Recruitment of the 40S ribosomal subunit by the West Nile virus 3' UTR promotes the cross-talk between the viral genomic ends for translation regulation.

Flaviviral RNA genomes are composed of discrete RNA structural units arranged in an ordered fashion and grouped into complex folded domains that regulate essential viral functions, e.g. replication and translation. This is achieved by adjusting the overall structure of the RNA genome via the establishment of inter- and intramolecular interactions. Translation regulation is likely the main process controlling flaviviral gene expression. Although the genomic 3' UTR is a key player in this regulation, little is known about the molecular mechanisms underlying this role. The present work provides evidence for the specific recruitment of the 40S ribosomal subunit by the 3' UTR of the West Nile virus RNA genome, showing that the joint action of both genomic ends contributes the positioning of the 40S subunit at the 5' end. The combination of structural mapping techniques revealed specific conformational requirements at the 3' UTR for 40S binding, involving the highly conserved SL-III, 5'DB, 3'DB and 3'SL elements, all involved in the translation regulation. These results point to the 40S subunit as a bridge to ensure cross-talk between both genomic ends during viral translation and support a link between 40S recruitment by the 3' UTR and translation control.

Inter- and Intramolecular RNA-RNA Interactions Modulate the Regulation of Translation Mediated by the 3' UTR in West Nile Virus.

RNA viruses rely on genomic structural elements to accomplish the functions necessary to complete the viral cycle. These elements participate in a dynamic network of RNA-RNA interactions that determine the overall folding of the RNA genome and may be responsible for the fine regulation of viral replication and translation as well as the transition between them. The genomes of members of the genus Flavivirus are characterized by a complexly folded 3' UTR with a number of RNA structural elements that are conserved across isolates of each species. The present work provides evidence of intra- and intermolecular RNA-RNA interactions involving RNA structural elements in the 3' UTR of the West Nile virus genome. The intermolecular interactions can be visualized in vitro by the formation of molecular dimers involving the participation of at least the SLI and 3'DB elements. Certainly, the 3' UTR of dengue virus, which lacks the SLI element, forms molecular dimers in lower quantities via a single interaction site, probably 3'DB. The functional analysis of sequence or deletion mutants revealed an inverse relationship between 3' UTR dimerization and viral translation efficiency in cell cultures. A network of RNA-RNA interactions involving 3' UTR structural elements might therefore exist, helping to regulate viral translation.

The Genomic 3' UTR of Flaviviruses Is a Translation Initiation Enhancer.

Viruses rely on the cellular machinery of host cells to synthesize their proteins, and have developed different mechanisms enabling them to compete with cellular mRNAs for access to it. The genus Flavivirus is a large group of positive, single-stranded RNA viruses that includes several important human pathogens, such as West Nile, Dengue and Zika virus. The genome of flaviviruses bears a type 1 cap structure at its 5' end, needed for the main translation initiation mechanism. Several members of the genus also use a cap-independent translation mechanism. The present work provides evidence that the WNV 5' end also promotes a cap-independent translation initiation mechanism in mammalian and insect cells, reinforcing the hypothesis that this might be a general strategy of flaviviruses. In agreement with previous reports, we show that this mechanism depends on the presence of the viral genomic 3' UTR. The results also show that the 3' UTR of the WNV genome enhances translation of the cap-dependent mechanism. Interestingly, WNV 3' UTR can be replaced by the 3' UTR of other flaviviruses and the translation enhancing effect is maintained, suggesting a molecular mechanism that does not involve direct RNA-RNA interactions to be at work. In addition, the deletion of specific structural elements of the WNV 3' UTR leads to increased cap-dependent and cap-independent translation. These findings suggest the 3' UTR to be involved in a fine-tuned translation regulation mechanism.

In Vitro Methods to Decipher the Structure of Viral RNA Genomes.

RNA viruses encode essential information in their genomes as conserved structural elements that are involved in efficient viral protein synthesis, replication, and encapsidation. These elements can also establish complex networks of RNA-RNA interactions, the so-called RNA interactome, to shape the viral genome and control different events during intracellular infection. In recent years, targeting these conserved structural elements has become a promising strategy for the development of new antiviral tools due to their sequence and structural conservation. In this context, RNA-based specific therapeutic strategies, such as the use of siRNAs have been extensively pursued to target the genome of different viruses. Importantly, siRNA-mediated targeting is not a straightforward approach and its efficiency is highly dependent on the structure of the target region. Therefore, the knowledge of the viral structure is critical for the identification of potentially good target sites. Here, we describe detailed protocols used in our laboratory for the in vitro study of the structure of viral RNA genomes. These protocols include DMS (dimethylsulfate) probing, SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) analysis, and HMX (2'-hydroxyl molecular interference). These methodologies involve the use of high-throughput analysis techniques that provide extensive information about the 3D folding of the RNA under study and the structural tuning derived from the interactome activity. They are therefore a good tool for the development of new RNA-based antiviral compounds.