Poster abstracts
Poster number 108 submitted by Joshua-Paolo Reyes
Regulatory elements in the HIV-1 5´UTR modulate Gag binding specificity
Joshua-Paolo C. Reyes (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210), Erik D. Olson (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210)
Abstract:
The 5ʹUTR of the 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 copies 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 downstream regulatory element counteracts the upstream element and restores high-affinity binding1,2. 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 interactions3. Using a 356-nt RNA construct that now includes the upstream negative regulatory element in addition to Psi, we observed a loss in Gag binding specificity and an increase in electrostatic interactions. However, a 400-nt construct that additionally contains the positive element, restored highly specific binding and reduced the electrostatic interactions, similar to the 109-nt Psi RNA. Interestingly, Gag binding specificity remained high even in constructs wherein the 44 nt of the downstream positive regulatory element were scrambled. Thus, while the presence of downstream sequences appears to be important for high-specificity Gag binding, their identity is not. Taken together, these data are consistent with a mechanism whereby the negative and positive regulatory elements flanking Psi modulate Gag binding but call into question the long-range interaction model.
References:
1Abd El-Wahab, E. W., Smyth, R. P., Mailler, E., Bernacchi, S., Vivet-Boudou, V., Hijnen, M. et al. Specific recognition of the HIV-1 genomic RNA by the Gag precursor. (2014) Nat Commun 5, 4304.
2Bernacchi, S., Abd El-Wahab, E. W., Dubois, N., Hijnen, M. E. W., Smyth, R. P. et al. HIV-1 Pr55Gag binds genomic and spliced RNAs with different affinity and stoichiometry. (2017) RNA Biology 14:1, 90-103
3Webb J. A., Jones C. P., Parent L. J., Rouzina I, Musier-Forsyth K. Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: implications for viral genomic RNA packaging. (2013) RNA 19, 1078-88.
Keywords: HIV-1 Gag, HIV-1 Psi, gRNA Packaging