HIV-1 Transactivation Response RNA Research Articles

Co-crystal structures of HIV TAR RNA bound to lab-evolved proteins show key roles for arginine relevant to the design of cyclic peptide TAR inhibitors

RNA-protein interfaces control key replication events during the HIV-1 life cycle. The viral trans-activator of transcription (Tat) protein uses an archetypal arginine-rich motif (ARM) to recruit the host positive transcription elongation factor b (pTEFb) complex onto the viral trans-activation response (TAR) RNA, leading to activation of HIV transcription. Efforts to block this interaction have stimulated production of biologics designed to disrupt this essential RNA-protein interface. Here, we present four co-crystal structures of lab-evolved TAR-binding proteins (TBPs) in complex with HIV-1 TAR. Our results reveal that high-affinity binding requires a distinct sequence and spacing of arginines within a specific β2-β3 hairpin loop that arose during selection. Although loops with as many as five arginines were analyzed, only three arginines could bind simultaneously with major-groove guanines. Amino acids that promote backbone interactions within the β2-β3 loop were also observed to be important for high-affinity interactions. Based on structural and affinity analyses, we designed two cyclic peptide mimics of the TAR-binding β2-β3 loop sequences present in two high-affinity TBPs (KD values of 4.2 ± 0.3 and 3.0 ± 0.3 nm). Our efforts yielded low-molecular weight compounds that bind TAR with low micromolar affinity (KD values ranging from 3.6 to 22 μm). Significantly, one cyclic compound within this series blocked binding of the Tat-ARM peptide to TAR in solution assays, whereas its linear counterpart did not. Overall, this work provides insight into protein-mediated TAR recognition and lays the ground for the development of cyclic peptide inhibitors of a vital HIV-1 RNA-protein interaction.

Structural Characterization of a Peptide Derived from a Lab-Evolved Protein that Targets HIV-1 TAR RNA

Innovative approaches are needed to create new therapeutics that target HIV, especially those that act on novel facets of the viral life cycle. The HIV-1 TAR (trans-activation response) element RNA is a validated drug target that resists mutations to maintain interactions with the viral protein Tat and the host pTEFb complex, giving rise to a protein-RNA complex that is essential for efficient proviral transcription. So far, TAR has evaded discovery of compounds with sufficient affinity and selectivity to warrant pharmaceutical development. To meet this challenge, we pursued a ‘semi-design and protein evolution’ approach that yielded many high-affinity TAR Binding Proteins (TBPs) derived from RRM1 of the U1A spliceosomal protein. Here we describe the lab-evolution results and present the structural analysis of TBP6.7 in complex with TAR at 1.8Å. TBP6.7 recognizes conserved guanines in the TAR major groove using a constellation of arginines within the beta2-beta3 loop. We demonstrate the feasibility of reducing TBPs to short a cyclic peptide (peptide 1) that retains TAR binding and inhibit Tat-mediated transcription in HeLa cell nuclear lysate. In addition, TBP6.7(Q48T) shows that a mutation in the beta2-beta3 loop yields additional interactions between K20, K22 and N15 of the β1-α1 loop and the TAR bulge. Concurrently, we also present a crystallization module validated by isothermal titration calorimetry (ITC), which will be used for co-crystallizing TAR RNA with peptides and small molecules. Future efforts will involve optimization of peptide 1, biophysical characterization of optimized peptides and co-crystallization with TAR RNA.

Exosomes derived from HIV-1-infected cells promote growth and progression of cancer via HIV TAR RNA

People living with HIV/AIDS on antiretroviral therapy have increased risk of non-AIDS-defining cancers (NADCs). However, the underlying mechanism for development and progression of certain NADCs remains obscure. Here we show that exosomes released from HIV-infected T cells and those purified from blood of HIV-positive patients stimulate proliferation, migration and invasion of oral/oropharyngeal and lung cancer cells. The HIV transactivation response (TAR) element RNA in HIV-infected T-cell exosomes is responsible for promoting cancer cell proliferation and inducing expression of proto-oncogenes and Toll-like receptor 3 (TLR3)-inducible genes. These effects depend on the loop/bulge region of the molecule. HIV-infected T-cell exosomes rapidly enter recipient cells through epidermal growth factor receptor (EGFR) and stimulate ERK1/2 phosphorylation via the EGFR/TLR3 axis. Thus, our findings indicate that TAR RNA-containing exosomes from HIV-infected T cells promote growth and progression of particular NADCs through activation of the ERK cascade in an EGFR/TLR3-dependent manner.

