RNA SECONDARY STRUCTURE AND BINDING-SITES FOR GAG GENE-PRODUCTS IN THE 5'-PACKAGING SIGNAL OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1

The selective encapsidation of retroviral RNA requires sequences in th e Gag protein, as well as a cis-acting RNA packaging signal (Psi W sit e) near the 5' end of the genomic transcript. Gag protein of human imm unodeficiency virus type 1 (HIV-1) has recently been found to bind spe cifically to the HIV-1 Psi element in vitro. Here we report studies ai med at mapping features within the genetically defined yr locus that a re required for binding of HIV-1 Gag or of its processed nucleocapsid derivative. The full-length HIV-1 Gag (p55) and nucleocapsid (p15) seq uences were expressed as glutathione S-transferase (GST) fusion protei ns in Escherichia coli. In a gel shift assay containing excess competi tor tRNA, affinity-purified GST-p15 and GST-p55 proteins bound to a 20 6-nucleotide Psi RNA element spanning the major splice donor and gag s tart codons but did not bind to antisense Psi transcripts. Quantitativ e filter-binding assays revealed that both GST-p55 and GST-p15 bound t o this RNA sequence with identical affinities (apparent K-d similar or equal to 5 x 10(-8) M), indicating that all major determinants of Psi binding affinity reside within the nucleocapsid portion of Gag. Chemi cal and RNase accessibility mapping, coupled with computerized sequenc e analysis, suggested a model for Psi RNA structure comprising four in dependent stem-loops. Filter-binding studies revealed that RNAs corres ponding to three of these hypothetical stem-loops can each function as a independent Gag binding site and that each is bound with approximat ely fourfold-lower apparent affinity than the full-length Psi locus. I nteraction of Gag with these regions is likely to play a major role in directing HIV-1 RNA encapsidation in vivo.