The HIV-1 transactivator of transcription (Tat) is a protein essential for both viral gene expression and virus replication. Tat is an RNA-binding protein that, in cooperation with host cell factors cyclin T1 and cyclin-dependent kinase 9, regulates transcription at the level of elongation. Tat also interacts with numerous other intracellular and extracellular proteins, and is implicated in a number of pathogenic processes. The Tat protein is encoded by two exons and is 101 residues in length. The first exon encodes a 72-residue molecule that activates transcription with the same proficiency as the full-length protein. The physico-chemical properties of Tat make it a particularly challenging target for structural studies: Tat contains seven cysteine residues, six of which are essential for transactivation, and is highly susceptible to oxidative cross-linking and aggregation. In addition, a basic segment (residues 48-57) gives the protein a high net positive charge of +12 at pH 7, endowing it with a high affinity for anionic polymers and surfaces. In order to study the structure of Tat, both alone and in complex with partner molecules, we have developed a system for the bacterial expression and purification of polyhistidine-tagged and isotopically enriched (in 15N and 15N /13C) recombinant HIV-1 Tat1-72 (BH10 isolate) that yields large amounts of protein. These preparations have facilitated the assignment of 95% of the non-proline backbone resonances using heteronuclear 3-dimensional nuclear magnetic resonance (NMR) spectroscopy. Analysis by mass spectrometry and NMR demonstrate that the cysteine-rich Tat protein is unambiguously reduced and monomeric in aqueous solution at pH 4. NMR chemical shifts and coupling constants suggest that it exists in a disordered conformation. Line broadening and multiple peaks in the cysteine-rich and core regions suggest that transient folding occurs in two of the five sequence domains. NMR relaxation parameters were measured and analysed by spectral density and model-free approaches both confirming the lack of structure throughout the length of the molecule. The absence of a fixed conformation and the observation of fast dynamics are consistent with the ability of the Tat protein to interact with a wide variety of proteins and nucleic acid lending further support to the concept that Tat exists as an intrinsically disordered protein. / October 2007

<p>HIV-1 relies on both viral and cellular host factors for expression of its genome. Tat specific factor 1 (Tat-SF1) was identified as a cellular cofactor required for enhanced transcription of HIV-1 <italic>in vitro</italic>. Insight into the role of Tat-SF1 in the HIV-1 lifecycle has previously been limited to immunodepletions and <italic>in vitro</italic> analyses or transient overexpression experiments. Here, we present studies that utilize RNA interference (RNAi) to reevaluate Tat-SF1's role in Tat transactivation and HIV-1 replication <italic>in vivo</italic>. We report that although Tat-SF1 depletion reduces HIV-1 infectivity, it does not affect Tat transactivation <italic>in vivo</italic>. However, Tat-SF1 depletion changes the levels of unspliced and spliced RNAs. We propose that Tat-SF1 has a novel role of post-transcriptionally regulating HIV-1 gene expression, possibly through alternative splicing.</p><p>The functions of Tat-SF1 in cellular gene expression are not well understood, so we utilized the stable cell lines constructed for our HIV-1 studies to investigate the cellular functions of Tat-SF1. To identify target genes of Tat-SF1, we employed a combination of RNAi and human exon arrays. These arrays, which survey both transcript-level and exon-level changes genome-wide, revealed approximately 1,400 genes with alternative exon usage after Tat-SF1 depletion (p&le;0.01). In contrast, 500 genes showed significant transcript-level changes (p&le;0.01), all with minimal fold changes. Computational analyses showed that genes with alternative exon usage after Tat-SF1 depletion were over-represented in the insulin signaling and ubiquitin mediated proteolysis biological pathways. Furthermore, there was approximately 2-fold enrichment of Tat-SF1 target genes among previously reported HIV-1 dependency factors. The type of exon choice affected by Tat-SF1 depletion exhibited a strong 5&rsquo; bias. Finally, a novel Tat-SF1 binding motif, GACGGG, was found to be over-represented among target genes and may play a functional role in first exon choice. Together, these data are the strongest evidence to date of Tat-SF1 functioning in both transcription and splicing of cellular genes.</p> / Dissertation

Tat1-72, is an intrinsically disordered protein at pH 4.1 as previously indicated by NMR chemical shifts and coupling constants, and confirmed by 15N-relaxation parameters. The presence of SDS elicits a conformational change to α-helicity in Tat1-72. In the presence of the non-ionic DDM detergent and zinc, Tat was found to be soluble at pH 4 when bound to TAR RNA; TAR binding also elicits a conformational shift to α-helicity in Tat1-72. The β-sheet content of Tat1-72 is increased in the presence of NaCl. In similar conditions, Tat1-72 aggregates stained with Congo Red displayed a yellow-green birefringence and a red-shift in the Congo Red absorbance that is typical of β-amyloid fibril. The web-based algorithm “WALTZ” identifies the majority of the Tat1-72 hydrophobic core region as amyloidogenic. The helical propensity of Tat1-72 in TFE was determined by two-dimensional NMR spectroscopy.

