A novel combined RNA-protein interaction analysis distinguishes HIV-1 Gag protein binding sites from structural change in the viral RNA leader

Abstract

RNA-protein interactions govern many viral and host cell processes. Conventional ‘footprinting’ to examine RNA-protein complex formation often cannot distinguish between sites of RNA-protein interaction and sites of RNA structural remodelling. We have developed a novel technique combining photo crosslinking with RNA 2′ hydroxyl reactivity (‘SHAPE’) that achieves rapid and hitherto unachievable resolution of both RNA structural changes and the sites of protein interaction within an RNA-protein complex. ‘XL-SHAPE’ was validated using well-characterized viral RNA-protein interactions: HIV-1 Tat/TAR and bacteriophage MS2 RNA/Coat Binding Protein. It was then used to map HIV-1 Gag protein interactions on 2D and 3D models of the viral RNA leader. Distinct Gag binding sites were identified on exposed RNA surfaces corresponding to regions identified by mutagenesis as important for genome packaging. This widely applicable technique has revealed a first view of the stoichiometry and structure of the initial complex formed when HIV captures its genome.

Human T-cell leukemia virus type 1 Gag domains have distinct RNA-binding specificities with implications for RNA packaging and dimerization

“…5A). This is similar to the destabilization observed throughout the HIV-1 5Ј leader upon HIV-1 Gag binding as elucidated by XL-SHAPE (48). BLV MA-gRNA interaction sites have previously been mapped to regions spanning the PBS to the 5Ј-terminal 60 residues in the gag coding region (29,79), which are very similar to the probing results for HTLV-1 MA reported here.…”

“…Cross-linking with SHAPE (XL-SHAPE) combines two complementary techniques, SHAPE and UV cross-linking, to allow simultaneous identification of protein-binding sites as well as any concurrent RNA conformational changes associated with the interaction (48). SHAPE allows for identification of secondary structure changes or sites of increased or decreased flexibility upon protein binding, whereas UV cross-linking identifies specific nt that are in very close proximity to a bound protein.…”

HIV-1 Pr55Gagbinds genomic and spliced RNAs with different affinity and stoichiometry

“…This result together with previous findings clearly points out how long-range interactions are necessary for gRNA specific encapsidation. 20,21,23,24,32,[39][40][41][42]69,79,107,108 Even though further analysis will be necessary to identify the nucleotides that are involved, our model indicates that this interaction is not possible in svRNAs (which differ from gRNA downstream of SL2, Fig. 2), and this could impact on Pr55 Gag discrimination between unspliced gRNA and svRNAs.…”

“…66,67,73 A major difficulty in deciphering the mechanisms by which Pr55 Gag selects HIV-1 gRNA has been the expression and purification of full-length Pr55 Gag , which is notoriously sensitive to proteolytic cleavage during bacterial expression. 47 Most previous studies were therefore performed using truncated forms of Pr55 Gag lacking the p6 domain (GagDp6) 51,64,[74][75][76][77][78][79] and/or fused to GST 2,3,65,66,78,80 or the isolated NC domain. 64,[81][82][83] We recently succeeded in purifying large amounts of intact full length Pr55 Gag , 84 allowing us to characterize its interaction with a large panel of vRNA fragments.…”

An Improved Prediction Model for Zinc-Binding Sites in Proteins Based on Bayesian Method

“…The products of the interaction, known as metalloproteins, have important biological functions. The research into this interaction can greatly promote disease treatment and drug development [3,4]. In the Protein Data Bank (PDB) [5], more than one third of proteins combine with metal ions.…”