HIV-1 Transactivator Of Transcription Research Articles

TAT38 and TAT38 mimics potently inhibit adipogenesis by repressing C/EBPα, PPARγ, Pi-PPARγ, and SREBP1 expression

Antiretroviral therapy-naive people living with HIV possess less fat than people without HIV. Previously, we found that HIV-1 transactivator of transcription (TAT) decreases fat in ob/ob mice. The TAT38 (a.a. 20–57) is important in the inhibition of adipogenesis and contains three functional domains: Cys-ZF domain (a.a. 20–35 TACTNCYCAKCCFQVC), core-domain (a.a. 36–46, FITKALGISYG), and protein transduction domain (PTD)(a.a. 47–57, RAKRRQRRR). Interestingly, the TAT38 region interacts with the Cyclin T1 of the P-TEFb complex, of which expression increases during adipogenesis. The X-ray crystallographic structure of the complex showed that the Cys-ZF and the core domain bind to the Cyclin T1 via hydrophobic interactions. To prepare TAT38 mimics with structural and functional similarities to TAT38, we replaced the core domain with a hydrophobic aliphatic amino acid (from carbon numbers 5 to 8). The TAT38 mimics with 6-hexanoic amino acid (TAT38 Ahx (C6)) and 7-heptanoic amino acid (TAT38 Ahp (C7)) inhibited adipogenesis of 3T3-L1 potently, reduced cellular triglyceride content, and decreased body weight of diet-induced obese (DIO) mice by 10.4–11 % in two weeks. The TAT38 and the TAT38 mimics potently repressed the adipogenic transcription factors genes, C/EBPα, PPARγ, and SREBP1. Also, they inhibit the phosphorylation of PPARγ. The TAT peptides may be promising candidates for development into a drug against obesity or diabetes.

Acute Administration of HIV-1 Tat Protein Drives Glutamatergic Alterations in a Rodent Model of HIV-Associated Neurocognitive Disorders.

HIV-1-associated neurocognitive disorders (HAND) are a major comorbidity of HIV-1 infection, marked by impairment of executive function varying in severity. HAND affects nearly half of people living with HIV (PLWH), with mild forms predominating since the use of anti-retroviral therapies (ART). The HIV-1 transactivator of transcription (Tat) protein is found in the cerebrospinal fluid of patients adherent to ART, and its administration or expression in animals causes cognitive symptoms. Studies of Tat interaction with the N-methyl-D-aspartate receptor (NMDAR) suggest that glutamate toxicity contributes to Tat-induced impairments. To identify changes in regional glutamatergic circuitry underlying cognitive impairment, we injected recombinant Tat86 or saline to medial prefrontal cortex (mPFC) of male Sprague-Dawley rats. Rats were assessed with behavioral tasks that involve intact functioning of mPFC including the novel object recognition (NOR), spatial object recognition (SOR), and temporal order (TO) tasks at 1 and 2 postoperative weeks. Following testing, mPFC tissue was collected and analyzed by RT-PCR. Results showed Tat86 in mPFC-induced impairment in SOR, and upregulation of Grin1 and Grin2a transcripts. To further understand the mechanism of Tat toxicity, we assessed the effects of full-length Tat101 on gene expression in mPFC by RNA sequencing. The results of RNAseq suggest that glutamatergic effects of Tat86 are maintained with Tat101, as Grin2a was upregulated in Tat101-injected tissue, among other differentially expressed genes. Spatial learning and memory impairment and Grin2a upregulation suggest that exposure to Tat protein drives adaptation in mPFC, altering the function of circuitry supporting spatial learning and memory.

Transcriptional regulation of the HIV-1 inhibitory factor human mannose receptor 1 by the myeloid-specific transcription factor PU.1.

