IN-VITRO SELECTION OF HIV-1 TAR VARIANTS BY THE TAT PROTEIN

Starting from a pool of 10(13) RNA sequences, vie isolated a number of TAR RNA variants after nine rounds of selection by binding to recombi nant Tat in vitro (SELEX procedure). Sequence analysis of part of the selected molecular species indicated that two TAR variants (clones A a nd B) were, respectively, represented five and four times. These two g roups of sequences constituted approximately 25% of the total number o f analyzed clones (9/34). As far as the primary and presumptive second ary structures of the wild-type TAR are concerned, the selected A and B variants showed an almost complete sequence conservation of the Tat- binding domain, but the configuration of this nucleotide region differ ed within the secondary structure. Despite this difference, as verifie d by gel retardation and filter binding assays, both the A and B varia nts bound Tat in vitro with an affinity that was very close to that of the wild-type TAR. Conversely, neither variant sustained Tat-mediated transactivation in vivo when they replaced the wild-type TAR inside t he long terminal repeat of HIV-1. Taken together, our results suggest that these TAR variants have lost the ability to bind cell factor(s) i n vivo and may therefore represent useful decoys for the inhibition of HIV-1 replication. (C) 1998 Elsevier Science B.V. All rights reserved .