NONCANONICAL INTERACTIONS IN A KISSING LOOP COMPLEX - THE DIMERIZATION INITIATION SITE OF HIV-1 GENOMIC RNA

Retroviruses encapsidate two molecules of genomic RNA that are noncova lently Linked close to their 5' ends in a region called the dimer link age structure (DLS). The dimerization initiation site (DIS) of human i mmunodeficiency virus type 1 (HIV-1) constitutes the essential part of the DLS in vitro and is crucial for efficient HIV-1 replication in ce ll culture. We previously identified the DIS as a hairpin structure, l ocated upstream of the major splice donor site, that contains in the l oop a six-nucleotide self-complementary sequence preceded and followed by two and one purines, respectively: Two RNA monomers form a kissing loop complex via intermolecular interactions of the six nucleotide se lf-complementary sequence. Here, we introduced compensatory mutations in the self-complementary sequence and/or a mutation in the flanking p urines. We determined the kinetics of dimerization, the thermal stabil ities and the apparent equilibrium dissociation constants of wild-type and mutant dimers and used chemical probing to obtain structural info rmation. Our results demonstrate the importance of the 5'-flanking pur ine and of the two central bases of the self-complementary sequence in the dimerization process. The experimental data are rationalized by t riple interactions between these residues in the deep groove of the ki ssing helix and are incorporated into a three-dimensional model of the kissing loop dimer. In addition, chemical probing and molecular model ing favor the existence of a non-canonical interaction between the con served adenine residues at the first and last positions in the DIS loo p. Furthermore, we show that destabilization of the kissing loop compl ex at the DIS can be compensated by interactions involving sequences l ocated downstream of the splice donor site of the HIV-1 genomic RNA. ( C) 1997 Academic Press Limited.