The protein coding-information only represents a small portion of the genetic load of a living organism. It is well established that essential information codes functional RNAs, called non-coding RNAs (ncRNAs), which play key roles in the essential biological processes of the cell life. Many mRNAs also act as truly ncRNAs besides being translated into proteins. Therefore, the repertoire of potential drug targets to fight diseases goes beyond proteins. Viral RNA genomes encode all the information for completion of the infectious cycle. They are multifunctional molecules, which act as replication templates and mRNAs. Further, defined structural domains in viral RNA genomes play key functions for the completion of the viral cycle and the regulation of the essential processes; these domains have also been involved in virulence. The West Nile Virus (WNV) genome consists in a single stranded RNA molecule, which contains a single ORF flanked by untranslated regions (UTRs). The 3’UTR is required for efficient translation, but the mechanisms involved in this regulation are still obscure. In this work, we show evidences that the WNV-3’UTR specifically recruits the 40S ribosomal subunit. We have localized two potential binding sites of the 40S. Binding of the 40S induced conformational changes in highly conserved structural domains within the WNV-3’UTR. Functional assays support the hypothesis that recruitment of the 40S particle by the 3’UTR is required for an efficient translation. Interfering with the 40S recruitment, by targeting the WNV-3’UTR binding sites, constitutes a potential antiviral strategy by the development of new therapeutic compounds.