THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 ENCAPSIDATION SITE IS A MULTIPARTITE RNA ELEMENT COMPOSED OF FUNCTIONAL HAIRPIN STRUCTURES

We analyzed the leader region of human immunodeficiency virus type 1 ( HIV-1) RNA to decipher the nature of the cis-acting E/Psi element requ ired for encapsidation of viral RNA into virus particles. Our data ind icate that, for RNA encapsidation, there are at least two functional s ubregions in the leader region. One subregion is located at a position immediately proximal to the major splice donor, and the second is loc ated between the splice donor and the beginning of the gag gene. This suggests that at least two discrete cis-acting elements are recognitio n signals for encapsidation. To determine whether specific putative RN A secondary structures serve as the signal(s) for encapsidation, we co nstructed primary base substitution mutations that would be expected t o destabilize these potential structures and second-site compensatory mutations that would restore secondary structure. Analysis of these mu tants allowed the identification of two discrete hairpins that facilit ate RNA encapsidation in vivo. Thus, the HIV-1 E/Psi region is a multi partite element composed of specific and functional RNA secondary stru ctures. Compensation of the primary mutations by the second-site mutat ions could not be attained in trans. This indicates that interstrand b ase pairing between these two stem regions within the hairpins does no t appear to be the basis for HIV-1 RNA dimer formation. Comparison of the hypothetical RNA secondary structures from 10 replication-competen t HIV-1 strains suggests that a subset of the hydrogen-bonded base pai rs within the stems of the hairpins is likely to be required for funct ion in cis.