Identification of a high affinity nucleocapsid protein binding element within the Moloney murine leukemia virus Psi-RNA packaging signal: Implications for genome recognition

Murine leukemia virus (MLV) is currently the most widely used gene delivery system in gene therapy trials. The simple retrovirus packages two copies o f its RNA genome by a mechanism that involves interactions between the nucl eocapsid (NC) domain of a virally-encoded Gag polyprotein and a segment of the RNA genome located just upstream of the Gag initiation codon, known as the psi -site. Previous studies indicated that the MLV psi -site contains t hree stem loops (SLB-SLD), and that stem loops SLC and SLD play prominent r oles in packaging. We have developed a method for the preparation and purif ication of large quantities of recombinant Moloney MLV NC protein, and have studied its interactions with a series of oligoribonucleotides that contai n one or more of the psi -RNA stem loops, At RNA concentrations above simil ar to0.3 mM, isolated stem loop SLB forms a duplex and stem loops SL-C and SL-D form kissing complexes, as expected from previous studies. However, ne ither the monomeric nor the dimeric forms of these isolated stem loops bind s NC with significant affinity. Longer constructs containing two stem loops (SL-BC and SL-CD) also exhibit low affinities for NC. However, NC binds wi th high affinity and stoichiometrically to both the monomeric and dimeric f orms of an RNA construct that contains all three stem loops (SL-BCD; K-d = 132(+/- 55) nM). Titration of SL-BCD with NC also shifts monomer-dimer equi librium toward the dimer. Mutagenesis experiments demonstrate that the cons erved GACG tetraloops of stem loops C and D do not influence the monomer-di mer equilibrium of SL-BCD, that the tetraloop of stem loop B does not parti cipate directly in NC binding, and that the tetraloops of stem loops C and D probably also do not bind to NC. These surprising results differ consider ably from those observed for HIV-1, where NC binds to individual stem loops with high affinity via interactions with exposed residues of the tetraloop s. The present results indicate that MLV NC binds to a pocket or surface th at only exists in the presence of all three stem loops. (C) 2001 Academic P ress.