Like other flaviviruses, West Nile virus (WNV) have disseminated through the entire world in the last two decades with significant morbidity and mortality. Few advances regarding human vaccines or treatments have since been made. Like other RNA viruses, flaviviruses encode part of their genetic information in highly conserved structural domains present in their RNA genome. Specifically, the flaviviral 3’UTR region acts as a multivalent element that plays essential roles in viral replication, encapsidation and translation, making it a potential target for the development of specific pharmacological antiviral treatments. We have observed that the 3’UTR of WNV is capable of recruiting the 40S ribosomal subunit in the absence of protein factors in a specific manner. This phenomenon also occurs in other flaviviruses. By using a molecular interference strategy, it has been detected a deep structural reorganization event as result of the 40S recruitment at the essential 3’DB domain, suggesting that conformational changes could underlie the translation-enhancing effect mediated by the 3’UTR, which has been reported by several authors. Further, the recruitment of the 40S subunit is strictly dependent on RNA-protein interactions, with a 15 kDa undisclosed protein being intimately involved in this interaction. We also provide the proof of concept that the interference with the recruitment of the 40S subunit by blocking the binding sites in the 3’UTR region constitutes a potential therapeutic strategy against WNV. Our findings thus open the way to the development of new drugs specialized in the binding to specific structural RNA motifs.