BENT PSEUDOKNOTS AND NOVEL RNA INHIBITORS OF TYPE-1 HUMAN-IMMUNODEFICIENCY-VIRUS (HIV-1) REVERSE-TRANSCRIPTASE

The reverse transcriptase (RT) of the human immunodeficiency virus (HI V) is a proven target for therapeutic intervention of HIV infections. We have found several new RNA inhibitors of HIV-1 RT that differ signi ficantly from the pseudoknot ligands found previously, along with a wi de variety of pseudoknot variants. One pseudoknot variant and three no vel ligands were studied in more detail. Each specifically inhibits DN A polymerization by HIV RT (half-maximal inhibition at 0.3 to 20 nM in hibitor), but not that of RTs derived from MMLV or AMV. The minimal bi nding element of each isolate was determined by deletion analysis and by gel electrophoresis of protein-bound, partially alkaline-hydrolyzed RNA. Truncations of three of the isolates bound nearly as well as (or better than) the parental sequences, while most deletions in the four th caused substantial disruption of binding. The truncated versions of two isolates were subjected to six rounds of secondary SELEX after re synthesizing them mutagenically. Patterns of conserved and covarying n ucleotides yielded structural models consistent with 5' and 3' boundar y determinations for these molecules. Among the four isolates studied in detail, the first is confirmed as being a pseudoknot, albeit with s ubstantial structural differences as compared to the canonical pseudok nots identified previously. The second forms a stem-loop structure wit h additional flanking sequences required for binding. Tentative struct ural models for the other two isolates are presented. The minimal full y active truncations of each of these four isolates compete with each other and with a classical RNA pseudoknot for binding to HIV RT, sugge sting that they all recognize the same or overlapping sites on the pro tein, in spite of their apparently dissimilar structures. We model the ir interactions with RT as mimicking the 40 to 45 degree bend in dsDNA co-crystallized with RT. (C) 1996 Academic Press Limited