Abstract 5060: Exosomes derived from HIV-infected T cells promote cancer cell growth and progression via the HIV TAR RNA

Abstract Background: Residual and persistent HIV replication plays key roles in the development and progression of non-AIDS-defining cancers (NADCs), including head and neck as well as lung cancers. However, the underlying mechanisms remain elusive. HIV-infected T cells produce immunologically active exosomes to influence intercellular communication and regulate immune response at both local and distant sites, thus potentially contribute to enhanced risk for tumorigenesis. We investigated the role of exosomes derived from HIV-infected T cells in growth and progression of head and neck cancer (HNC) and lung cancer cells. Methods: Exosomes were isolated from conditioned media of HIV-1-infected J1.1 cells and non-infected control Jurkat cells using the ultracentrifugation protocol. Exosomes were validated by western blotting for tetraspanins (CD9, CD63 and CD81) and transmission electron microscope. The acetyl-CoA acetylcholinesterase (AChE) enzyme activity was used to quantify exosomes. HNC cells UM-SCC-104, TR146, HSC3, SCC9 and lung cancer cells NCI-H1299, NCI-H1437 were treated with exosomes for cell growth, migration and gene expression in vitro and in vivo. Exosome HIV RNA was determined by quantitative RT-PCR. Results: Exosomes isolated from HIV-1-infected T cells, but not those from normal control T cells, significantly stimulated HNC and lung cancer cell proliferation, migration and invasion in vitro. Exosomes purified from plasma of HIV-positive individuals also stimulated proliferation of cancer cells. HNC and lung cancer cells co-inoculation with exosomes from HIV-1-infected T cells into nude mice promoted tumor growth compared to those from non-HIV control T cells. Mechanistically, we found that the HIV trans-activation response (TAR) element RNA, which exists in excess of other HIV RNAs in exosomes from HIV-1-infected T cells, enhanced cancer cell proliferation and stimulated EGF- and TLR3-inducible gene expression. The TAR RNA mutant with nucleotide replacements in the loop/bulge region failed to induce gene expression. Moreover, co-transfection of the TAR RNA aptamer blocked induction of gene expression induced by HIV-positive T-cell exosomes. Conclusion: HIV TAR RNA-containing exosomes derived from HIV-1-infected T cells promote growth and progression of NADCs through interaction with EGFR and TLR3. Citation Format: Lechuang chen, Zhimin Feng, Hong Yue, Scott Sieg, Bingcheng Wang, Alex Y. Huang, Ge Jin. Exosomes derived from HIV-infected T cells promote cancer cell growth and progression via the HIV TAR RNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5060.

Molecular dynamics and MM/GBSA-integrated protocol probing the correlation between biological activities and binding free energies of HIV-1 TAR RNA inhibitors

The interaction of HIV-1 transactivator protein Tat with its cognate transactivation response (TAR) RNA has emerged as a promising target for developing antiviral compounds and treating HIV infection, since it is a crucial step for efficient transcription and replication. In the present study, molecular dynamics (MD) simulations and MM/GBSA calculations have been performed on a series of neamine derivatives in order to estimate appropriate MD simulation time for acceptable correlation between ΔGbind and experimental pIC50 values. Initially, all inhibitors were docked into the active site of HIV-1 TAR RNA. Later to explore various conformations and examine the docking results, MD simulations were carried out. Finally, binding free energies were calculated using MM/GBSA method and were correlated with experimental pIC50 values at different time scales (0–1 to 0–10 ns). From this study, it is clear that in case of neamine derivatives as simulation time increased the correlation between binding free energy and experimental pIC50 values increased correspondingly. Therefore, the binding energies which can be interpreted at longer simulation times can be used to predict the bioactivity of new neamine derivatives. Moreover, in this work, we have identified some plausible critical nucleotide interactions with neamine derivatives that are responsible for potent inhibitory activity. Furthermore, we also provide some insights into a new class of oxadiazole-based back bone cyclic peptides designed by incorporating the structural features of neamine derivatives. On the whole, this approach can provide a valuable guidance for designing new potent inhibitors and modify the existing compounds targeting HIV-1 TAR RNA.

