Design and preparation of the RNAs used in this study. (A) 5′LP mimics the packaging conformation of the HIV-1 gRNA 5′ leader. It is derived from nucleotides 105–344 of the NL4-3 strain genome and consists of U5, DIS, SD, Ψ, and AUG (U5: unique 5′; DIS: dimerization initiation site; SD: splice donor; Ψ: packaging signal stem–loop; AUG: start codon of gag). The palindromic sequence at DIS was mutated to GAGA to prevent dimerization. (B) 5′LT mimics the translation conformation. This RNA does not contain AUG and cannot form the U5:AUG interaction required for stabilizing the packaging conformation. The DIS region is retained to interact with U5. (Nonnative residues are in red.) (C) 5′LP and 5′LT showed good homogeneity on urea-PAGE and were eluted as monomers by size exclusion chromatography (SEC). A variant of 5′LP (5′LP-dimer) that retains the palindromic sequence was eluted as a dimer on SEC, as expected.