The HIV-1 transactivator of transcription (Tat) is a protein essential for both viral gene expression and virus replication. Tat is an RNA-binding protein that, in cooperation with host cell factors cyclin T1 and cyclin-dependent kinase 9, regulates transcription at the level of elongation. Tat also interacts with numerous other intracellular and extracellular proteins, and is implicated in a number of pathogenic processes. The Tat protein is encoded by two exons and is 101 residues in length. The first exon encodes a 72-residue molecule that activates transcription with the same proficiency as the full-length protein. The physico-chemical properties of Tat make it a particularly challenging target for structural studies: Tat contains seven cysteine residues, six of which are essential for transactivation, and is highly susceptible to oxidative cross-linking and aggregation. In addition, a basic segment (residues 48-57) gives the protein a high net positive charge of +12 at pH 7, endowing it with a high affinity for anionic polymers and surfaces. In order to study the structure of Tat, both alone and in complex with partner molecules, we have developed a system for the bacterial expression and purification of polyhistidine-tagged and isotopically enriched (in 15N and 15N /13C) recombinant HIV-1 Tat1-72 (BH10 isolate) that yields large amounts of protein. These preparations have facilitated the assignment of 95% of the non-proline backbone resonances using heteronuclear 3-dimensional nuclear magnetic resonance (NMR) spectroscopy. Analysis by mass spectrometry and NMR demonstrate that the cysteine-rich Tat protein is unambiguously reduced and monomeric in aqueous solution at pH 4. NMR chemical shifts and coupling constants suggest that it exists in a disordered conformation. Line broadening and multiple peaks in the cysteine-rich and core regions suggest that transient folding occurs in two of the five sequence domains. NMR relaxation parameters were measured and analysed by spectral density and model-free approaches both confirming the lack of structure throughout the length of the molecule. The absence of a fixed conformation and the observation of fast dynamics are consistent with the ability of the Tat protein to interact with a wide variety of proteins and nucleic acid lending further support to the concept that Tat exists as an intrinsically disordered protein.

The HIV-1 transactivator of transcription (Tat) is a protein essential for both viral gene expression and virus replication. Tat is an RNA-binding protein that, in cooperation with host cell factors cyclin T1 and cyclin-dependent kinase 9, regulates transcription at the level of elongation. Tat also interacts with numerous other intracellular and extracellular proteins, and is implicated in a number of pathogenic processes. The Tat protein is encoded by two exons and is 101 residues in length. The first exon encodes a 72-residue molecule that activates transcription with the same proficiency as the full-length protein. The physico-chemical properties of Tat make it a particularly challenging target for structural studies: Tat contains seven cysteine residues, six of which are essential for transactivation, and is highly susceptible to oxidative cross-linking and aggregation. In addition, a basic segment (residues 48-57) gives the protein a high net positive charge of +12 at pH 7, endowing it with a high affinity for anionic polymers and surfaces. In order to study the structure of Tat, both alone and in complex with partner molecules, we have developed a system for the bacterial expression and purification of polyhistidine-tagged and isotopically enriched (in 15N and 15N /13C) recombinant HIV-1 Tat1-72 (BH10 isolate) that yields large amounts of protein. These preparations have facilitated the assignment of 95% of the non-proline backbone resonances using heteronuclear 3-dimensional nuclear magnetic resonance (NMR) spectroscopy. Analysis by mass spectrometry and NMR demonstrate that the cysteine-rich Tat protein is unambiguously reduced and monomeric in aqueous solution at pH 4. NMR chemical shifts and coupling constants suggest that it exists in a disordered conformation. Line broadening and multiple peaks in the cysteine-rich and core regions suggest that transient folding occurs in two of the five sequence domains. NMR relaxation parameters were measured and analysed by spectral density and model-free approaches both confirming the lack of structure throughout the length of the molecule. The absence of a fixed conformation and the observation of fast dynamics are consistent with the ability of the Tat protein to interact with a wide variety of proteins and nucleic acid lending further support to the concept that Tat exists as an intrinsically disordered protein.

