Trans-activation Responsive Region Research Articles

Structural Determinants of HIV-1 Nucleocapsid Protein for cTAR DNA Binding and Destabilization, and Correlation with Inhibition of Self-primed DNA Synthesis

The nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) is formed of two highly conserved CCHC zinc fingers flanked by small basic domains. NC is required for the two obligatory strand transfers in viral DNA synthesis through its nucleic acid chaperoning properties. The first DNA strand transfer relies on NC's ability to bind and destabilize the secondary structure of complementary transactivation response region (cTAR) DNA, to inhibit self-priming, and to promote the annealing of cTAR to TAR RNA. To further investigate NC chaperone properties, our aim was to identify by fluorescence spectroscopy and gel electrophoresis, the NC structural determinants for cTAR binding and destabilization, and for the inhibition of self-primed DNA synthesis on a model system using a series of NC mutants and HIV-1 reverse transcriptase. NC destabilization and self-priming inhibition properties were found to be supported by the two fingers in their proper context and the basic (29)RAPRKKG(35) linker. The strict requirement of the native proximal finger suggests that its hydrophobic platform (Val13, Phe16, Thr24 and Ala25) is crucial for binding, destabilization and inhibition of self-priming. In contrast, only partial folding of the distal finger is required, probably for presenting the Trp37 residue in an appropriate orientation. Also, Trp37 and the hydrophobic residues of the proximal finger appear to be essential for the propagation of the melting from the cTAR ends up to the middle of the stem. Finally, both N-terminal and C-terminal basic domains contribute to cTAR binding but not to its destabilization.

A Model of N‐Terminal Cyclin T1 Based on FRET Experiments

Human Cyclin T1 is the cyclin partner of kinase CDK9 in the positive transcription elongation factor b (P‐TEFb). P‐TEFb is recruited by Tat, the transactivator of the human immunodeficiency virus type 1 (HIV‐1), to the viral promoter by direct interactions between Tat, Cyclin T1 and the cis‐acting transactivation‐responsive region (TAR) present at the 5′‐end of each viral mRNA. At present, no structural data for Cyclin T1 are available. Here, we build a structural model of an N‐terminus portion of Cyclin T1 (aa 27–263) based on the X‐ray structure of Cyclin H. The model is compared with site directed mutagenesis data from the literature and validated by fluorescence resonance energy transfer (FRET) using Tat as a probe in living cells. This model provides a first step towards the structural characterization of the CDK9–CycT1–Tat‐TAR complex, which is crucial for HIV‐1 replication and may constitute a promising target for pharmaceutical intervention.

Capillary Zone Electrophoretic Separation of Four Novel Compounds Active Against Human Immunodeficiency Virus Type 1

The separation of four novel small molecules active in the treatment of acquired immunodeficiency syndrome (AIDS), is presented. The molecules were synthesized by computer-aided design with the property of inhibiting the binding of the human immunodeficiency virus type 1 trans-activator of transcription (HIV-1 Tat) protein to the trans-activation response region (TAR) RNA required for HIV-1 transcription, and thus blocking of the HIV replication cycle. The active compounds were separated to baseline by capillary zone electrophoresis (CZE) under optimized conditions with 0.1 mol L−1 ammonium acetate solution (pH 3.8) as running buffer and UV detection. The method will be used study the interactions between the anti-HIV-1 active molecules and HIV TAR RNA and for rapid screening of these potential drugs for early drug discovery.

Secondary Structure and Secondary Structure Dynamics of DNA Hairpins Complexed with HIV-1 NC Protein

Reverse transcription of the HIV-1 RNA genome involves several complex nucleic acid rearrangement steps that are catalyzed by the HIV-1 nucleocapsid protein (NC), including for example, the annealing of the transactivation response (TAR) region of the viral RNA to the complementary region (TAR DNA) in minus-strand strong-stop DNA. We report herein single-molecule fluorescence resonance energy transfer measurements on single immobilized TAR DNA hairpins and hairpin mutants complexed with NC (i.e., TAR DNA/NC). Using this approach we have explored the conformational distribution and dynamics of the hairpins in the presence and absence of NC protein. The data demonstrate that NC shifts the equilibrium secondary structure of TAR DNA hairpins from a fully “closed” conformation to essentially one specific “partially open” conformation. In this specific conformation, the two terminal stems are “open” or unwound and the other stems are closed. This partially open conformation is arguably a key TAR DNA intermediate in the NC-induced annealing mechanism of TAR DNA.

