Regulatory Elements in the HIV-1 5'UTR that Modulate Gag Binding Specificity
Loading...
Date
2018-05
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
The Ohio State University
Abstract
The 5'UTR of the HIV-1 genomic RNA (gRNA) contains a structured RNA element (termed Psi) that is specifically recognized through the interaction with the nucleocapsid (NC) domain of HIV-1 Gag, ensuring that two copies of gRNA are packaged into newly assembled virions. However, the mechanism by which Gag recognizes gRNA over other cellular RNAs and spliced RNAs is not well understood. Exposed guanosine residues within the loops and bulges of Psi have been shown to be high affinity NC binding sites, providing a selective advantage for gRNA. Furthermore, a recent study suggested that a negative regulatory element upstream of Psi reduces high-affinity Gag binding, and a positive downstream regulatory element counteracts the upstream element and restores high-affinity binding. These elements are proposed to form a long-range interaction that promotes packaging of only full-length gRNA and excludes packaging of spliced RNAs. Using a fluorescence anisotropy-based salt-titration binding assay, which measures the electrostatic and nonelectrostatic (i.e., specific) components of protein binding RNA, we have previously shown that Gag interacts with a 109 nt Psi RNA construct with high specificity and relatively few electrostatic interactions. High specificity of this Psi construct is maintained when guanosine residues in the bulges of SL2 are mutated, but guanosine mutations in the bulges of SL1 and regions downstream of SL3 greatly diminish this high specificity. When using a 356-nt RNA construct that now includes the upstream negative regulatory element in addition to Psi, we observed similar Gag binding specificity and electrostatic interactions to the 109 nt WT Psi construct. However, a 400 nt construct that additionally contains the positive element, increased specific binding and reduced the electrostatic interactions. Interestingly, Gag binding specificity remained high even in constructs 3 wherein the 44 nt of the downstream positive regulatory element were scrambled. Thus, while the presence of downstream sequences is important for high-specificity Gag binding, their identity is not. Furthermore, the secondary structures of spliced RNA constructs were probed via selective 2สน hydroxyl acylation analyzed by primer extension (SHAPE) to study how they prevent specific packaging. In a construct composed of the first 400 nt of the Vpr mRNA, destabilization of the polyA hairpin was observed which has been shown to be critical for RNA packaging and viral replication. Meanwhile, the secondary structure of a construct containing the first 400 nt of the Tat mRNA displays a sequestered primer binding site (PBS) which has been shown to be necessary for tRNALys3 annealing and initiation of reverse transcription. Altogether, these results allow us to better understand the mechanism by which genomic RNA packaging occurs.
Description
2017 Rustbelt RNA Conference Undergraduate Poster Presentation Honorable Mention
Keywords
HIV-1, 5'UTR, RNA Biology, HIV-1 Gag