HIV Tat protein downregulates surface expression of the interleukin-7 receptor alpha-chain (CD127) on CD8 T cells resulting in impaired T cell proliferation and cytolytic capacity. Once taken up by CD8 T cells, Tat binds directly to the cytoplasmic tail of CD127 inducing receptor internalization and degradation. Given the important roles of CD127 in proper immune function, the Tat/CD127 interactions were characterized and the mechanisms required to induce receptor loss from the surface of CD8 T cells were investigated. Tat deletion mutants were generated each sequentially lacking a region of the protein. CD8 T cells isolated from HIV negative volunteers were exposed to exogenous or intracellular Tat proteins before surface CD127 expression was analyzed by flow cytometry. To characterize Tat/CD127 physical interactions, wild type Tat and Tat mutants were incubated with lysates from a CD127+ Jurkat cell line followed by CD127/Tat co-immunoprecipitation. The effect of Tat on CD127 post-translational modifications was also investigated. Removal of the N-terminus of Tat (aa 1-10 or aa 17-21) prevented Tat from downregulating CD127 and prevented Tat from binding CD127 as assessed by co-immunoprecipitation. Deletion of the basic region (aa 48-59) also prevented Tat from downregulating CD127 but did not prevent Tat from interacting physically as demonstrated by co-immunoprecipitation. Strikingly, endogenously expressed Basic Tat acted as a dominant negative mutant, causing an accumulation of CD127 at the cell surface. These observations suggest that Tat may bind CD127 via its N-terminus to disrupt the normal recycling of the receptor, and then recruit cellular endocytic machinery to the receptor via it’s basic region, to remove the receptor from the cell surface and target it for degradation. Furthermore, Tat encourages the ubiquitination of CD127 by recruiting the cytokine-inducible SH2 containing (CIS) protein to the receptor, possibly leading to accelerated CD127 internalization and proteasomal degradation. I propose a model whereby Tat binds CD127 via its N-terminal region then recruits CIS via its basic region. CIS in turn recruits a cellular E3 ubiquitin ligase to ubiquitin tag the receptor for internalization and proteasome degradation. This research may lead to novel treatments designed to maintain IL-7 signalling and strengthen CD8 T cell function in HIV+ persons.

Tat1-72, is an intrinsically disordered protein at pH 4.1 as previously indicated by NMR chemical shifts and coupling constants, and confirmed by 15N-relaxation parameters. The presence of SDS elicits a conformational change to α-helicity in Tat1-72. In the presence of the non-ionic DDM detergent and zinc, Tat was found to be soluble at pH 4 when bound to TAR RNA; TAR binding also elicits a conformational shift to α-helicity in Tat1-72. The β-sheet content of Tat1-72 is increased in the presence of NaCl. In similar conditions, Tat1-72 aggregates stained with Congo Red displayed a yellow-green birefringence and a red-shift in the Congo Red absorbance that is typical of β-amyloid fibril. The web-based algorithm “WALTZ” identifies the majority of the Tat1-72 hydrophobic core region as amyloidogenic. The helical propensity of Tat1-72 in TFE was determined by two-dimensional NMR spectroscopy.

Spontaneous and evoked potentials (EPs) were recorded with cross-cortical microelectrode arrays following partial occlusion of the MCA and ACA in urethane-anaesthetised rats. The control group received no occlusion, while the treatment group was injected with anti-stroke peptide Tat-NR2B9c 5min before ischemia. Spontaneous EEG power significantly decreased in the stroke-only group when compared to controls (p<0.001). A greater loss of EEG power was observed on anterior electrodes closer to the occluded area versus posterior contacts in stroke-only rats (p<0.05). The Tat-NR2B9c+stroke group lost significantly less power when compared to stroke-only animals (p<0.05). EP amplitude in the stroke-only group was significantly reduced following ischemia when compared to control and Tat-NR2B9c+stroke animals (p<0.001). Epileptiform discharges were observed in 8/10 untreated stroke rats and 3/5 stroke rats treated with Tat-NR2B9c. The characteristic features of spontaneous and evoked potentials validate this rat focal stroke model for in vivo testing of pharmacological agents.