Inhibition of HIV-1 TAR RNA–Tat peptide complexation using poly(acrylic acid)

HIV-1 is regulated at the transcriptional level by the interaction of Tat protein with the transactivation responsive region (TAR) RNA, a 59-base stem–loop structure located at the 5 ′-end of all nascent HIV-1 transcripts. Here, by targeting the Tat peptide, we found that negatively charged poly(acrylic acid) (PAA) had high affinity with Tat peptide and could inhibit the interaction of TAR with Tat. Therefore, PAA could block HIV replication by binding to Tat not to TAR RNA, providing a new thinking for the design of novel anti-HIV drugs.

The HIV‐1 Tat Transactivator Protein: a Therapeutic Target?

The human immunodeficiency virus-1 (HIV-1), the causative agent of autoimmune deficiency syndrome (AIDS) is a major health problem world-wide. Central to HIV infection is the transactivator protein Tat, that plays a critical role in the nucleus during the HIV infectious cycle, by binding the transactivation-responsive region (TAR) and thereby enhancing transcriptional elongation. Tat appears to gain nuclear entry through a novel mechanism, independent of the normal cellular importin/Ran-dependent pathways, and regulated by a cytoplasmic retention mechanism. Since blocking Tat nuclear import is likely to prevent HIV infection, detailed delineation of Tat's nuclear import pathway is critical to assessing its viability as a therapeutic target. Other feasible anti-HIV therapies include approaches to inhibit Tat-TAR interaction.

Targeting the HIV trans‐Activation Responsive Region — Approaches Towards RNA‐Binding Drugs

AbstractFor Abstract see ChemInform Abstract in Full Text.

Alterations in HIV-1 LTR promoter activity during AIDS progression

HIV-1 variants evolving in AIDS patients frequently show increased replicative capacity compared to those present during early asymptomatic infection. It is known that late stage HIV-1 variants often show an expanded coreceptor tropism and altered Nef function. In the present study we investigated whether enhanced HIV-1 LTR promoter activity might also evolve during disease progression. Our results demonstrate increased LTR promoter activity after AIDS progression in 3 of 12 HIV-1-infected individuals studied. Further analysis revealed that multiple alterations in the U3 core-enhancer and in the transactivation-response (TAR) region seem to be responsible for the enhanced functional activity. Our findings show that in a subset of HIV-1-infected individuals enhanced LTR transcription contributes to the increased replicative potential of late stage virus isolates and might accelerate disease progression.

Targeting the HIV trans-activation responsive region--approaches towards RNA-binding drugs.

Targeting the HIV trans-activation responsive region--approaches towards RNA-binding drugs.

Design and Synthesis of Guanidinoglycosides Directed against the TAR RNA of HIV‐1

AbstractReplication of human immunodeficiency virus type 1 (HIV‐1) requires specific interactions of the Tat protein with the transactivation responsive region (TAR) RNA. Tat‐TAR RNA Interaction is mediated by a short arginine‐rich domain of the protein. Disruption of this interaction could, in theory, create a state of complete viral latency. Here, four novel 6‐amino‐6‐deoxytrehalose guanidinoglycoside derivatives (10 and 13–15) as target molecules have been designed to bind to TAR RNA for blocking the interaction of Tat‐TAR RNA. They were obtained by coupling 6‐amino‐6‐deoxy‐α,α‐trehalose (6) with the protected amino acids, deprotection by catalytic hydrogenation, followed by guanidinylated with S‐methylisothiourea sulfate. Their abilities to inhibit Tat‐TAR RNA interaction were determined by a Tat‐dependent HIV‐1 long terminal repeats (LTR)‐driven chloramphenicol acetyltransferase (CAT) assays.

