title: Structure and stability of wild-type and mutant RNA internal loops from the SL-1 domain of the HIV-1 packaging signal.
abstract: The packaging signal (Psi) of the human immunodeficiency virus type 1 (HIV-1) enables encapsidation of the full-length genomic RNA against a background of a vast excess of cellular mRNAs. The core HIV-1 Psi is approximately 109 nucleotides and contains sequences critical for viral genomic dimerisation and splicing, in addition to the packaging signal. It consists of a series of stem-loops (termed SL-1 to SL-4), which can be arranged in a cloverleaf secondary structure. Using a combination of NMR spectroscopy, UV melting experiments, molecular modeling and phylogenetic analyses, we have explored the structure of two conserved internal loops proximal to the palindromic sequence of SL-1. Internal loop A, composed of six purines, forms a flexible structure that is strikingly similar to the Rev responsive element motif when bound to Rev protein. This result suggests that it may function as a protein-binding site. The absolutely conserved four-purine internal loop B is instead conformationally and thermodynamically unstable, and exhibits multiple conformations in solution. By introducing a double AGG to GGA mutation within this loop, its conformation is stabilised to form a new intra-molecular G:A:G base-triplet. The structure of the GGA mutant explains the relative instability of the wild-type loop. In a manner analogous to SL-3, we propose that conformational flexibility at this site may facilitate melting of the structure during Gag protein capture or genomic RNA dimerisation.
authors.affiliation: Department of Medicine, University of Cambridge, Level 5, Addenbrookes Hospital, Hills Road, CB2 2QQ, Cambridge, UK.
authors.forename: Jane
authors.initials: J
authors.lastname: Greatorex
authors.forename: José
authors.initials: J
authors.lastname: Gallego
authors.forename: Gabriele
authors.initials: G
authors.lastname: Varani
authors.forename: Andrew
authors.initials: A
authors.lastname: Lever
mesh_terms: D020029:Base Pairing; D001665:Binding Sites; D015683:Gene Products, gag / Q000378:metabolism; D016679:Genome, Viral; D015497:HIV-1 / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011687:Purines / Q000378:metabolism; D020871:RNA Stability; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D019065:Virus Assembly
chemical_list: D015683:Gene Products, gag; D011687:Purines; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase; D012260:Ribonucleases