Structure of a Conserved Retroviral RNA Packaging Element by NMR Spectroscopy and Cryo-Electron Tomography

The 5'-untranslated regions of all gammaretroviruses contain a conserved double-hairpin motif (Psi(CD)) that is required for genome packaging Both hairpins (SL-C and SL-D) contain GACG tetraloops that, in isolated RNAs, are capable of forming kissing interactions stabilized by two intermolecular G C base pairs We have determined the three-dimensional structure of the double hairpin from the Moloney murine leukemia virus ([Psi(CD)](2), 132 nt, 428 kDa) using a H-2-edited NMR-spectroscopy-based approach This approach enabled the detection of H-1-H-1 dipolar interactions that were not observed in previous studies of isolated SL-C and SL-D hairpin RNAs using traditional H-1-H-1 correlated and H-1-C-13-edited NMR methods The hairpins participate in intermolecular cross-kissing interactions (SL-C to SL-D' and SLC' to SL-D) and stack in an end-to-end manner (SL-C to SL-D and SL-C' to SL-D') that gives rise to an elongated overall shape (ca 95 angstrom x 45 angstrom x 25 angstrom) The global structure was confirmed by cryo-electron tomography (cryo-ET), making [Psi(CD)]2 simultaneously the smallest RNA to be structurally characterized to date by cryo-ET and among the largest to be determined by NMR Our findings suggest that, in addition to promoting dimenzahon, [Psi(CD)](2) functions as a scaffold that helps initiate virus assembly by exposing a cluster of conserved UCUG elements for binding to the cognate nucleocapsid domains of assembling viral Gag proteins (C) 2010 Elsevier Ltd All nghts reserved