Destabilization of the HIV-1 Complementary Sequence of TAR by the Nucleocapsid Protein Through Activation of Conformational Fluctuations

The nucleocapsid protein NCp7 of HIV-1 possesses nucleic acid chaperone properties that are critical for the two obligatory strand transfer reactions required for the synthesis of a complete proviral DNA by reverse transcriptase. The first DNA strand transfer relies on the destabilization by NCp7 of double-stranded segments of the transactivation response region (TAR) sequence at the 3′ end of the genomic RNA and the complementary sequence cTAR at the 3′ terminus of minus strong-stop DNA, the early product of reverse transcription. In order to determine the dynamics of NCp7-mediated nucleic acid destabilization, we investigated by time-resolved fluorescence spectroscopy and two photon fluorescence correlation spectroscopy, the interaction of a doubly labeled cTAR sequence with NC(12–55) containing NCp7 CCHC zinc fingers and flanking basic amino acid residues. From the chemical rates and the activation energy associated with the conformational fluctuations observed in the absence of NC, it is concluded that such fluctuations are associated with the opening and closing of the double-stranded terminal segments of cTAR. The destabilizing activity of NC(12–55) occurs mainly through a major increase of the opening rate constant of cTAR. Moreover, NC appears to augment the number of pathways between the open and closed states of cTAR, suggesting that it initiates melting of base-pairs at different locations within the terminal segments of cTAR. This activity of NC on the dynamics of cTAR secondary structure is thought to be critical for the formation of the cTAR–TAR complex, which is essential for the specificity and extent of proviral DNA synthesis by reverse transcriptase.

Binding affinity and inhibitory potency of neomycin and streptomycin on the Tat peptide interaction with HIV-1 TAR RNA detected by on-line acoustic wave sensor.

The binding of two aminoglycoside antibiotics, neomycin and streptomycin, to a segment of the transactivation responsive region (TAR) RNA of the human immunodeficiency virus, and their inhibitory potency to disrupt the interaction of the RNA with a regulatory Tat protein-derived peptide, have been studied using a flow-through acoustic wave detector system. Binding affinity is directly correlated with the inhibitory potency of these molecules and the acoustic wave detection system shows that neomycin exhibits at least a ten-fold greater affinity for TAR RNA and that it is also a more potent inhibitor than streptomycin. These results are in agreement with previous studies. However, unlike the time-consuming batch-based assays, use of the flow-through format offers considerable potential for the rapid screening of the chemistry of relatively small-molecule-nucleic acid binding events.

Nucleic Acid Conformational Changes Essential for HIV-1 Nucleocapsid Protein-mediated Inhibition of Self-priming in Minus-strand Transfer

Reverse transcription of the HIV-1 genome is a complex multi-step process. HIV-1 nucleocapsid protein (NC) is a nucleic acid chaperone protein that has been shown to greatly facilitate the nucleic acid rearrangements that precede the minus-strand transfer step in reverse transcription. NC destabilizes the highly structured transactivation response region (TAR) present in the R region of the RNA genome, as well as a complementary hairpin structure (“TAR DNA”) at the 3′-end of the newly synthesized minus-strand strong-stop DNA ((−) SSDNA). Melting of the latter structure inhibits a self-priming (SP) reaction that competes with the strand transfer reaction. In an in vitro minus-strand transfer system consisting of a (−) SSDNA mimic and a TAR-containing acceptor RNA molecule, we find that when both nucleic acids are present, NC facilitates formation of the transfer product and the SP reaction is greatly reduced. In contrast, in the absence of the acceptor RNA, NC has only a small inhibitory effect on the SP reaction. To further investigate NC-mediated inhibition of SP, we developed a FRET-based assay that allows us to directly monitor conformational changes in the TAR DNA structure upon NC binding. Although the majority (∼71%) of the TAR DNA molecules assume a folded hairpin conformation in the absence of NC, two minor “semi-folded” and “unfolded” populations are also observed. Upon NC binding to the TAR DNA alone, we observe a modest shift in the population towards the less-folded states. In the presence of the RNA acceptor molecule, NC binding to TAR DNA results in a shift of the majority of molecules to the unfolded state. These measurements help to explain why acceptor RNA is required for significant inhibition of the SP reaction by NC, and support the hypothesis that NC-mediated annealing of nucleic acids is a concerted process wherein the unwinding step occurs in synchrony with hybridization.

