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

Abstract

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.

The mRNA encoding the JUND tumor suppressor detains nuclear RNA-binding proteins to assemble polysomes that are unaffected by mTOR

“…Published protocols were used to perform density sedimentation and A254 spectrometry (58). Briefly, cells were transfected for 24 h and treated with Torin-1 (50 nM) for 18 h. The culture medium was supplemented with 0.1 mg/mL cycloheximide (CHX) for 10 min.…”

The 5′ Untranslated Region of the Major Immediate Early mRNA Is Necessary for Efficient Human Cytomegalovirus Replication

“…RNA binding proteins (RBPs) can recognize specific sequences in the 5= UTR and facilitate ribosome recruitment (51) to enhance translation. Alternatively, RNA structures in the 5= UTR can impede ribosomal subunit scanning or translation start site recognition (52)(53)(54)(55)(56) or serve as binding sites for RBPs that suppress translation (50,(57)(58)(59). Experimentally determining the impact of a 5= UTR on mRNA translation is therefore critical for understanding the regulation of protein expression.…”

An epitranscriptomic switch at the 5′-UTR controls genome selection during HIV-1 genomic RNA packaging

“…However, the full-length RNA also serves as mRNA for the synthesis of Gag and Gag-Pol precursors and thus, translation and packaging are expected to be two mutually exclusive processes. Although the adoption of a branched multiple hairpin (BMH) conformation of the 5′-UTR was initially proposed to favor dimerization and packaging over translation 12–14 , it was later demonstrated that translation of the full-length RNA is under positive selection and thus, not regulated by a conformational switch of the 5′-UTR 18, 19 . Additional structural studies carried out in cells and virions also argued against structural rearrangements as drivers of the transition between translation and packaging of the HIV-1 full-length RNA 16, 17, 28 .…”

“…In such models, the 5′-UTR alternates in conformations that occlude the dimerization initiation signal (DIS) or the Gag start codon thus, favoring translation or dimerization and packaging, respectively 3, 8 . However, chemical probing performed in cells and purified viral particles showed that a single structure, in which DIS is accessible for dimerization and packaging, predominates in these biological states 16, 17 Moreover, the packaging prone structure does not interfere with full-length RNA translation suggesting that other factors rather than structural rearrangements are involved in the regulation of the cytoplasmic sorting of the HIV-1 full-length RNA 18, 19 .…”