Ultraslow Domain Motions in HIV-1 TAR RNA Revealed by Solid-State Deuterium NMR.

Intrinsic motions may allow HIV-1 transactivation response (TAR) RNA to change its conformation to form a functional complex with the Tat protein, which is essential for viral replication. Understanding the dynamic properties of TAR necessitates determining motion on the intermediate nanosecond-to-microsecond time scale. To this end, we performed solid-state deuterium NMR line-shape and T1Z relaxation-time experiments to measure intermediate motions for two uridine residues, U40 and U42, within the lower helix of TAR. We infer global motions at rates of ∼105 s-1 in the lower helix, which are much slower than those in the upper helix (∼106 s-1), indicating that the two helical domains reorient independently of one another in the solid-state sample. These results contribute to the aim of fully describing the properties of functional motions in TAR RNA.

Development of Small Molecules with a Noncanonical Binding Mode to HIV-1 Trans Activation Response (TAR) RNA.

Small molecules that bind to RNA potently and specifically are relatively rare. The study of molecules that bind to the HIV-1 transactivation response (TAR) hairpin, a cis-acting HIV genomic element, has long been an important model system for the chemistry of targeting RNA. Here we report the synthesis, biochemical, and structural evaluation of a series of molecules that bind to HIV-1 TAR RNA. A promising analogue, 15, retained the TAR binding affinity of the initial hit and displaced a Tat-derived peptide with an IC50 of 40 μM. NMR characterization of a soluble analogue, 2, revealed a noncanonical binding mode for this class of compounds. Finally, evaluation of 2 and 15 by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) indicates specificity in binding to TAR within the context of an in vitro-synthesized 365-nt HIV-1 5'-untranslated region (UTR). Thus, these compounds exhibit a novel and specific mode of interaction with TAR, providing important suggestions for RNA ligand design.

ChemInform Abstract: NMR Studies of Dynamic Biomolecular Conformational Ensembles

AbstractReview: 173 refs.

Specific Binding of the Nucleocapsid Protein Transforms the Folding Landscape of the HIV-1 TAR RNA Hairpin

Retroviral nucleocapsid (NC) proteins are nucleic acid chaperones that play a key role in the viral life cycle. During reverse transcription, HIV-1 NC destabilizes nucleic acids, including the transactivation response (TAR) hairpin to facilitate their structural re-arrangement into the lowest free energy conformations. By combining single molecule optical tweezers measurements with mfold-based free energy calculations of the unfolding landscape, we determine the equilibrium TAR stability and characterize the unfolding transition state in both the presence and absence of NC. Our results show that protein binding occurs specifically at guanines that border structural defects in the upper part of TAR hairpin. This results in preferential destabilization by NC of this part of TAR, thereby shifting the transition state closer to the bottom of the TAR stem, leading to a shorter critical unfolding length. Thus, NC facilitates TAR RNA annealing to its complementary DNA hairpin while having little effect on the stability of the final annealed duplexes. These results provide the first direct measurement of alterations in an RNA folding landscape that are induced by protein-RNA interactions, and are a novel case study of the effect of a protein that specifically destabilizes particular elements of the nucleic acid secondary structure.

