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

Abstract

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.

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

“…To test whether the recruitment of the 40S ribosomal subunit could be a molecular mechanism underlying the 3′ UTR mediated translation regulation, in vitro binding assays of the 40S subunit to the WNV 3′ UTR RNA were performed. Briefly, trace amounts of the 32 P-internally labeled WNV 3′ UTR transcript, which encompasses the full-length 3′ UTR of WNV (nucleotides 10,400–11,029) ( Romero-López et al, 2023 ), were incubated with increasing concentrations of purified 40S subunit. The resulting complexes were separated by differential retention filter assays, as it has been widely used for this purpose ( Angulo et al, 2016 ; Bai et al, 2013 ; Bhattacharyya et al, 2015 ; Deforges et al, 2017 ; Gao et al, 2013 ; Tallet-Lopez et al, 2003 ).…”

“…To identify the structural requirements for the 40S recruitment in the WNV 3′ UTR, hydroxyl molecular interference assays (HMX) with NMIA were performed essentially as previously described ( Romero-López et al, 2017 ; 2023 ). Briefly, the WNV 3′ UTR RNA was modified with NMIA under subsaturating and denaturing conditions.…”

“…This finding suggests that the 3′DB element is a dynamic structural unit, which can modify its folding in response to different conditions. Interestingly, we have previously demonstrated that the WNV 3′ UTR forms dimeric units, being the 3′DB element a key player for this process ( Romero-López et al, 2023 ). Further, a clear correlation between 3′DB and translation efficiency was observed, showing that this element is required to achieve translational enhancement mediated by the WNV 3′ UTR ( Romero-López et al, 2023 ).…”

“…Interestingly, we have previously demonstrated that the WNV 3′ UTR forms dimeric units, being the 3′DB element a key player for this process ( Romero-López et al, 2023 ). Further, a clear correlation between 3′DB and translation efficiency was observed, showing that this element is required to achieve translational enhancement mediated by the WNV 3′ UTR ( Romero-López et al, 2023 ). Again, the present data point that this effect could be mediated by the implication of the 3′DB in the recruitment of the 40S subunit and its subsequent delivery to the 5′ UTR.…”

“…DNA template encoding for the WNV 3′ UTR was obtained as described ( Romero-López et al, 2023 ). DNA encoding deletion mutants 3′WNV_10,400–10,900, 3′WNV_10,828–11,029, 3′WNV_10,742–10,950, 3′WNV_10,742–11,029 and 3′WNV_10,583–11,029 were obtained by PCR amplification from the plasmid pGLWNV ( Berzal-Herranz et al, 2022 ).…”