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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.

SUBMITTER: Osmer PS

PROVIDER: S-EPMC7650772 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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

Osmer Patrick S PS Singh Gatikrushna G Boris-Lawrie Kathleen K

Viruses 20200929 10

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 ...[more]

PMID: 33003639

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Project description:The development of modern and affordable sequencing technologies has allowed the study of viral populations to an unprecedented depth. This is of particular interest for the study of within-host RNA viral populations, where variation due to error-prone polymerases can lead to immune escape, antiviral resistance and adaptation to new host species. Methods to sequence RNA virus genomes include reverse transcription (RT) and polymerase chain reaction (PCR). RT-PCR is a molecular biology technique widely used to amplify DNA from an RNA template. The method itself relies on the in vitro synthesis of copy DNA from RNA followed by multiple cycles of DNA amplification. However, this method introduces artefactual errors that can act as confounding factors when the sequence data are analysed. Although there are a growing number of published studies exploring the intra- and inter-host evolutionary dynamics of RNA viruses, the complexity of the methods used to generate sequences makes it difficult to produce probabilistic statements about the likely sources of observed sequence variants. This complexity is further compounded as both the depth of sequencing and the length of the genome segment of interest increase. Here we develop a bayesian method to characterise and differentiate between likely structures for the background viral population. This approach can then be used to identify nucleotide sites that show evidence of change in the within-host viral population structure, either over time or relative to a reference sequence (e.g. an inoculum or another source of infection), or both, without having to build complex evolutionary models. Identification of these sites can help to inform the design of more focussed experiments using molecular biology tools, such as site-directed mutagenesis, to assess the function of specific amino acids. We illustrate the method by applying to datasets from experimental transmission of equine influenza, and a pre-clinical vaccine trial for HIV-1.

Dataset Information

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

Publications

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

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