Simultaneous recognition of HIV-1 TAR RNA bulge and loop sequences by cyclic peptide mimics of Tat protein

Abstract

The interaction of the HIV-1 transactivator protein Tat with its transactivation response (TAR) RNA is an essential step in viral replication and therefore an attractive target for developing antivirals with new mechanisms of action. Numerous compounds that bind to the 3-nt bulge responsible for binding Tat have been identified in the past, but none of these molecules had sufficient potency to warrant pharmaceutical development. We have discovered conformationally-constrained cyclic peptide mimetics of Tat that are specific nM inhibitors of the Tat-TAR interaction by using a structure-based approach. The lead peptides are nearly as active as the antiviral drug nevirapine against a variety of clinical isolates in human lymphocytes. The NMR structure of a peptide-RNA complex reveals that these molecules interfere with the recruitment to TAR of both Tat and the essential cellular cofactor transcription elongation factor-b (P-TEFb) by binding simultaneously at the RNA bulge and apical loop, forming an unusually deep pocket. This structure illustrates additional principles in RNA recognition: RNA-binding molecules can achieve specificity by interacting simultaneously with multiple secondary structure elements and by inducing the formation of deep binding pockets in their targets. It also provides insight into the P-TEFb binding site and a rational basis for optimizing the promising antiviral activity observed for these cyclic peptides.NMR ͉ transcription elongation factor-b ͉ antiviral ͉ Tat-TAR interaction ͉ RNA recognition T ranscription of the HIV-1 RNA in infected cells is strongly activated by the complex between the viral Tat protein and its cognate transactivation response (TAR) RNA, a 59-nt RNA found at the 5Ј end of all nascent viral transcripts (Fig. 1A). Tat and its cellular cofactor, the transcription elongation factor-b (P-TEFb), are recruited to the elongating RNA polymerase II (RNAP II) through interactions with TAR and are required for reactivation of the integrated proviral genome in latently infected cells (1). The cooperative binding of Tat and P-TEFb to TAR activates the CDK9 kinase of P-TEFb that phosphorylates RNAP II and the repressive NELF factors, leading to greatly enhanced RNAP II processivity (1-3).The Tat-TAR complex is an attractive target for developing new antivirals because the interaction between Tat and TAR is unique to the virus, whereas P-TEFb is used widely for transcription of most host genes. Furthermore, TAR is extremely conserved among viral isolates and P-TEFb plays a key role in promoting infectivity through the Tat-TAR complex and in the emergence from latency. These considerations have led to the synthesis and evaluation of numerous small-molecule and peptidic inhibitors of the Tat-TAR interaction during the last 15 years (4-7). However, none of these molecules had sufficient potency or selectivity to progress into preclinical studies and, indeed, inhibiting the Tat-TAR interaction has proven challenging. First, there is little precedent for the pharmacological disruption...

Invisible RNA state dynamically couples distant motifs

“…1A). The TAR bulge and apical loop form two distinct flexible sites for binding a variety of proteins (27, 28) as well as small molecules that are being developed as anti-HIV therapeutics (29,30). In the GS, apical loop residues C30, U31, G32, and A35 are exposed and available to interact with proteins (25, 26) (Fig.…”

Structure of a low-population binding intermediate in protein-RNA recognition

“…The HIV TAR-Tat interaction has been a subject of major attention in the past two decades, both for understanding the mechanism of transactivation and for development of anti-HIV therapeutics (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)30), and as a paradigm for the mechanism underlying protein-RNA recognition and signaling observed in a wide range of posttranscriptional regulatory processes. Our results reveal the structure of an intermediate in this interaction, illustrating how the use of RDCs as structural restraints in RAM simulations, particularly with further experimental validation through structure-based mutant design, provides a general strategy for obtaining high-resolution structures of low-population intermediates of RNA-protein complexes, which are very challenging for more conventional structure determination or dynamic techniques.…”

“…As a starting point for the calculations, and as a reference conformation to analyze the results, we used a previously determined structure of the HIV-1 TAR bound to a 14-residue, cyclic peptidomimetic of the HIV-1 Tat protein (PDB ID code 2KDQ) (20). In the absence of other binding (14,20).…”

“…As a starting point for the calculations, and as a reference conformation to analyze the results, we used a previously determined structure of the HIV-1 TAR bound to a 14-residue, cyclic peptidomimetic of the HIV-1 Tat protein (PDB ID code 2KDQ) (20). In the absence of other binding (14,20). Three distinct minima are observed: State I is the ground state of bound TAR with about 75% of the total sampled conformations, whereas states II and III are intermediate states with 15% and 7%, respectively, of the sampled conformations.…”

“…S1) with a highly dynamic structure (10,12,13). The NMR structures of free TAR (14)(15)(16) and of TAR bound to peptide fragments of Tat and to peptide mimetics of Tat in HIV (16)(17)(18)(19)(20) and other lentiviruses (21,22) revealed the conformational properties of TAR in its free and bound states, and demonstrated that this RNA molecule undergoes significant dynamic rearrangements associated with its functions. Although the TAR-Tat complex has become a paradigm for the widespread occurrence of conformational rearrangements and molecular adaptation in protein-RNA recognition, the pathway and intermediates linking the free and bound states of TAR are still unknown.…”