vitro polymerase assays, we defined two helical regions, a side stem-loop, a top loop, and a U bulge within SLA as crucial elements for viral replication. The determinants for SLA-polymerase recognition were found to be common in different DENV serotypes. In addition, structural elements within the SLA required for DENV RNA replication were also conserved among different mosquito- and tick-borne flavivirus genomes, suggesting possible common strategies for polymerase-promoter recognition in flaviviruses. Furthermore, a conserved oligo(U) track present downstream of the SLA was found to modulate RNA synthesis in transfected cells. In vitro polymerase assays indicated that a sequence of at least 10 residues following the SLA, upstream of the 5′UAR, was necessary for efficient RNA synthesis using the viral 3′UTR as template

RNA Sequences and Structures Required for the Recruitment and Activity of the Dengue Virus Polymerase*

Dengue virus RNA-dependent RNA polymerase specifically binds to the viral genome by interacting with a promoter element known as stem-loop A (SLA). Although a great deal has been learned in recent years about the function of this promoter in dengue virus-infected cells, the molecular details that explain how the SLA interacts with the polymerase to promote viral RNA synthesis remain poorly understood. Using RNA binding and polymerase activity assays, we defined two elements of the SLA that are involved in polymerase interaction and RNA synthesis. Mutations at the top of the SLA resulted in RNAs that retained the ability to bind the polymerase but impaired promoter-dependent RNA synthesis. These results indicate that protein binding to the SLA is not sufficient to induce polymerase activity and that specific nucleotides of the SLA are necessary to render an active polymerase-promoter complex for RNA synthesis. We also report that protein binding to the viral RNA induces conformational changes downstream of the promoter element. Furthermore, we found that structured RNA elements at the 3′ end of the template repress dengue virus polymerase activity in the context of a fully active SLA promoter. Using assays to evaluate initiation of RNA synthesis at the viral 3′-UTR, we found that the RNA-RNA interaction mediated by 5′-3′-hybridization was able to release the silencing effect of the 3′-stem-loop structure. We propose that the long range RNA-RNA interactions in the viral genome play multiple roles during RNA synthesis. Together, we provide new molecular details about the promoter-dependent dengue virus RNA polymerase activity

A 5′ RNA element promotes dengue virus RNA synthesis on a circular genome

The mechanisms of RNA replication of plus-strand RNA viruses are still unclear. Here, we identified the first promoter element for RNA synthesis described in a flavivirus. Using dengue virus as a model, we found that the viral RdRp discriminates the viral RNA by specific recognition of a 5′ element named SLA. We demonstrated that RNA–RNA interactions between 5′ and 3′ end sequences of the viral genome enhance dengue virus RNA synthesis only in the presence of an intact SLA. We propose a novel mechanism for minus-strand RNA synthesis in which the viral polymerase binds SLA at the 5′ end of the genome and reaches the site of initiation at the 3′ end via long-range RNA–RNA interactions. These findings provide an explanation for the strict requirement of dengue virus genome cyclization during viral replication

RNA recombination at Chikungunya virus 3'UTR as an evolutionary mechanism that provides adaptability.

The potential of RNA viruses to adapt to new environments relies on their ability to introduce changes in their genomes, which has resulted in the recent expansion of re-emergent viruses. Chikungunya virus is an important human pathogen transmitted by mosquitoes that, after 60 years of exclusive circulation in Asia and Africa, has rapidly spread in Europe and the Americas. Here, we examined the evolution of CHIKV in different hosts and uncovered host-specific requirements of the CHIKV 3'UTR. Sequence repeats are conserved at the CHIKV 3'UTR but vary in copy number among viral lineages. We found that these blocks of repeated sequences favor RNA recombination processes through copy-choice mechanism that acts concertedly with viral selection, determining the emergence of new viral variants. Functional analyses using a panel of mutant viruses indicated that opposite selective pressures in mosquito and mammalian cells impose a fitness cost during transmission that is alleviated by recombination guided by sequence repeats. Indeed, drastic changes in the frequency of viral variants with different numbers of repeats were detected during host switch. We propose that RNA recombination accelerates CHIKV adaptability, allowing the virus to overcome genetic bottlenecks within the mosquito host. These studies highlight the role of 3'UTR plasticity on CHIKV evolution, providing a new paradigm to explain the significance of sequence repetitions

Nucleotide variations detected in experimentally adapted viruses correlate with mutations found in natural isolates.

<p><b>(A)</b> Adaptive mutations and deletions were mapped on the RNA structure of the variable region of the viral 3’UTR. Deletions and point mutations rescued from mosquito cell-adapted populations are indicated in grey and red, respectively. Mutation selected in mammalian cells is shown in blue. <b>(B)</b> Conservation plots comparing variable regions of cell adapted viruses and natural isolates. <b>(C)</b> Representation of SL-II RNA structures of DENV genomes from natural isolates corresponding to different serotypes. Sequence alignment plots and secondary RNA structure models are shown for DENV isolates from humans (top) and mosquitoes (bottom).</p

Sequence variations at the DENV 3’UTR during experimental host adaptation.

<p><b>(A)</b> Deep sequencing of viral populations after 10 and 20 passages (P10 and P20) in mosquito and mammalian cells. The area and color of circles represent the frequency and number of mutations of the viral variants, respectively. A schematic fan dendrogram indicating the distance between the variants for each population is also shown. For nucleotide sequences and frequencies of each viral variant also see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004604#ppat.1004604.s001" target="_blank">S1 Fig</a>. <b>(B)</b> Sequence of cloned amplicons corresponding to the complete 3’UTR. For each P10 population obtained, 20 individual clones were sequenced. Cells lines used for adaptation experiments are indicated (mosquito C6/36, mosquito U4.4, and human A549 cells). Mutations are indicated in red and deletions in grey. A conservation plot is presented at the bottom. The three independent experiments in C6/36 cells are indicated on the left (I, II and III).</p

Fitness advantage of RNA structure duplication during DENV host switching.

<p><b>(A)</b> Normalized luciferase levels expressed by reporter DENV with or without mutations resembling adaptations in mosquito or mammalian cells, respectively, carrying one or two SLs are shown for mammalian cells. The luciferase values are the mean +/- SD, n = 4. <b>(B)</b> The same as in <b>(A)</b> using mosquito cells. <b>(C)</b> Schematic representation of adaptable RNA structures emulating host switching of viruses with single or double SLs. Viruses with a single SL encounter a fitness barrier in the transition from mosquito to mammalian cells due to accumulation of mosquito adaptive mutations, while viruses with double SLs show robustness during host switching.</p