A New Approach to 3D Modeling of Inhomogeneous Populations of Viral Regulatory RNA

Abstract

Tertiary structure (3D) is the physical context of RNA regulatory activity. Retroviruses are RNA viruses that replicate through the proviral DNA intermediate transcribed by hosts. Proviral transcripts form inhomogeneous populations due to variable structural ensembles of overlapping regulatory RNA motifs in the 5′-untranslated region (UTR), which drive RNAs to be spliced or translated, and/or dimerized and packaged into virions. Genetic studies and structural techniques have provided fundamental input constraints to begin predicting HIV 3D conformations in silico. Using SimRNA and sets of experimentally-determined input constraints of HIVNL4-3 trans-activation responsive sequence (TAR) and pairings of unique-5′ (U5) with dimerization (DIS) or AUG motifs, we calculated a series of 3D models that differ in proximity of 5′-Cap and the junction of TAR and PolyA helices; configuration of primer binding site (PBS)-segment; and two host cofactors binding sites. Input constraints on U5-AUG pairings were most compatible with intramolecular folding of 5′-UTR motifs in energetic minima. Introducing theoretical constraints predicted metastable PolyA region drives orientation of 5′-Cap with TAR, U5 and PBS-segment helices. SimRNA and the workflow developed herein provides viable options to predict 3D conformations of inhomogeneous populations of large RNAs that have been intractable to conventional ensemble methods.

HIV-1 hypermethylated guanosine cap licenses specialized translation unaffected by mTOR

“…Our results suggest that the 5′-UTR structures control epigenetic modification of the m 7 G-cap and sort them into the separate translation pathways to ensure productive synthesis of HIV-1 structural/enzymatic/accessory proteins when hosts shutdown global translation in response to mTOR inhibition. Our results support the hypothesis that PBS segment regions are metastable determinants important for sorting MS mRNAs for eIF4E-dependent translation of regulatory proteins and US/SS transcripts for CBP80/NCBP3-RHA–dependent translation of virion proteins ( 56 , 57 , 60 , 61 ).…”

Anomalous HIV-1 RNA, How Cap-Methylation Segregates Viral Transcripts by Form and Function

“…Using experimentally determined 5′-UTR input constraints and 30 million iterative Monte Carlo Simulations, tertiary (3D) models have been predicted on the simRNA platform [ 174 ]. Molecular dynamic visualization of the conformational differences between Cap-G and Cap-GGG were reviewed.…”

“…Rotations of the three-dimensional visualizations show differences in the orientation of the HIV-1 5′-Cap with the junction of the helical TAR, PolyA, U5 segments. Zoom-in taken from the model of the Dimer-prone 5′-UTR (356 nucleotides) [ 174 ] on the Cap site (magenta) near the junction of TAR (magenta); PolyA (blue); U5 (green). G104, blue atom and U105, green atom (Left) Cap-G. Cap appended to the transcription start site (Cap-G) was positioned close to the central hub of TAR-PolyA-U5 helices and engaged in Watson-Crick base pairing (G-C), as summarized in Figure 1 ; (Right) Cap-GGG.…”