Spontaneous and evoked potentials (EPs) were recorded with cross-cortical microelectrode arrays following partial occlusion of the MCA and ACA in urethane-anaesthetised rats. The control group received no occlusion, while the treatment group was injected with anti-stroke peptide Tat-NR2B9c 5min before ischemia. Spontaneous EEG power significantly decreased in the stroke-only group when compared to controls (p<0.001). A greater loss of EEG power was observed on anterior electrodes closer to the occluded area versus posterior contacts in stroke-only rats (p<0.05). The Tat-NR2B9c+stroke group lost significantly less power when compared to stroke-only animals (p<0.05). EP amplitude in the stroke-only group was significantly reduced following ischemia when compared to control and Tat-NR2B9c+stroke animals (p<0.001). Epileptiform discharges were observed in 8/10 untreated stroke rats and 3/5 stroke rats treated with Tat-NR2B9c. The characteristic features of spontaneous and evoked potentials validate this rat focal stroke model for in vivo testing of pharmacological agents.

The transactivator of transcription protein (Tat) of human immunodeficiency virus (HIV) plays an important role in both viral replication and AIDS pathogenesis. Tat is responsible for enhancing HIV-specific transcription, without which production of infectious virus is severely limited. Ongoing research, however, has identified a plethora of additional functions attributable to Tat including regulating additional HIV processes, altering the homeostasis of both infected and uninfected cells, and being directly involved in AIDS-related neuropathology and cancer. Tat thus plays a diverse role in both viral replication and pathogenesis. What is the relative importance of Tat’s additional functions compared to its principle function of transactivation? This question is explored in Chapter 2 in which tat clones from an epidemiologicallylinked transmission cohort were functionally tested. tat sequences were isolated and cloned from the cohort members at various time points and assessed for transactivation potential. The data revealed that transmission of HIV resulted in the selection of mutations in tat that were host specific and that impacted on the Tat clones’ abilities to transactivate. No correlation, however, was observed between the transactivation potentials of the Tat clones and the AIDS progression statuses of the hosts. The host-specific tat mutations may instead reflect the selection of variants fitter in one or more of Tat’s other functions. This therefore leads to the hypothesis that Tat functions in addition to transactivation are influential in AIDS pathogenesis and are subject to selective pressures during HIV transmission. Tat’s flexibility and diversity of functions are mediated by defined functional domains. One of the most important of these is the basic domain, a highly conserved region encompassing a unique nuclear localisation signal (NLS). The NLS allows Tat to localise to the cell nucleus to participate in transactivation. However, many of Tat’s additional functions require Tat to be outside of the nucleus or even outside of the cell. It is currently unknown how the NLS is modulated to enable Tat nuclear egress, but it may involve post-translational modifications of NLS residues. In Chapter 3, protein arginine methyltransferase 6 (PRMT6) is demonstrated to alter the localisation of Tat within the nucleus and increase the half-life of Tat within the cell. Both of these effects require the methylation activity of PRMT6 and two NLS residues, arginines 52 and 53, previously described to be substrates for PRMT6 methylation. The results suggest that PRMT6 primes Tat for functions outside of the nucleus by altering its subcellular localisation and increasing its stability. The critical role of the basic domain in Tat function is highlighted in Chapter 4 in which mutation of the basic domain led to dysregulation of HIV replication. The mutant, referred to as Nullbasic, has characteristics similar to transdominant Tat mutants, such as suppressing transactivation and localising to the cell cytoplasm. Surprisingly, Nullbasic potently inhibits HIV infectivity by strongly downregulating the expression of envelope, the key molecule involved in cell attachment and entry. Part of this downregulation is attributable to Nullbasic interfering with the nuclear export of envelope-encoding mRNA as a result of altering the subcellular localisation of Rev. However, an additional mechanism of interference is required to fully explain the strong suppression of envelope protein observed in cells. The data allude to a role for Tat in promoting envelope mRNA translation, a role subverted by mutating the basic domain. Taken together, these studies highlight the importance of a diversity of Tat functions to HIV replication and AIDS pathogenesis. Further, they emphasise the critical role of the basic domain to Tat function. Modifications of the basic domain not only influence function, but also regulate Tat spatially and temporally. Dysregulation of the basic domain by mutation not only subverts Tat function, but also elicits antiviral activity at multiple steps of the HIV lifecycle. The study of Tat and its basic domain is crucial in order to understand its role in HIV/AIDS and to recognise Tat as a pathogenic agent in its own right.