A new class of RNA-binding oligomers: peptoid amide and ester analogues.

Replication of human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5'-end of all HIV mRNAs. Here we report the design, synthesis and in vitro activities of oligopeptoid amide and ester analogues which bind TAR RNA with high affinities. These results show that we have identified a new class of unnatural oligomers for RNA targeting.

Eilatin Ru(II) complexes display anti-HIV activity and enantiomeric diversity in the binding of RNA.

Eilatin-containing octahedral ruthenium complexes inhibit HIV-1 replication in CD4+ HeLa cells and in human peripheral blood monocytes with IC(50) values of approximately 1 microM. Similar metal complexes that lack eilatin display 15-100-fold lower anti-HIV activities. [Ru(bpy)(2)"pre-eilatin"](2+), a complex that contains a nonplanar analogue of eilatin, shows significantly lower nucleic acid binding and lower anti-HIV activity than eilatin complexes. This result indicates that the extended planar surface presented by eilatin is important for both activities. Rev peptide and ethidium bromide displacement assays are used to probe the nucleic acid affinity and specificity of Lambda- and Delta-[Ru(bpy)(2)eilatin](2+). Two HIV-1 RNA sites are compared and a significant binding preference for the Rev response element over the transactivation response region is found. Simple DNA duplexes show a consistent selectivity for Lambda-[Ru(bpy)(2)eilatin](2+) compared to Delta-[Ru(bpy)(2)eilatin](2+), while RNAs show more diverse enantiomeric selectivities.

TAR RNA loop: a scaffold for the assembly of a regulatory switch in HIV replication.

Replication of HIV requires the Tat protein, which activates elongation of RNA polymerase II transcription at the HIV-1 promoter by interacting with the cyclin T1 (CycT1) subunit of the positive transcription elongation factor complex b (P-TEFb). The transactivation domain of Tat binds directly to the CycT1 subunit of P-TEFb and induces loop sequence-specific binding of P-TEFb onto nascent HIV-1 trans-activation responsive region (TAR) RNA. We used systematic RNA-protein photocross-linking, Western blot analysis, and protein footprinting to show that residues 252-260 of CycT1 interact with one side of the TAR RNA loop and enhance interaction of Tat residue K50 to the other side of the loop. Our results show that TAR RNA provides a scaffold for two protein partners to bind and assemble a regulatory switch in HIV replication. RNA-mediated assembly of RNA-protein complexes could be a general mechanism for stable ribonucleoprotein complex formation and a key step in regulating other cellular processes and viral replication.

Tat Acetyl-acceptor Lysines Are Important for Human Immunodeficiency Virus Type-1 Replication

The human immunodeficiency virus type-1 trans-activator Tat is a transcription factor that activates the HIV-1 promoter through binding to the trans-activation-responsive region (TAR) localized at the 5'-end of all viral transcripts. We and others have recently shown that Tat is directly acetylated at lysine 28, within the activation domain, and lysine 50, in the TAR RNA binding domain, by Tat-associated histone acetyltransferases p300, p300/CBP-associating factor, and hGCN5. Here, we show that mutation of acetyl-acceptor lysines to arginine or glutamine affects virus replication. Interestingly, mutation of lysine 28 and lysine 50 differentially affected Tat trans-activation of integrated versus nonintegrated long terminal repeat. Our results highlight the importance of lysine 28 and lysine 50 of Tat in virus replication and Tat-mediated trans-activation.

