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Native mass spectrometry reveals the initial binding events of HIV-1 rev to RRE stem II RNA.

ABSTRACT: Nuclear export complexes composed of rev response element (RRE) ribonucleic acid (RNA) and multiple molecules of rev protein are promising targets for the development of therapeutic strategies against human immunodeficiency virus type 1 (HIV-1), but their assembly remains poorly understood. Using native mass spectrometry, we show here that rev initially binds to the upper stem of RRE IIB, from where it is relayed to binding sites that allow for rev dimerization. The newly discovered binding region implies initial rev recognition by nucleotides that are not part of the internal loop of RRE stem IIB RNA, which was previously identified as the preferred binding region. Our study highlights the unique capability of native mass spectrometry to separately study the binding interfaces of RNA/protein complexes of different stoichiometry, and provides a detailed understanding of the mechanism of RRE/rev association with implications for the rational design of potential drugs against HIV-1 infection.

SUBMITTER: Schneeberger EM

PROVIDER: S-EPMC7666190 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Native mass spectrometry reveals the initial binding events of HIV-1 rev to RRE stem II RNA.

Schneeberger Eva-Maria EM Halper Matthias M Palasser Michael M Heel Sarah Viola SV Vušurović Jovana J Plangger Raphael R Juen Michael M Kreutz Christoph C Breuker Kathrin K

Nature communications 20201113 1

Nuclear export complexes composed of rev response element (RRE) ribonucleic acid (RNA) and multiple molecules of rev protein are promising targets for the development of therapeutic strategies against human immunodeficiency virus type 1 (HIV-1), but their assembly remains poorly understood. Using native mass spectrometry, we show here that rev initially binds to the upper stem of RRE IIB, from where it is relayed to binding sites that allow for rev dimerization. The newly discovered binding regi ...[more]

PMID: 33188169

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Project description:A series of compounds that target reactive metal chelates to the HIV-1 Rev response element (RRE) mRNA have been synthesized. Dissociation constants and chemical reactivity toward HIV RRE RNA have been determined and evaluated in terms of reduction potential, coordination unsaturation, and overall charge associated with the metal-chelate-Rev complex. Ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) were linked to a lysine side chain of a Rev-derived peptide by either EDC/NHS or isothiocyanate coupling. The resulting chelate-Rev (EDTA-Rev, DTPA-Rev, NTA-Rev, and DOTA-Rev) conjugates were used to form coordination complexes with Fe(2+), Co(2+), Ni(2+), and Cu(2+) such that the arginine-rich Rev peptide could mediate localization of the metal chelates to the Rev peptide's high-affinity mRNA binding partner, RRE stem loop IIB. Metal complexes of the extended peptides GGH-Rev and KGHK-Rev, which also contain N-terminal peptidic chelators (ATCUN motifs), were studied for comparison. A fluorescence titration assay revealed high-affinity RRE RNA binding by all 22 metal-chelate-Rev species, with K(D) values ranging from ~0.2 to 16 nM, indicating little to no loss of RNA affinity due to the coupling of the metal chelates to the Rev peptide. Dissociation constants for binding at a previously unobserved low-affinity site are also reported. Rates of RNA modification by each metal-chelate-Rev species were determined and varied from ~0.28 to 4.9 nM/min but were optimal for Cu(2+)-NTA-Rev. Metal-chelate reduction potentials were determined and varied from -228 to +1111 mV vs NHE under similar solution conditions, allowing direct comparison of reactivity with redox thermodynamics. Optimal activity was observed when the reduction potential for the metal center was poised between those of the two principal co-reagents for metal-promoted formation of reactive oxygen species: E°(ascorbate/ascorbyl radical) = -66 mV and E°(H(2)O(2)/hydroxyl radical) = 380 mV. Given the variety of oxidative activities of these metal complexes and their high-affinity binding to the targeted RRE mRNA following coupling to the Rev peptide, this class of metal-chelate-Rev derivatives constitutes a promising step toward development of multiple-turnover reagents for selective eradication of HIV-1 RRE mRNA.

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Native mass spectrometry reveals the initial binding events of HIV-1 rev to RRE stem II RNA.

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Native mass spectrometry reveals the initial binding events of HIV-1 rev to RRE stem II RNA.

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