HIV-1 infection of human macrophages leads to the downmodulation of human mannose receptor 1 (hMRC1), a cell-surface glycoprotein that is involved in the host innate immune response. We previously reported that downmodulation of hMRC1 involves the transactivator of transcription (Tat)-dependent transcriptional silencing of the hMRC1 promoter. However, the inhibitory effect of Tat on hMRC1 transcription was indirect and involved inhibition of the transcriptional activator PU.1, which normally upregulates hMRC1 expression in macrophages and other myeloid cells. We cloned a 284-bp fragment of the hMRC1 promoter, and within it, we identified four PU.1 box elements. We assessed the relative contribution of each of the four PU.1 boxes to PU.1-dependent transcriptional regulation and, surprisingly, found that only one of the four PU.1 boxes [PU.1(b)] was critically required for PU.1-mediated upregulation of luciferase expression. Transfer of this PU.1 box to a heterologous promoter conferred PU.1 responsiveness to an otherwise PU.1 insensitive promoter. Electrophoretic mobility shift assays identified this PU.1 box as a direct binding site for PU.1 both in the context of the hMRC1 promoter and the heterologous promoter. Furthermore, mutational analysis of the PU.1 protein identified the C-terminal DNA-binding domain in PU.1 as the region responsible for interaction with the PU.1 box. Recombinant HIV-1 Tat protein did not bind to the hMRC1 promoter element but efficiently interfered with the binding of PU.1 protein to the hMRC1 promoter. Thus, Tat is likely to inhibit the formation of active PU.1 transcription complexes, presumably by binding to and depleting common transcriptional cofactors.IMPORTANCEHIV-1 infection of cells results in the modulation of cellular gene expression by virus-encoded proteins in a manner that benefits the virus. We reported that HIV-1 transactivator of transcription (Tat) dysregulates the expression of the human mannose receptor 1 (hMRC1). hMRC1 is involved in the innate immune response of macrophages to foreign pathogens. Tat does not act directly on the hMRC1 promoter but instead inhibits PU.1, a cellular transcription factor regulating hMRC1 gene expression. Here, we characterize the PU.1-dependent regulation of hMRC1 expression. We identified four potential PU.1 binding sites in the hMRC1 promoter region but found that only one, PU.1(b), functioned as a true binding site for PU.1. Transfer of the PU.1(b) box to a heterologous promoter did not activate this promoter per se but rendered it responsive to PU.1. Our results support the view that PU.1 acts as a transcriptional co-factor whose activity can be regulated by HIV-1 Tat.

HIV Tat-mediated altered oligodendrocyte maturation involves autophagy-lysosomal dysfunction

Abstract Objectives The current study was undertaken to understand the underlying molecular mechanism(s) by which HIV Transactivator of transcription (Tat) alters oligodendrocyte maturation through the generation of reactive oxygen species (ROS), impairment of lysosomal functioning, and dysregulation of autophagy. Methods We exposed primary rat immature oligodendrocytes to HIV Tat and utilized various experimental techniques to assess its effects on oligodendrocytes maturation. We measured ROS levels, assessed lysosomal membrane potential, determined cathepsin D activity, and analyzed the expression of autophagy-related markers. Furthermore, we investigated the potential of ROS scavengers and lysosomal protectants to mitigate the damaging effects of HIV Tat on oligodendrocytes maturation. Results Exposure of primary rat immature oligodendrocytes to HIV Tat significantly increased ROS levels, indicating the induction of oxidative stress. This oxidative stress impaired lysosomal functioning, as evidenced by a substantial increase in lysosomal membrane potential and a decrease in cathepsin D activity. Compromised lysosomal function resulted in dysregulated autophagy, which was confirmed by increased expression of SQSTM1. However, the administration of ROS scavengers and lysosomal protectants effectively attenuated the detrimental effects of HIV Tat on oligodendrocytes maturation. Conclusions Our findings demonstrate that HIV Tat exposure induces oxidative stress, impairs lysosomal functioning, and dysregulates autophagy in oligodendrocytes. These molecular changes likely contribute to the altered maturation of oligodendrocytes observed in HIV-infected individuals. Understanding these underlying mechanisms provides valuable insights into the pathogenesis of HIV-associated neurocognitive disorders and highlights the potential of therapeutic strategies targeting ROS scavenging and lysosomal protection as adjunctive approaches for managing such complications in HIV +ve individuals.

HIV-1 Tat impairs the establishment and maintenance of latency in a quiescent T cell line model of HIV infection

Abstract Human Immunodeficiency Virus (HIV) persists as a transcriptionally silent, latent provirus despite optimal therapy. Latently infected cells avoid immune detection and viral cytopathic effects becoming a long-lived reservoir of HIV. To elucidate factors responsible for the establishment of latency, our lab developed two novel HIV-1 reporter constructs (89.6 VT1 and 454 VT2). Studies using these reporters demonstrated that the multiplicity of infection (MOI) (the ratio of viral particles to cells) strongly correlated with the proportion of active versus latent infection in a “quiescent” T cell line. This observation suggested the hypothesis that at low viral inoculum, the availability of a viral factor, such as the HIV transactivator of transcription (Tat), was limiting and affected the likelihood that active versus latent infection was established. To test this, we (1) examined relative tat levels in reporter viruses that established different proportions of latent versus active infection (2) measured active versus latent infection in a cell line that constitutively overexpressed tat and (3) modified the dual reporter 89.6 VT1 to overexpress tat after an IRES element. In every case, higher tat levels correlated with increased active infection and a reduction in dependency of active infection on the amount of inoculum. Ongoing studies will test the extent to which Tat levels vary in native HIV proviral genomes. These studies provide insight into the molecular mechanisms that govern latency establishment at high versus low viral infection conditions and may shed light on new approaches to eradicate latently infected cells. Supported by grants from NIH/ NIAID (R01 AI148084 S1)

Novel Allosteric Modulator Southern Research Institute-32743 Reverses HIV-1 Transactivator of Transcription-Induced Increase in Dopamine Release in the Caudate Putamen of Inducible Transactivator of Transcription Transgenic Mice.

