Altered‐specificity mutants of the HIV Rev arginine‐rich motif‐RRE IIB interaction. Issue 6 (10th January 2020)

• Record Type: Journal Article
• Authors: Raad, Nicole G.; Ghattas, Ingrid R.; Amano, Ryo; Watanabe, Natsuki; Sakamoto, Taiichi; Smith, Colin A.
• Is Part Of: Journal of molecular recognition. Volume 33:Issue 6(2020)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2020-01-10
• Subjects: altered specificity -- arginine‐rich motif -- disordered protein -- fitness landscape -- HIV Rev -- neutral evolution -- RNA‐protein interaction; Molecular recognition -- Periodicals; Models, Molecular -- Periodicals; Molecular Conformation -- Periodicals; Molecular Sequence Data -- Periodicals; Molecular Structure -- Periodicals; Carrier Proteins -- Periodicals; 572.8
• DOI: 10.1002/jmr.2833
• Languages: English
• ISSNs: 0952-3499

Abstract

Abstract: Arginine‐rich motifs (ARMs) bind RNA structures with high affinity and specificity, and the human immunodeficiency virus (HIV) exploits ARM‐RNA interactions to regulate its lifecycle. The expression of HIV structural genes relies on recognition between the ARM of its Rev protein and its primary binding site, an internal loop in the viral RNA, the Rev‐response element region IIB (IIB). Many functional variants of the Rev ARM‐IIB interaction have been discovered, yet how easily it can evolve new specificities is poorly explored. A double mutant of Rev ARM, R35G‐N40 V, uses an unknown strategy to recognize IIB. Here, isothermal titration calorimetry and gel shift assays show that the R35G‐N40V‐IIB interaction has high affinity and specificity in vitro and a larger unfavorable entropy change upon binding than that of wild‐type Rev ARM‐IIB. In stark contrast with the critical dependence of wild‐type Rev on Arg35, Arg39, Asn40, and Arg44, mutational profiling shows R35G‐N40V is highly mutable at positions 40 and 44 and dependent on Gly35, Arg38, Arg39, Arg42, and Arg43. Affinity measurements in vitro and reporter assay measurements in vivo are consistent with the wild‐type Rev ARM and R35G‐N40V maintaining their recognition strategies when binding IIB mutants specific to wild‐type Rev ARM and R35G‐N40V, respectively. Some single amino acid mutants of wild‐type Rev ARM and R35G‐N40V have enhanced specificity, recognizing mutant IIBs yet not wild‐type IIB. These results provide another example of viral ARM‐RNA interactions evolving new specificities with few mutations, consistent with neutral theories of evolution. Abstract : Isothermal titration calorimetry and gel shift assays show that the interaction of HIV Rev R35G‐N40V and RRE IIB has high affinity and specificity in vitro. In stark contrast with the critical dependence of wild‐type Rev on Arg35, Arg39, Asn40, and Arg44, R35G‐N40V is mutable at positions 40 and 44 and dependent on Gly35, Arg38, Arg39, Arg42, and Arg43. Some single amino acid mutants of wild‐type Rev ARM and R35G‐N40V have enhanced specificity, recognizing mutant IIBs yet not wild‐type IIB.