Conserved nucleotides in the TAR RNA stem of human immunodeficiency virus type 1 are critical for Tat binding and trans activation: model for TAR RNA tertiary structure

Abstract

Interaction between the human immunodeficiency virus type 1 (HIV-1) trans-activator Tat and its cis-acting responsive RNA element TAR is necessary for activation of HIV-1 gene expression. We investigated the hypothesis that the essential uridine residue at position 23 in the bulge of TAR RNA is involved in intramolecular hydrogen bonding to stabilize an unique RNA structure required for recognition by Tat. Nucleotide substitutions in the two base pairs of the TAR stem directly above the essential trinucleotide bulge that maintain base pairing but change sequence prevent complex formation with Tat in vitro. Corresponding mutations tested in a trans-activation assay strongly affect the biological activity of TAR in vivo, suggesting an important role for these nucleotides in the Tat-TAR interaction. On the basis of these data, a model is proposed which implicates uridine 23 in a stable tertiary interaction with the GC pair directly above the bulge. This interaction would cause widening of the major groove of the RNA, thereby exposing its hydrogen-bonding surfaces for possible interaction with Tat. The model also predicts a gap between uridine 23 and the first base pair in the stem above, which would require one or more unpaired nucleotides to close, but does not predict any other role for such nucleotides. In accordance with this prediction, synthetic propyl phosphate linkers of equivalent length to 1 or 2 nucleotides, were found to be fully acceptable substitutes in the bulge above uridine 23, demonstrating that neither the bases nor the ribose moieties at these positions are implicated in the recognition of TAR RNA by Tat.

Evidence for a base triple in the free HIV-1 TAR RNA

“…It is therefore unlikely that these mutations have a severe impact on the interaction of TAR with Tat. Indeed, none of the mutants have a defect in binding of the Tat peptide, which is consistent with previous studies Crothers 1991, 1992;Delling et al 1992;Churcher et al 1993). This suggests that the bent TAR conformation with the (A22-U40)U31 triple affects steps in HIV-1 gene expression other than Tat-mediated trans-activation.…”

“…Additional titrations with this peptide and an Escherichia coli-expressed recombinant form of the Tat protein did not yield detectable differences in Tat-binding affinity (results not shown). This is consistent with previous studies in which similar TAR mutants were shown to have essentially no effect on Tat binding Crothers 1991, 1992;Delling et al 1992;Churcher et al 1993). Thus, we cannot correlate the defects in LTR promoter activity with reduced Tat affinity of the TAR mutants.…”

Direct interactions between autoantigen La and human immunodeficiency virus leader RNA

“…The trans-activation response element (TAR) is a leader RNA found in all human immunodeficiency virus (HIV) transcripts. It has a stem-bulge-loop secondary structure which is necessary for function (6,8,17). During the life cycle of HIV, the primary role for TAR is to serve as a high-affinity RNAtethering site for the viral transcription activating factor, Tat (10,15,19,20,75,86).…”

Face-time with TAR: Portraits of an HIV-1 RNA with diverse modes of effector recognition relevant for drug discovery

“…html) (30, 31) have led to a resurgence in efforts to target the transactivation response (TAR) 2 element. This 59-nucleotide RNA is located in the 5Ј-LTR of all viral transcripts and features a conserved hairpin that harbors an apical loop and pyrimidinerich bulge that are each indispensable for transactivation (33)(34)(35)(36)(37)(38)(39)(40). TAR interacts with the viral Tat protein, which recruits the host pTEFb complex away from inactivating HEXIM-7SK RNA complexes (Fig.…”