Development of neurocognitive disorder in human immunodeficiency virus (HIV)-infected patients has been linked to dysregulation of dopamine by the HIV-1 transactivator of transcription (Tat) protein, a negative allosteric modulator of dopamine transporter (DAT). Using fast scan cyclic voltammetry, the present study determined the effects of in vivo Tat expression on dopamine release in the caudate putamen of inducible Tat transgenic (iTat-tg) mice and the impact of a novel DAT allosteric modulator, Southern Research Institute (SRI)-32743, on the Tat effect. We found that 7- or 14-day doxycycline (Dox)-induced Tat expression in iTat-tg mice resulted in a 2-fold increase in phasic but not tonic stimulated baseline dopamine release relative to saline control mice. To determine whether the Tat-induced increase in dopamine release is mediated by DAT regulation, we examined the effect of an in vitro applied DAT inhibitor, nomifensine, on the dopamine release. Nomifensine (1 nM-10 µM) concentration-dependently enhanced phasic stimulated dopamine release in both saline- and Dox-treated iTat-tg mice, while the magnitude of the nomifensine-mediated dopamine release was unchanged between saline and Dox treatment groups. A single systemic administration of SRI-32743 prior to animal sacrifice reversed the increased dopamine release in the baseline of phasic dopamine release and nomifensine-augmented dopamine levels in Dox-treated iTat-tg mice, while SRI-32743 alone did not alter baseline of dopamine release. These findings suggest that Tat expression induced an increase in extracellular dopamine levels by not only inhibiting DAT-mediated dopamine transport but also stimulating synaptic dopamine release. Thus, DAT allosteric modulators may serve as a potential therapeutic intervention for HIV infection-dysregulated dopamine system observed in HIV-1 positive individuals. SIGNIFICANCE STATEMENT: HIV infection-induced dysregulation of the dopaminergic system has been implicated in the development of neurocognitive impairments observed in HIV positive patients. Understanding the mechanisms underlying HIV-1 Tat protein-induced alteration of extracellular dopamine levels will provide insights into the development of molecules that can attenuate Tat interaction with targets in the dopaminergic system. Here, we determined whether Tat alters dopamine release and how the novel DAT allosteric modulator, SRI-32743, impacts dopamine neurotransmission to attenuate Tat-induced effects on extracellular dopamine dynamics.

Mutations of tyrosine 467 in the human norepinephrine transporter attenuate HIV-1 Tat-induced inhibition of dopamine transport while retaining physiological function.

Dysregulation of dopaminergic transmission induced by the HIV-1 transactivator of transcription (Tat) has been implicated as a central factor in the development of HIV-1 associated neurocognitive disorders (HAND). We have demonstrated that the tyrosine470 residue of the human dopamine transporter (hDAT) plays a critical role in Tat-hDAT interaction. Based on the computational modeling predictions, the present study sought to examine the mutational effects of the tyrosine467 residue of the human norepinephrine transporter (hNET), a corresponding residue of the hDAT tyrosine470, on Tat-induced inhibition of reuptake of dopamine through the hNET. Mutations of the hNET tyrosine467 to a histidine (Y467H) or a phenylalanine (Y467F) displayed similar kinetic properties of reuptake of [3H]dopamine and [3H]norepinephrine in PC12 cells expressing wild-type hNET and its mutants. Compared to wild-type hNET, neither of Y467H or Y467F altered Bmax and Kd values of [3H]WIN35,428 binding, whereas Y467H but not Y467F decreased the Bmax of [3H]nisoxetine binding without changes in Kd. Y467H also increased the affinity of nisoxetine for inhibiting [3H]dopamine uptake relative to wild-type hNET. Recombinant Tat1-86 (140 nM) induced a significant reduction of [3H]dopamine uptake in wild-type hNET, which was attenuated in both Y467H and Y467F. Compared to wild-type hNET, neither Y467H or Y467F altered [3H]dopamine efflux in CHO cells expressing WT hNET and mutants, whereas Y467F but not Y467H decreased [3H]MPP+ efflux. These results demonstrate tyrosine467 as a functional recognition residue in the hNET for Tat-induced inhibition of dopamine transport and provide a novel insight into the molecular basis for developing selective compounds that target Tat-NET interactions in the context of HAND.

SRI-32743, a novel allosteric modulator, attenuates HIV-1 Tat protein-induced inhibition of the dopamine transporter and alleviates the potentiation of cocaine reward in HIV-1 Tat transgenic mice

