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

Eva-Maria Schneeberger,Matthias Halper,Raphael Plangger,Jovana Vušurović,Sarah Viola Heel,Michael Palasser,Christoph Kreutz,Kathrin Breuker,Michael Juen

doi:10.1038/s41467-020-19144-7

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.

Highlights

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
The RRE RNA constructs RRE-II-0, RRE-IIB-0, and RRE-TR-0 (Fig. 1) instead of fulllength RRE RNA (~350 nt) and rev arginine-rich motif (ARM) peptide[24] instead of rev protein (116 residues) were used in this study to limit the mass of the RRE/rev complexes, such that the signals of all fragments from collisionally activated dissociation (CAD) were isotopically resolved on our 7 T FT-ICR mass spectrometer for unambiguous assignment of fragment identity
Our mass spectrometry (MS) data provide meaningful insight into the assembly of the functional RRE/rev ribonucleoprotein complex as the interaction of rev protein with RRE RNA is modular, i.e., rev ARM peptide molecules bind to RRE RNA with the same affinity as rev protein monomer, and requires the same amino acids for recognition[31]

Summary

Introduction

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. 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]. Both tat and rev are critical to the expression of viral proteins and HIV-1 replication[17]. The as yet unresolved challenge to correlate binding regions with individual rev molecules that successively bind to RRE RNA complicates the rational design of highly efficient drugs for the treatment of HIV-1 infection

Methods

Results

Conclusion