Here, we have characterized a step in translation initiation of viral and cellular mRNAs that contain RNA secondary structures immediately at the vicinity of their m7GTP cap. This is mediated by the DEAD‐box helicase DDX3 which can directly bind to the 5′ of the target mRNA where it clamps the entry of eIF4F through an eIF4G and Poly A‐binding protein cytoplasmic 1 (PABP) double interaction. This could induce limited local strand separation of the secondary structure to allow 43S pre‐initiation complex attachment to the 5′ free extremity of the mRNA. We further demonstrate that the requirement for DDX3 is highly specific to some selected transcripts, cannot be replaced or substituted by eIF4A and is only needed in the very early steps of ribosome binding and prior to 43S ribosomal scanning. Altogether, these data define an unprecedented role for a DEAD‐box RNA helicase in translation initiation.

Here, we show a novel molecular mechanism promoted by the DEAD-box RNA helicase DDX3 for translation of the HIV-1 genomic RNA. This occurs through the adenosine triphosphate-dependent formation of a translation initiation complex that is assembled at the 5′ m⁷GTP cap of the HIV-1 mRNA. This is due to the property of DDX3 to substitute for the initiation factor eIF4E in the binding of the HIV-1 m⁷GTP 5′ cap structure where it nucleates the formation of a core DDX3/PABP/eIF4G trimeric complex on the HIV-1 genomic RNA. By using RNA fluorescence in situ hybridization coupled to indirect immunofluorescence, we further show that this viral ribonucleoprotein complex is addressed to compartmentalized cytoplasmic foci where the translation initiation complex is assembled.