Cocaine abuse increases the incidence of HIV-1-associated neurocognitive disorders. We have demonstrated that HIV-1 transactivator of transcription (Tat) allosterically modulates dopamine (DA) reuptake through the human DA transporter (hDAT), potentially contributing to Tat-induced cognitive impairment and potentiation of cocaine conditioned place preference (CPP). This study determined the effects of a novel allosteric modulator of DAT, SRI-32743, on the interactions of HIV-1 Tat, DA, cocaine, and [3H]WIN35,428 with hDAT in vitro. SRI-32743 (50 nM) attenuated Tat-induced inhibition of [3H]DA uptake and decreased the cocaine-mediated dissociation of [3H]WIN35,428 binding in CHO cells expressing hDAT, suggesting a SRI-32743-mediated allosteric modulation of the Tat-DAT interaction. In further in vivo studies utilizing doxycycline-inducible Tat transgenic (iTat-tg) mice, 14 days of Tat expression significantly reduced the recognition index by 31.7% in the final phase of novel object recognition (NOR) and potentiated cocaine-CPP 2.7-fold compared to responses of vehicle-treated control iTat-tg mice. The Tat-induced NOR deficits and potentiation of cocaine-CPP were not observed in saline-treated iTat-tg or doxycycline-treated G-tg (Tat-null) mice. Systemic administration (i.p.) of SRI-32743 prior to behavioral testing ameliorated Tat-induced impairment of NOR (at a dose of 10 mg/kg) and the Tat-induced potentiation of cocaine-CPP (at doses of 1 or 10 mg/kg). These findings demonstrate that Tat and cocaine interactions with DAT may be regulated by compounds interacting at the DAT allosteric modulatory sites, suggesting a potential therapeutic intervention for HIV-infected patients with concurrent cocaine abuse.

Age-related neuroendocrine, cognitive, and behavioral co-morbidities are promoted by HIV-1 Tat expression in male mice.

In the U.S. about half of the HIV-infected individuals are aged 50 and older. In men living with HIV, secondary hypogonadism is common and occurs earlier than in seronegative men, and its prevalence increases with age. While the mechanisms(s) are unknown, the HIV-1 trans-activator of transcription (Tat) protein disrupts neuroendocrine function in mice partly by dysregulating mitochondria and neurosteroidogenesis. We hypothesized that conditional Tat expression in middle-aged male transgenic mice [Tat(+)] would promote age-related comorbidities compared to age-matched controls [Tat(−)]. We expected Tat to alter steroid hormone milieu consistent with behavioral deficits. Middle-aged Tat(+) mice had lower circulating testosterone and progesterone than age-matched controls and greater circulating corticosterone and central allopregnanolone than other groups. Young Tat(+) mice had greater circulating progesterone and estradiol-to-testosterone ratios. Older age or Tat exposure increased anxiety-like behavior (open field; elevated plus-maze), increased cognitive errors (radial arm water maze), and reduced grip strength. Young Tat(+), or middle-aged Tat(−), males had higher mechanical nociceptive thresholds than age-matched counterparts. Steroid levels correlated with behaviors. Thus, Tat may contribute to HIV-accelerated aging.

Memantine Attenuates Cocaine and neuroHIV Neurotoxicity in the Medial Prefrontal Cortex.

Individuals with substance use disorder are at a higher risk of contracting HIV and progress more rapidly to AIDS as drugs of abuse, such as cocaine, potentiate the neurotoxic effects of HIV-associated proteins including, but not limited to, HIV-1 trans-activator of transcription (Tat) and the envelope protein Gp120. Neurotoxicity and neurodegeneration are hallmarks of HIV-1-associated neurocognitive disorders (HANDs), which are hypothesized to occur secondary to excitotoxicity from NMDA-induced neuronal calcium dysregulation, which could be targeted with NMDA antagonist drugs. Multiple studies have examined how Gp120 affects calcium influx and how cocaine potentiates this influx; however, they mostly focused on single cells and did not analyze effects in neuronal and vascular brain networks. Here, we utilize a custom multi-wavelength imaging platform to simultaneously study the neuronal activity (detected using genetically encoded Ca2+ indicator, GcaMP6f, expressed in neurons) and hemodynamic changes (measured by total hemoglobin and oxygenated hemoglobin within the tissue) in the prefrontal cortex (PFC) of HIV-1 Tg rats in response to cocaine and evaluate the effects of the selective NMDA antagonist drug memantine on cocaine and HIV neurotoxicity compared to those of non-HIV-1 Tg animals (controls). Our results show that memantine improved cocaine-induced deficit in cerebral blood volume while also attenuating an abnormal increase of the neuronal calcium influx and influx duration in both control rats and HIV-1 Tg rats. Cocaine-induced neuronal and hemodynamic dysregulations were significantly greater in HIV-1 Tg rats than in control rats. With memantine pretreatment, HIV-1 Tg rats showed attenuated cocaine’s effects on neuronal and hemodynamic responses, with responses similar to those observed in control rats. These imaging results document an enhancement of neuronal Ca2+ influx, hypoxemia, and ischemia with cocaine in the PFC of HIV-1 Tg rats that were attenuated by memantine pretreatment. Thus, the potential utility of memantine in the treatment of HAND and of cocaine-induced neurotoxicity deserves further investigation.

Molecular Modeling of Subtype-Specific Tat Protein Signatures to Predict Tat-TAR Interactions That May Be Involved in HIV-Associated Neurocognitive Disorders.