Modeling of HIV-1 TAR RNA-Ligand Complexes

Trans activation response (TAR) region is an RNA target of considerable importance in controlling the replication cycle of the human immunodeficiency virus (HIV). At a transcriptional level, HIV-1 is regulated by means of the interaction between Tat protein and TAR RNA. The TAR-Tat complex is an attractive target for developing novel antiviral drugs. Herein, the recognition modes of 8 structurally different ligands, as mimics of Tat protein, in complex with a TAR RNA are investigated using the DOCK 6.4 flexible docking protocol in association with the newly-implemented scoring function AMBER including solvation implicitly through the generalized Born solvent-accessible surface area (GB/SA) continuum model. The TAR RNA-ligand interactions are further characterized and contrasted using the nature of separate contributions to the stability of the complexes. Several interesting implications for the key challenge, the development of low molecular weight ligands binding to HIV-1 TAR RNA with high affinity and specificity, are discussed.

A Small-Molecule Probe Induces a Conformation in HIV TAR RNA Capable of Binding Drug-Like Fragments

The HIV-1 transactivation response (TAR) element–Tat interaction is a potentially valuable target for treating HIV infection, but efforts to develop TAR-binding antiviral drugs have not yet yielded a successful candidate for clinical development. In this work, we describe a novel approach toward screening fragments against RNA that uses a chemical probe to target the Tat-binding region of TAR. This probe fulfills two critical roles in the screen: by locking the RNA into a conformation capable of binding other fragments, it simultaneously allows the identification of proximal binding fragments by ligand-based NMR. Using this approach, we have discovered six novel TAR-binding fragments, three of which were docked relative to the probe–RNA structure using experimental NMR restraints. The consistent orientations of functional groups in our data-driven docked structures and common electrostatic properties across all fragment leads reveal a surprising level of selectivity by our fragment-sized screening hits. These models further suggest linking strategies for the development of higher-affinity lead compounds for the inhibition of the TAR–Tat interaction.

Recognition of HIV TAR RNA by triazole linked neomycin dimers.

A series of neomycin dimers have been synthesized using 'click chemistry' with varying linker functionality and length to target the TAR RNA region of HIV virus. TAR (trans activation response) RNA region, a 59 base pair stem loop structure located at 5'-end of all nascent HIV-1 transcripts interacts with a key regulatory protein, Tat, and necessitates the replication of HIV-1 virus. Neomycin, an aminosugar, has been shown to exhibit more than one binding site with HIV TAR RNA. Multiple TAR binding sites of neomycin prompted us to design and synthesize a small library of neomycin dimers using click chemistry. The binding between neomycin dimers and HIV TAR RNA was characterized using spectroscopic techniques including FID (Fluorescent Intercalator Displacement) titration and UV-thermal denaturation. UV thermal denaturation studies demonstrate that neomycin dimer binding increase the melting temperature (T(m)) of the HIV TAR RNA up to 10°C. Ethidium bromide displacement titrations revealed nanomolar IC(50) between neomycin dimers and HIV TAR RNA, whereas with neomycin, a much higher IC(50) in the micromolar range is observed.

Essential structural requirements for specific recognition of HIV TAR RNA by peptide mimetics of Tat protein

The pharmacological disruption of the interaction between the HIV Tat protein and its cognate transactivation response RNA (TAR) would generate novel anti-viral drugs with a low susceptibility to drug resistance, but efforts to discover ligands with sufficient potency to warrant pharmaceutical development have been unsuccessful. We have previously described a family of structurally constrained β-hairpin peptides that potently inhibits viral growth in HIV-infected cells. The nuclear magnetic resonance (NMR) structure of an inhibitory complex revealed that the peptide makes intimate contacts with the 3-nt bulge and the upper helix of the RNA hairpin, but that a single residue contacts the apical loop where recruitment of the essential cellular co-factor cyclin T1 occurs. Attempting to extend the peptide to form more interactions with the RNA loop, we examined a library of longer peptides and achieved >6-fold improvement in affinity. The structure of TAR bound to one of the extended peptides reveals that the peptide slides down the major groove of the RNA, relative to our design, in order to maintain critical interactions with TAR. These conserved contacts involve three amino acid side chains and identify critical interaction points required for potent and specific binding to TAR RNA. They constitute a template of essential interactions required for inhibition of this RNA.

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

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.