Likewise, Le Grice stated that the sequence of events required for rev assembly and the positioning of individual rev molecules on the RRE remain unknown, and that the role of the three-way junction from which stem IIB originates in binding of the second rev molecule is unclear 15. However, as pointed out by Frankel, structural details on the formation of the ribonucleoprotein complex remain largely elusive, and one particularly important question is how the individual subunits of rev recognize the RRE 4.

The N-terminal ARM binding region (residues 34вЂ“50, 11 of which are arginine residues) of rev (116 residues) is framed by oligomerization domains (residues 9вЂ“26, 51вЂ“65) that stabilize the ribonucleoprotein complex 15. Previous studies have identified nucleotides in stem II of RRE RNA 18, 19, 20, 21 to which the arginine-rich motif (ARM) of rev protein binds 19 in the assembly of the functional RRE/rev ribonucleoprotein complex in which up to 8вЂ“10 rev molecules sequentially associate with a single RRE molecule 22, 23. Both tat and rev are thus critical to the expression of viral proteins and HIV-1 replication 17. While tat binding to TAR RNA increases the production of viral messenger RNA (mRNA) 14, rev protein binding to rev response element (RRE) segments of unspliced and singly spliced viral mRNA facilitates their nuclear export 15, 16. This native top-down MS approach relies on the preservation of electrostatic interactions between protonated peptides and deprotonated RNA at energies that are sufficiently high for phosphodiester backbone bond cleavage 6, for which as few as two arginine residues are sufficient 13. We have recently demonstrated that native electrospray ionization (ESI) 7, 8, 9, 10, 11, 12 provides time-resolved, stoichiometric information on complexes of transactivation responsive (TAR) RNA and trans-activating (tat) peptide from human immunodeficiency virus type 1 (HIV-1), and that collisionally activated dissociation (CAD) can reveal tat binding sites of TAR RNA at the single-nucleotide level 6. An emerging alternative that can complement these techniques, especially when RNA-protein complexes show unfavorable conformational dynamics that complicate NMR data interpretation 2, 3 or fail to crystallize properly 4, 5, is native top-down mass spectrometry (MS) 6. To gain insight into the biochemistry of retroviruses and further antiviral drug development, atomic-level details of RNA-protein complex structures and their binding interfaces can be studied by nuclear magnetic resonance (NMR) spectroscopy or X-ray crystallography. Interactions between proteins and ribonucleic acids (RNA) play important roles in biological processes including retroviral replication 1. 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. 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. 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.

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.

Stems 65 free native instruments