HIV-1 is responsible for a spectrum of neurocognitive deficits defined as HIV-associated neurocognitive disorders (HAND). The HIV transactivator of transcription (Tat) protein plays a key role in the neuropathophysiology of HAND. The Tat protein functions by transactivation of viral genes through its interaction with the transactivation response (TAR) RNA element. Subtype-specific Tat protein signatures including C31S, R57S and Q63E present in Tat subtype C has previously been linked to a lowered neuropathophysiology compared to Tat subtype B. In this study, we attempted to understand the molecular mechanism by which Tat subtype-specific variation, particularly, C31S, R57S, and Q63E influence the Tat-TAR interaction. We performed molecular modeling to generate accurate three-dimensional protein structures of the HIV-1 Tat subtypes C and B using the Swiss model webserver. Thereafter, we performed a molecular docking of the TAR RNA element to each of the Tat subtypes B and C protein structures using the HDOCK webserver. Our findings indicate that Tat subtype B had a higher affinity for the TAR RNA element compared to Tat subtype C based on a higher docking score of −187.37, a higher binding free energy value of −9834.63 ± 216.17 kJ/mol, and a higher number of protein–nucleotide interactions of 26. Furthermore, Tat subtype B displayed more flexible regions when bound to the TAR element and this flexibility could account for the stronger affinity of Tat subtype B to TAR. From the Tat signatures linked to neuropathogenesis, only R57/R57S are involved in Tat-TAR interaction. Due to the lack of electrostatic interactions observed between Tat subtype C and TAR, weaker affinity is observed, and this may contribute to a lower level of neuropathophysiology observed in subtype C infection.

Endolysosome iron restricts Tat-mediated HIV-1 LTR transactivation by increasing HIV-1 Tat oligomerization and β-catenin expression

HIV-1 transactivator of transcription (Tat) protein is required for HIV-1 replication, and it has been implicated in the pathogenesis of HIV-1-associated neurocognitive disorder (HAND). HIV-1 Tat can enter cells via receptor-mediated endocytosis where it can reside in endolysosomes; upon its escape from these acidic organelles, HIV-1 Tat can enter the cytosol and nucleus where it activates the HIV-1 LTR promoter. Although it is known that HIV-1 replication is affected by the iron status of people living with HIV-1 (PLWH), very little is known about how iron affects HIV-1 Tat activation of the HIV-1 LTR promoter. Because HIV-1 proteins de-acidify endolysosomes and endolysosome de-acidification affects subcellular levels and actions of iron, we tested the hypothesis that the endolysosome pool of iron is sufficient to affect Tat-induced HIV-1 LTR transactivation. Ferric (Fe3+) and ferrous (Fe2+) iron both restricted Tat-mediated HIV-1 LTR transactivation. Chelation of endolysosome iron with deferoxamine (DFO) and 2-2 bipyridyl, but not chelation of cytosolic iron with deferiprone and deferasirox, significantly enhanced Tat-mediated HIV-1 LTR transactivation. In the presence of iron, HIV-1 Tat increasingly oligomerized and DFO prevented the oligomerization. DFO also reduced protein expression levels of the HIV-1 restriction agent beta-catenin in the cytosol and nucleus. These findings suggest that DFO increases HIV-1 LTR transactivation by increasing levels of the more active dimeric form of Tat relative to the less active oligomerized form of Tat, increasing the escape of dimeric Tat from endolysosomes, and/or reducing beta-catenin protein expression levels. Thus, intracellular iron might play a significant role in regulating HIV-1 replication, and these findings raise cautionary notes for chelation therapies in PLWH.

HIV Tat Protein Induces Myocardial Fibrosis Through TGF-β1-CTGF Signaling Cascade: A Potential Mechanism of HIV Infection-Related Cardiac Manifestations.

Human immunodeficiency virus (HIV) infection is a risk factor of cardiovascular diseases (CVDs). HIV-infected patients exhibit cardiac dysfunction coupled with cardiac fibrosis. However, the reason why HIV could induce cardiac fibrosis remains largely unexplored. HIV-1 trans-activator of transcription (Tat) protein is a regulatory protein, which plays a critical role in the pathogenesis of various HIV-related complications. In the present study, recombinant Tat was administered to mouse myocardium or neonatal mouse cardiac fibroblasts in different doses. Hematoxylin-eosin and Masson's trichrome staining were performed to observe the histological changes of mice myocardial tissues. EdU staining and MTS assay were used to evaluate the proliferation and viability of neonatal mouse cardiac fibroblasts, respectively. Real-time PCR and western blot analysis were used to detect CTGF, TGF-β1, and collagen I mRNA and protein expression levels, respectively. The results showed that Tat promoted the occurrence of myocardial fibrosis in mice. Also, we found that Tat increased the proliferative ability and the viability of neonatal mouse cardiac fibroblasts. The protein and mRNA expression levels of TGF-β1 and CTGF were significantly upregulated both in Tat-treated mouse myocardium and neonatal mouse cardiac fibroblasts. However, co-administration of TGF-β inhibitor abrogated the enhanced expression of collagen I induced by Tat in neonatal mouse cardiac fibroblasts. In conclusion, Tat contributes to HIV-related cardiac fibrosis through enhanced TGF-β1-CTGF signaling cascade.

