| Retroviruses selectively package two sense-strand viral genomic RNAs that are non-covalently linked at their 5' ends to form a complex, but structurally unresolved dimer. Determining the molecular basis for dimerization is an initial step in understanding the role of this dimer in retroviral biology and also in devising novel anti-viral and retroviral vector therapies. Initially, I define a 170-nucleotide Minimal D&barbelow;imerization A&barbelow;ctive S&barbelow;equence (MiDAS) for a representative gamma retrovirus, the Moloney murine sarcoma virus, by stringent competitive dimerization. I then analyze, at single-nucleotide resolution, the secondary structure of the MiDAS monomer and final dimer states using quantitative S&barbelow;elective 2'-H&barbelow;ydroxyl A&barbelow;cylation and P&barbelow;rimer E&barbelow;xtension (SHAPE) chemistry. SHAPE analysis characterizes two novel structural features within the monomer and dimer states. (i) Both the monomer and dimer contain partially overlapping, but distinct flexible domains spanning 63 and 58 nucleotides, respectively. (ii) The well conserved SL1-SL2 domain that consists of two GACG-containing stem loop structures undergoes a modest conformational RNA switch in which SL1 is extended by four base-pairs. I then analyze the structural implications of both SL1-SL2 conformations and identify that this domain can mediate specific interactions between RNA strands in the final dimer by a two-step mechanism. Finally, using site-directed and solvent-based hydroxyl radical footprinting, I show that the MiDAS final dimer adopts a compact three-dimensional structure containing multiple long-range tertiary interactions. Moreover, within the dimer, the SL1-SL2 domain folds autonomously into a tertiary U-shaped structure stabilized by interdigitated interactions. |
