The basal translation rate of authentic HIV-1 RNA is regulated by 5'UTR nt-pairings at junction of R and U5.

The paradigm protein synthesis rate is regulated by structural complexity of the 5'untranslated region (UTR) derives from bacterial and other riboswitches. In-solution, HIV-1 5'UTR forms two interchangeable long-range nucleotide (nt) -pairings, one sequesters the gag start codon promoting dimerization while the other sequesters the dimer initiation signal preventing dimerization. While the effect of these nt-pairings on dimerization and packaging has been documented their effect on authentic HIV translation in cellulo has remained elusive until now. HIVNL4-3 5'UTR substitutions were designed to individually stabilize the dimer-prone or monomer-prone conformations, validated in-solution, and introduced to molecular clones. The effect of 5'UTR conformation on ribosome loading to HIV unspliced RNA and rate of Gag polypeptide synthesis was quantified in cellulo. Monomer- and dimer-prone 5'UTRs displayed equivalent, basal rate of translation. Gain-of-function substitution U103, in conjunction with previously defined nt-pairings that reorient AUG to flexible nt-pairing, significantly activated the translation rate, indicating the basal translation rate is under positive selection. The observed translation up-mutation focuses attention to nt-pairings at the junction of R and U5, a poorly characterized structure upstream of the characterized HIV riboswitch and demonstrates the basal translation rate of authentic HIV RNA is regulated independently of monomer:dimer equilibrium of the 5'UTR.