HIV Tat-Mediated Induction of Monocyte Transmigration Across the Blood–Brain Barrier: Role of Chemokine Receptor CXCR3

HIV trans-activator of transcription (Tat), one of the cytotoxic proteins secreted from HIV-infected cells, is also known to facilitate chemokine-mediated transmigration of monocytes into the brain leading, in turn, to neuroinflammation and thereby contributing to the development of HIV-associated neurocognitive disorders (HAND). The mechanism(s) underlying HIV Tat-mediated enhancement of monocyte transmigration, however, remain largely unknown. CXC chemokine receptor 3 (CXCR3) that is expressed by the peripheral monocytes is known to play a role in the monocyte influx and accumulation. In the present study, we demonstrate for the first time that exposure of human monocytes to HIV Tat protein resulted in upregulated expression of CXCR3 leading, in turn, to increased monocyte transmigration across the blood–brain barrier (BBB) both in the in vitro and in vivo model systems. This process involved activation of toll-like receptor 4 (TLR4), with downstream phosphorylation and activation of TANK-binding kinase 1 (TBK1), and subsequent phosphorylation and nuclear translocation of interferon regulatory factor 3 (IRF3), ultimately leading to enhanced expression of CXCR3 in human monocytes. These findings imply a novel molecular mechanism underlying HIV Tat-mediated increase of monocyte transmigration across the BBB, while also implicating a novel role of CXCR3-dependent monocyte transmigration in HIV Tat-mediated neuroinflammation.

Prevention of HIV-1 TAT Protein-Induced Peripheral Neuropathy and Mitochondrial Disruption by the Antimuscarinic Pirenzepine.

HIV-associated distal sensory polyneuropathy (HIV-DSP) affects about one third of people with HIV and is characterized by distal degeneration of axons. The pathogenesis of HIV-DSP is not known and there is currently no FDA-approved treatment. HIV trans-activator of transcription (TAT) is associated with mitochondrial dysfunction and neurotoxicity in the brain and may play a role in the pathogenesis of HIV-DSP. In the present study, we measured indices of peripheral neuropathy in the doxycycline (DOX)-inducible HIV-TAT (iTAT) transgenic mouse and investigated the therapeutic efficacy of a selective muscarinic subtype-1 receptor (M1R) antagonist, pirenzepine (PZ). PZ was selected as we have previously shown that it prevents and/or reverses indices of peripheral neuropathy in multiple disease models. DOX alone induced weight loss, tactile allodynia and paw thermal hypoalgesia in normal C57Bl/6J mice. Conduction velocity of large motor fibers, density of small sensory nerve fibers in the cornea and expression of mitochondria-associated proteins in sciatic nerve were unaffected by DOX in normal mice, whereas these parameters were disrupted when DOX was given to iTAT mice to induce TAT expression. Daily injection of PZ (10 mg/kg s.c.) prevented all of the disorders associated with TAT expression. These studies demonstrate that TAT expression disrupts mitochondria and induces indices of sensory and motor peripheral neuropathy and that M1R antagonism may be a viable treatment for HIV-DSP. However, some indices of neuropathy in the DOX-inducible TAT transgenic mouse model can be ascribed to DOX treatment rather than TAT expression and data obtained from animal models in which gene expression is modified by DOX should be accompanied by appropriate controls and treated with due caution.

Mutations of Human DopamineTransporter at Tyrosine88, Aspartic Acid206, and Histidine547 Influence Basal and HIV-1 Tat\u2010inhibited Dopamine Transport

HIV-1 transactivator of transcription (Tat) has a great impact on the development of HIV-1 associated neurocognitive disorders through disrupting dopamine transmission. This study determined the mutational effects of human dopamine transporter (hDAT) on basal and Tat-induced inhibition of dopamine transport. Compared to wild-type hDAT, the maximal velocity (Vmax) of [3H]dopamine uptake was decreased in D381L and Y88F/D206L/H547A, increased in D206L/H547A, and unaltered in D206L. Recombinant TatR1 − 86 inhibited dopamine uptake in wild-type hDAT, which was attenuated in either DAT mutants (D206L, D206L/H547A, and Y88F/D206L/H547A) or mutated TatR1 − 86 (K19A and C22G), demonstrating perturbed Tat-DAT interaction. Mutational effects of hDAT on the transporter conformation were evidenced by attenuation of zinc-induced increased [3H]WIN35,428 binding in D206L/H547A and Y88F/D206A/H547A and enhanced basal MPP+ efflux in D206L/H547A. H547A-induced outward-open transport conformational state was further validated by enhanced accessibility to MTSET ([2-(trimethylammonium)ethyl]-methanethiosulfonate) of an inserted cysteine (I159C) on a hDAT background.. Furthermore, H547A displayed an increase in palmitoylation inhibitor-induced inhibition of dopamine uptake relative to wide-type hDAT, indicating a change in basal palmitoylation in H547A. These results demonstrate that Y88F, D206L, and H547A attenuate Tat inhibition while preserving DA uptake, providing insights into identifying targets for improving DAT-mediated dopaminergic dysregulation.Graphical HIV-1 Tat inhibits dopamine uptake through human dopamine transporter (hDAT) on the presynaptic terminal through a direct allosteric interaction. Key hDAT residues D-H547, D-Y88, and D-D206 are predicted to be involved in the HIV-1 Tat-DAT binding. Mutating these residues attenuates this inhibitory effect by disrupting the Tat-hDAT interaction

