In vitro selection of HIV-1 TAR variants by the Tat protein.
Starting from a pool of 10(13) RNA sequences, we isolated a number of TAR RNA variants after nine rounds of selection by binding to recombinant Tat in vitro (SELEX procedure). Sequence analysis of part of the selected molecular species indicated that two TAR variants (clones A and B) were, respectively, represented five and four times. These two groups of sequences constituted approximately 25% of the total number of analyzed clones (9/34). As far as the primary and presumptive secondary 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 differed within the secondary structure. Despite this difference, as verified by gel retardation and filter binding assays, both the A and B variants bound Tat in vitro with an affinity that was very close to that of the wild-type TAR. Conversely, neither variant sustained Tat-mediated trans-activation in vivo when they replaced the wild-type TAR inside the long terminal repeat of HIV_1. Taken together, our results suggest that these TAR variants have lost the ability to bind cell factor(s) in vivo and may therefore represent useful decoys for the inhibition of HIV-1 replication.
Marozzi
A
A
Dipartimento di Biologia e Genetica per le Scienze Mediche, Università di Milano, Italy.
Meneveri
R
R
Giacca
M
M
Gutierrez
M I
MI
Siccardi
A G
AG
Ginelli
E
E
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001709:Biotechnology; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D015696:Gene Products, tat / Q000235:genetics* / Q000378:metabolism; D014644:Genetic Variation; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection; D054322:tat Gene Products, Human Immunodeficiency Virus
D017931:DNA Primers; D015696:Gene Products, tat; D012367:RNA, Viral; D054322:tat Gene Products, Human Immunodeficiency Virus