Positive and Negative Regulatory Elements in the HIV-1 5'UTR Control Specific Recognition by Gag
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Date
2017-03-29
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Abstract
The 5ʹ untranslated region (5ʹUTR) of the human immunodeficiency virus type 1 (HIV-1) genomic RNA (gRNA) contains a structured RNA element (termed Psi) that is specifically recognized by the HIV-1 Gag polyprotein, ensuring that two strands of gRNA are packaged into newly assembled virions. However, the mechanism by which Gag recognizes gRNA over other cellular RNAs and spliced viral RNAs is not well understood. A recent study suggested that a negative regulatory element upstream of Psi reduces high-affinity Gag binding, and a positive regulatory element downstream of Psi counteracts the upstream element and restores high-affinity binding. The aim of this study is to determine how these elements affect the specificity and mode of Gag binding. Using a fluorescence anisotropy-based salt-titration binding assay, the electrostatic and nonelectrostatic (i.e., specific) components of binding were measured. We have previously shown that Gag interacts with a 109-nucleotide (nt) Psi RNA construct that lacks the putative regulatory elements with high specificity and relatively few electrostatic interactions. Using a 356-nt RNA construct that includes the negative regulatory element in addition to Psi, we observed a loss in Gag binding specificity and an increase in electrostatic interactions. Interestingly, a 400-nt construct that contains the positive and negative elements flanking Psi restored highly specific binding and reduced the electrostatic interactions made with the RNA. Furthermore, a construct wherein the 40-nt positive regulatory element was appended to Psi, demonstrated the same specificity as Psi alone. Taken together, these data are consistent with a mechanism whereby the negative and positive regulatory elements flanking Psi modulate Gag binding mode and specificity.
Description
Biological Sciences (The Ohio State University Denman Undergraduate Research Forum)
Keywords
HIV-1, Gag, 5'UTR, Psi Packaging Element