TAT-modified serum albumin nanoparticles for sustained-release of tetramethylpyrazine and improved targeting to spinal cord injury

BackgroundSpinal Cord injury (SCI) is a kind of severe traumatic disease. The inflammatory response is a significant feature after SCI. Tetramethylpyrazine (TMP), a perennial herb of umbelliferae, is an alkaloid extracted from ligustici. TMP can inhibit the production of nitric oxide and reduce the inflammatory response in peripheral tissues. It can be seen that the therapeutic effect of TMP on SCI is worthy of affirmation. TMP has defects such as short half-life and poor water-solubility. In addition, the commonly used dosage forms of TMP include tablets, dropping pills, injections, etc., and its tissue and organ targeting is still a difficult problem to solve. To improve the solubility and targeting of TMP, here, we developed a nanotechnology-based drug delivery system, TMP-loaded nanoparticles modified with HIV trans-activator of transcription (TAT-TMP-NPs).ResultsThe nanoparticles prepared in this study has integrated structure. The hemolysis rate of each group is less than 5%, indicating that the target drug delivery system has good safety. The results of in vivo pharmacokinetic studies show that TAT-TMP-NPs improves the bioavailability of TMP. The quantitative results of drug distribution in vivo show that TAT-TMP-NPs is more distributed in spinal cord tissue and had higher tissue targeting ability compared with other treatment groups.ConclusionsThe target drug delivery system can overcome the defect of low solubility of TMP, achieve the targeting ability, and show the further clinical application prospect.

Escalating morphine dosing in HIV-1 Tat transgenic mice with sustained Tat exposure reveals an allostatic shift in neuroinflammatory regulation accompanied by increased neuroprotective non-endocannabinoid lipid signaling molecules and amino acids

BackgroundHuman immunodeficiency virus type-1 (HIV-1) and opiates cause long-term inflammatory insult to the central nervous system (CNS) and worsen disease progression and HIV-1-related neuropathology. The combination of these proinflammatory factors reflects a devastating problem as opioids have high abuse liability and continue to be prescribed for certain patients experiencing HIV-1-related pain.MethodsHere, we examined the impact of chronic (3-month) HIV-1 transactivator of transcription (Tat) exposure to short-term (8-day), escalating morphine in HIV-1 Tat transgenic mice that express the HIV-1 Tat protein in a GFAP promoter-regulated, doxycycline (DOX)-inducible manner. In addition to assessing morphine-induced tolerance in nociceptive responses organized at spinal (i.e., tail-flick) and supraspinal (i.e., hot-plate) levels, we evaluated neuroinflammation via positron emission tomography (PET) imaging using the [18F]-PBR111 ligand, immunohistochemistry, and cytokine analyses. Further, we examined endocannabinoid (eCB) levels, related non-eCB lipids, and amino acids via mass spectrometry. ResultsTat-expressing [Tat(+)] transgenic mice displayed antinociceptive tolerance in the tail withdrawal and hot-plate assays compared to control mice lacking Tat [Tat(−)]. This tolerance was accompanied by morphine-dependent increases in Iba-1 ± 3-nitrotryosine immunoreactive microglia, and alterations in pro- and anti-inflammatory cytokines, and chemokines in the spinal cord and striatum, while increases in neuroinflammation were absent by PET imaging of [18F]-PBR111 uptake. Tat and morphine exposure differentially affected eCB levels, non-eCB lipids, and specific amino acids in a region-dependent manner. In the striatum, non-eCB lipids were significantly increased by short-term, escalating morphine exposure, including peroxisome proliferator activator receptor alpha (PPAR-α) ligands N-oleoyl ethanolamide (OEA) and N-palmitoyl ethanolamide (PEA), as well as the amino acids phenylalanine and proline. In the spinal cord, Tat exposure increased amino acids leucine and valine, while morphine decreased levels of tyrosine and valine but did not affect eCBs or non-eCB lipids.ConclusionOverall results demonstrate that 3 months of Tat exposure increased morphine tolerance and potentially innate immune tolerance evidenced by reductions in specific cytokines (e.g., IL-1α, IL-12p40) and microglial reactivity. In contrast, short-term, escalating morphine exposure acted as a secondary stressor revealing an allostatic shift in CNS baseline inflammatory responsiveness from sustained Tat exposure.

