An RNA structural switch regulates diploid genome packaging by Moloney murine leukemia virus.
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 (Psi(WT); residues 147-623) also exhibits a dimerization-dependent affinity for NC, with the native dimer ([Psi(WT)](2)) binding 12+/-2 NC molecules with high affinity (K(d)=17+/-7 nM) and with the monomer, stabilized by substitution of dimer-promoting loop residues with hairpin-stabilizing sequences (Psi(M)), binding 1-2 NC molecules. Identical dimer-inhibiting mutations in MoMuLV-based vectors significantly inhibit genome packaging in vivo (approximately 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.
Miyazaki
Yasuyuki
Y
Howard Hughes Medical Institute and Department of Chemistry and Biochemistry, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, USA.
Garcia
Eric L
EL
King
Steven R
SR
Iyalla
Kilali
K
Loeliger
Kelsey
K
Starck
Patrice
P
Syed
Sameera
S
Telesnitsky
Alice
A
Summers
Michael F
MF
D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D019281:Dimerization; D004171:Diploidy; D016679:Genome, Viral / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000235:genetics* / Q000502:physiology*; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D019251:Nucleocapsid / Q000235:genetics; D020871:RNA Stability; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics*; D013696:Temperature; D014158:Transcription, Genetic; D019065:Virus Assembly / Q000235:genetics*
D012367:RNA, Viral