Enhancement of Nuclear Factor-κB Acetylation by Coactivator p300 and HIV-1 Tat Proteins

Nuclear factor (NF)-kappaB transcription factors are involved in the control of a large number of normal cellular and organismal processes, such as immune and inflammatory responses, developmental processes, cellular growth, and apoptosis. Transcription of the human immunodeficiency virus type 1 (HIV-1) genome depends on the intracellular environment where the integrate viral DNA is regulated by a complex interplay among viral regulatory proteins, such as Tat, and host cellular transcription factors, such as NF-kappaB, interacting with the viral long terminal repeat region. CBP (CREB-binding protein) and p300, containing an intrinsic histone acetyltransferase (HAT) activity, have emerged as coactivators for various DNA-binding transcription factors. Here, we show that the p50 subunit as well as the p50/p65 of NF-kappaB, and not other factors such as SP1, TFIIB, polymerase II, TFIIA, or p65, can be acetylated by CBP/p300 HAT domain. Acetylation of p50 was completely dependent on the presence of both HAT domain and Tat proteins, implying that Tat influences the transcription machinery by aiding CBP/p300 to acquire new partners and increase its functional repertoire. Three lysines, Lys-431, Lys-440, and Lys-441 in p50 were all acetylated in vitro, and a sequence similarity among p50, p53, Tat, and activin receptor type I on these particular lysines was observed. All proteins have been shown to be acetylated by the CBP/p300 HAT domain. Acetylated p50 increases its DNA binding properties, as evident by streptavidin/biotin pull-down assays when using labeled NF-kappaB oligonucleotides. Increased DNA binding on HIV-1 long terminal repeat coincided with increases in the rate of transcription. Therefore, we propose that acetylation of the DNA binding domain of NF-kappaB aids in nuclear translocation and enhanced transcription and also suggest that the substrate specificity of CBP/p300 can be altered by small peptide molecules, such as HIV-encoded Tat.

Full-length sequences of two CRF01_ae (subtype e) HIV type 1 isolates from 1995 samples of patients with sexually transmitted diseases in Thailand.

We isolated two CRF01_AE human immunodeficiency virus type 1 (95TNIH022 and 95TNIH047) from the 1995 blood samples derived from asymptomatic carriers in Ubonratchatani province of northeastern Thailand. Both isolates can replicate in peripheral blood mononuclear cells, but not in several T cell lines examined. The full-length sequences recovered from proviruses in infected cells by long-range polymerase chain reaction were determined. Phylogenetic analyses of these sequences at individual genes showed them to be closely related to those of reported CRF01_AE HIV-1, such as 1990 isolate CM240 and 1993 isolate 93TH253. Two isolates in this study also showed a similar pattern of CRF01_AE mosaicism and a similar structure at the long terminal repeat, i.e., a copy number of NF-kappaB binding sites, sequence at the TATA box, and the putative secondary structure of stem-loop in the transactivation response region. Our results showed that 1995 Thai E isolates could contribute to our understanding of the epidemiology, pathogenesis, and diagnostics of HIV-1 CRF01_AE and further to vaccine development.

Probing Tat peptide-TAR RNA interactions by psoralen photo-cross-linking.

Replication of human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5'-end of all HIV mRNAs. We have used a site-specific cross-linking method based on psoralen photochemistry to determine the effect of core residues from the Tat sequence on the protein orientation in the Tat-TAR complex and on the specificity of Tat-TAR binding. We synthesized two Tat fragments, Tat(42-72) and Tat(37-72), and incorporated a psoralen-modified amino acid at position 41 during solid-phase assembly of the peptides. We used these psoralen-Tat conjugates to form specific complexes with TAR RNA. Upon near-ultraviolet irradiation (360 nm), psoralen-Asp41-Tat(37-72) cross-linked to a single site in the TAR RNA sequence. The RNA-protein complex was purified and the cross-link site on TAR RNA was determined by primer extension analysis, which revealed that Asp41 of Tat is close to U42 of the lower stem region of TAR RNA. Specificity of the RNA-peptide cross-linking reactions was determined by competition experiments. Our results show that the addition of only four residues (Cys37-Thr40) from the Tat core region significantly enhanced the specificity of the Tat peptide-TAR interactions without altering the site or chemical nature of the cross-link. These studies provide new insights into RNA-protein recognition that could be useful in designing peptidomimetics for RNA targeting. Such psoralen-peptide conjugates provide a new class of probes for sequence-specific protein-nucleic acid interactions and could be used to selectively control gene expression or to induce site-directed mutations.