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Abstract
SummaryHIV-1 genomic RNA has a noncoding 5′ region containing sequential conserved structural motifs that control many parts of the life cycle. Very limited data exist on their three-dimensional (3D) conformation and, hence, how they work structurally. To assemble a working model, we experimentally reassessed secondary structure elements of a 240-nt region and used single-molecule distances, derived from fluorescence resonance energy transfer, between defined locations in these elements as restraints to drive folding of the secondary structure into a 3D model with an estimated resolution below 10 Å. The folded 3D model satisfying the data is consensual with short nuclear-magnetic-resonance-solved regions and reveals previously unpredicted motifs, offering insight into earlier functional assays. It is a 3D representation of this entire region, with implications for RNA dimerization and protein binding during regulatory steps. The structural information of this highly conserved region of the virus has the potential to reveal promising therapeutic targets.
Highlights
Effective treatments exist to suppress HIV replication, but high sequence variability and mutational escape contribute to the lack of an effective vaccine
By electrophoresis of nucleotides 104– 413 of the HIV-1 packaging signal RNA, we have demonstrated that dimerization occurs for our subsection length under the conditions used in our FRET experiments (Figure 1B, lane 2)
The first of these steps requires a prediction of the 2D organization of the sequence. This we achieved using SHAPE technology (Wilkinson et al, 2008), which relies on the fact that conformationally flexible nucleotides are preferentially reactive to N-methylisatoic anhydride (NMIA)
Summary
Effective treatments exist to suppress HIV replication, but high sequence variability and mutational escape contribute to the lack of an effective vaccine. Regions of high RNA sequence conservation provide attractive therapeutic targets One such sequence is the 50 untranslated region (UTR), present in all genomic HIV transcripts whose stringent conservation is attributable to the presence of many regulatory regions controlling reverse transcription (Aiyar et al, 1992), transcription (Aboulela et al, 1995), dimerization (Laughrea and Jette , 1994) (necessary for packaging; Russell et al, 2004), and splicing (Harrison and Lever, 1992). Small nucleotide perturbations of the sequence can cause catastrophic effects on viral infectivity (Harrison et al, 1998a), probably through effects on the global 50 RNA structure during folding
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