Positive and Negative Regulatory Elements in the HIV-1 5'UTR Control Specific Recognition by Gag
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Date
2016-09-15
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Abstract
The highly conserved 5ʹ untranslated region (5ʹUTR) of the human immunodeficiency virus type 1 (HIV-1) genomic RNA (gRNA) regulates numerous aspects of the viral lifecycle. The 5'UTR contains a structured RNA element (termed the Psi packaging signal) 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 specifically recognizes the gRNA over the myriad of other cellular RNAs and spliced viral RNAs is not well understood. A recent study suggested that a negative regulatory element exists upstream of Psi that blocks high-affinity Gag binding, and a positive regulatory element downstream of Psi counteracts the upstream element and restores high-affinity binding. This mechanism would help ensure the preferential recognition of gRNA over spliced viral RNAs by Gag. However, this study did not directly address how the upstream and downstream elements effect the specificity or conformation of Gag binding. To further elucidate the details of this mechanism, we employ a fluorescence anisotropy-based salt-titration binding assay to determine the electrostatic and nonelectrostatic (i.e., specific) components of binding. We have previously shown that Gag interacts with a 100-nucleotide Psi RNA construct that lacks the putative regulatory elements with high specificity and few electrostatic interactions. We now find that Gag binding to nucleotides 1-356 of the gRNA, which contains Psi and the negative regulatory element, is characterized by lower specificity and a greater number of electrostatic interactions, indicating an altered mode of binding. Interestingly, a 400-nucleotide RNA, which contains Psi and both the negative and positive regulatory elements, restores the highly specific Psi-like binding mode. Taken together, these data are consistent with the conclusion that positive and negative regulatory elements flank Psi and serve to modulate gRNA recognition by Gag.
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Biological and Biomedical Sciences
Keywords
HIV-1, 5'UTR, Gag, Psi Packaging Element