Retroviruses selectively package two copies of their RNA genomes via mechanisms that have yet to be fully deciphered. Recent studies with small fragments of the Moloney murine leukemia virus (MoMuLV) genome suggested that selection may be mediated by an RNA switch mechanism, in which conserved UCUG elements that are sequestered by base-pairing in the monomeric RNA become exposed upon dimerization to allow binding to the cognate nucleocapsid (NC) domains of the viral Gag proteins. Here we show that a large fragment of the MoMuLV 5′ untranslated region that contains all residues necessary for efficient RNA packaging (ΨWT; residues 147-623) also exhibits a dimerization-dependent affinity for NC, with the native dimer ([ΨWT]2) binding 12 ± 2 NC molecules with high affinity (Kd = 17 ± 7 nM) and with the monomer, stabilized by substitution of dimer-promoting loop residues with hairpin-stabilizing sequences (ΨM), binding 1-2 NC molecules. Identical dimer-inhibiting mutations in MoMuLV-based vectors significantly inhibit genome packaging in vivo (∼ 100-fold decrease), whereas a large deletion of nearly 200 nucleotides just upstream of the gag start codon has minimal effects. Our findings support the proposed RNA switch mechanism and further suggest that virus assembly may be initiated by a complex comprising as few as 12 Gag molecules bound to a dimeric packaging signal.
|Number of pages||12|
|Journal||Journal of Molecular Biology|
|State||Published - Feb 12 2010|
Bibliographical noteFunding Information:
Support from the National Institutes of Health (grant GM42561 to M.F.S. and grant CA069300 to A.T.) is gratefully acknowledged. P.S. and S.S. were supported by a Howard Hughes Medical Institute undergraduate education grant (to University of Maryland Baltimore County).
- RNA structure
- diploid genome packaging
- human cancer
- nucleocapsid protein
- retrovirus assembly
ASJC Scopus subject areas
- Structural Biology
- Molecular Biology