Abstract PO-31: HIV-1 transactivator of transcription deregulates key Burkitt lymphoma associated oncogenes at both the transcriptional and post-transcriptional level

Abstract Burkitt lymphoma (BL) is one of the most common HIV-associated lymphomas. This cancer represents one of the most frequent causes of mortality among HIV+ people in Southern Africa, which has the highest incidence of HIV/AIDS worldwide. As is the case for Kaposi sarcoma, recent reports associate an oncogenic function with HIV in lymphoma development. This study explores the oncogenic potential of HIV-1 protein Transactivator of transcription (Tat) in BL via its ability to manipulate the expression of c-MYC and AID, two key drivers of disease in BL. The ability of HIV-1 Tat to influence the activity of the full-length (FL) (2861 bp) c-MYC promoter was assessed using Dual Luciferase Reporter (DLR) assays. Using sequential deletions, the minimal promoter region mediating the effect was identified. Site-directed mutagenesis (SDM) was used to identify promoter elements mediating the response. Co-immunoprecipitation assays (Co-IP) were used to assess interactions between Tat and the AP-1 factor JunB. Chromatin Immunoprecipitation (ChIP) assays were done to confirm that JunB bound the promoter in vivo. To assess the ability of Tat to mediate the expression of miRNA-181b-5p, a known repressor of AICDA, Ramos cells were transfected with Tat followed by qRT-PCR. Corresponding changes in AID protein levels were determined using Western blot. To assess whether miRNA-181b-5p targets AICDA in B cells, the FL AICDA 3'UTR was cloned into the pGL3-Promoter vector and DLR assays were performed in the presence of miRNA-181b-5p mimic. Our data reveal that Tat enhances c-MYC promoter activity in a dose-dependent manner, with a maximum activity of 2.6-fold recorded when 500ng of pcDNA-Tat was used on 400ng of pGL3-c-MYC plasmid harboring the FL promoter. A loss of 35% activity was observed [1.7 (±0.05) fold] upon promoter deletion and two AP-1 sites (positions -1128 bp and -1376 bp) within the deleted region were found to mediate the Tat-dependent increase. A loss in activity of 20.6% was recorded when AP-1 site 1 was mutated, and 25% when AP-1 site 2 was mutated. A double mutant decreased the activity by 29%. Co-IP revealed a strong interaction between Tat and AP-1 factor JunB, and ChIP data showed that JunB was strongly recruited to those two AP-1 sites in the presence of Tat. Our result also reveals that the expression of the AICDA specific miRNA181b-5p is significantly reduced in the presence of Tat, which correlates with an increase in AID protein levels. We also confirm that the AICDA FL 3'UTR, which contains putative miRNA-181b-5p binding sites, is partially repressed by a miRNA-181b-5p mimic. Our study reveals that Tat potentiates oncogenesis by enhancing the expression of c-MYC and AID, two key lymphoma drivers. We show that Tat can enhance c-MYC expression by enhancing the recruitment of cellular TFs to the promoter. We also show that the same HIV protein can enhance the expression of AID by modulating the expression of miRNA-181b-5p. Citation Format: Leonardo Alves de Souza Rios, Nontlantla Mdletshe, Shaheen Mowla. HIV-1 transactivator of transcription deregulates key Burkitt lymphoma associated oncogenes at both the transcriptional and post-transcriptional level [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-31.

Voltage-dependent calcium channel β subunit-derived peptides reduce excitatory neurotransmission and arterial blood pressure

AimsVoltage-dependent calcium channels (VDCCs) play an important role in various physiological functions in the nervous system and the cardiovascular system. In L-, N-, P/Q-, and R-type VDCCs, β subunit assists the channels for membrane targeting and modulates channel properties. In this study, we investigated whether an inhibition of the β subunit binding to α subunit, the pore-forming main subunit of VDCCs, have any effect on channel activation and physiological functions. Main methodsPeptides derived from the specific regions of β subunit that bind to the α-interaction domain in I-II linker of α subunit were manufactured, presuming that the peptides interrupt α-β subunit interaction in the channel complex. Then, they were tested on voltage-activated Ca2+ currents recorded in acutely isolated trigeminal ganglion (TG) neurons, excitatory postsynaptic currents (EPSCs) in the spinal dorsal horn neurons, and arterial blood pressure (BP) recorded from the rat femoral artery. Key findingsWhen applied internally through patch pipettes, the peptides decreased the peak amplitudes of the voltage-activated Ca2+ currents. After fusing with HIV transactivator of transcription (TAT) sequence to penetrate cell membrane, the peptides significantly decreased the peak amplitudes of Ca2+ currents and the peak amplitudes of EPSCs upon the external application through bath solution. Furthermore, the TAT-fused peptides dose dependently reduced the rat BP when administered intravenously. SignificanceThese data suggest that an interruption of α-β subunit association in VDCC complex inhibits channel activation, thereby reducing VDCC-mediated physiological functions such as excitatory neurotransmission and arterial BP.