Enzyme Hinders HIV-1 Tat Viral Transport and Real-Time Measured with Nanopores.

HIV-1 Tat protein, an intercellular transporter with a determinant function of delivering "information-rich" molecules in viral multiplication, was tryptic-hydrolyzed and real-time single molecule-monitored in a transmembrane pore. The electrokinetic studies revealed the catalytic and inhibitory effects on enzymatic digestion associated with Ca2+ and Cu2+ ions, respectively, in response to binding interactions with trypsin. Our strategy permits accurate and distinguishable sensing of Ca2+ and Cu2+via an enzyme assay. In addition, considering the closer mimic of the real situation of HIV spread, measurements in the serum and on cells were also investigated. Transmembrane current measurements together with fluorescence microscopy imaging indicated the potential to perturb the Tat transport in the serum environment and on cells. Because the involved Tat proteolysis should prevent the occurrence of viral delivery, the presented method probably enables efficient hindrance to HIV-1 infection, in complementary to current traditional treatments.

Arginine Forks Are a Widespread Motif to Recognize Phosphate Backbones and Guanine Nucleobases in the RNA Major Groove.

RNA recognition by proteins is central to biology. Here we demonstrate the existence of a recurrent structural motif, the "arginine fork", that codifies arginine readout of cognate backbone and guanine nucleobase interactions in a variety of protein-RNA complexes derived from viruses, metabolic enzymes, and ribosomes. Nearly 30 years ago, a theoretical arginine fork model was posited to account for the specificity between the HIV-1 Tat protein and TAR RNA. This model predicted that a single arginine should form four complementary contacts with nearby phosphates, yielding a two-pronged backbone readout. Recent high-resolution structures of TAR-protein complexes have unveiled new details, including (i) arginine interactions with the phosphate backbone and the major-groove edge of guanine and (ii) simultaneous cation-π contacts between the guanidinium group and flanking nucleobases. These findings prompted us to search for arginine forks within experimental protein-RNA structures retrieved from the Protein Data Bank. The results revealed four distinct classes of arginine forks that we have defined using a rigorous but flexible nomenclature. Examples are presented in the context of ribosomal and nonribosomal interfaces with analysis of arginine dihedral angles and structural (suite) classification of RNA targets. When arginine fork chemical recognition principles were applied to existing structures with unusual arginine-guanine recognition, we found that the arginine fork geometry was more consistent with the experimental data, suggesting the utility of fork classifications to improve structural models. Software to analyze arginine-RNA interactions has been made available to the community.

Role of Divalent Cations in HIV-1 Replication and Pathogenicity.

Divalent cations are essential for life and are fundamentally important coordinators of cellular metabolism, cell growth, host-pathogen interactions, and cell death. Specifically, for human immunodeficiency virus type-1 (HIV-1), divalent cations are required for interactions between viral and host factors that govern HIV-1 replication and pathogenicity. Homeostatic regulation of divalent cations' levels and actions appear to change as HIV-1 infection progresses and as changes occur between HIV-1 and the host. In people living with HIV-1, dietary supplementation with divalent cations may increase HIV-1 replication, whereas cation chelation may suppress HIV-1 replication and decrease disease progression. Here, we review literature on the roles of zinc (Zn2+), iron (Fe2+), manganese (Mn2+), magnesium (Mg2+), selenium (Se2+), and copper (Cu2+) in HIV-1 replication and pathogenicity, as well as evidence that divalent cation levels and actions may be targeted therapeutically in people living with HIV-1.

Hindsiipropane B alleviates HIV-1 Tat-induced inflammatory responses by suppressing HDAC6-NADPH oxidase-ROS axis in astrocytes.

Human immunodeficiency virus-1 (HIV-1) transactivator of transcription (Tat) is an important viral factor in neuroinflammation. Hindsiipropane B, present in Celastrus hindsii, possesses various biological mechanisms including antiinflammatory activity. In this report, we explored the regulatory activity of hindsiipropane B on HIV-1 Tat-mediated chemokine production and its mode of action in astrocytes. Hindsiipropane B significantly alleviated HIV-1 Tat-mediated production of inflammatory chemokines, CCL2, CXCL8, and CXCL10. Hindsiipropane B inhibited expression of HDAC6, which is important regulator in HIV-1 Tat-mediated chemokine production. Hindsiipropane B diminished HIV-1 Tat-mediated reactive oxygen species (ROS) generation and NADPH oxidase activation/expression. Furthermore, hindsiipropane B inhibited HIV-1 Tat-mediated signaling cascades including MAPK, NF-κB, and AP-1. These data suggest that hindsiipropane B exerts its inhibitory effects on HIV-1 Tat-mediated chemokine production via down-regulating the HDAC6-NADPH oxidase-MAPK-NF-κB/AP-1 signaling axis, and could serve as a therapeutic lead compound against HIV-1 Tat-associated neuroinflammation. [BMB Reports 2018; 51(8): 394-399].

Dysregulation of mitochondrial bioenergetics and quality control by HIV-1 Tat in cardiomyocytes.

Cardiovascular disease remains a leading cause of morbidity and mortality in HIV-positive patients, even in those whose viral loads are well controlled with antiretroviral therapy. However, the underlying molecular events responsible for the development of cardiac disease in the setting of HIV remain unknown. The HIV-encoded Tat protein plays a critical role in the activation of HIV gene expression and profoundly impacts homeostasis in both HIV-infected cells and uninfected cells that have taken up released Tat via a bystander effect. Since cardiomyocyte function, including excitation-contraction coupling, greatly depends on energy provided by the mitochondria, in this study, we performed a series of experiments to assess the impact of Tat on mitochondrial function and bioenergetics pathways in a primary cell culture model derived from neonatal rat ventricular cardiomyocytes (NRVCs). Our results show that the presence of Tat in cardiomyocytes is accompanied by a decrease in oxidative phosphorylation, a decline in the levels of ATP, and an accumulation of reactive oxygen species (ROS). Tat impairs the uptake of mitochondrial Ca2+ ([Ca2+ ]m ) and the electrophysiological activity of cardiomyocytes. Tat also affects the protein clearance pathway and autophagy in cardiomyocytes under stress due to hypoxia-reoxygenation conditions. A reduction in the level of ubiquitin along with dysregulated degradation of autophagy proteins including SQSTM1/p62 and a reduction of LC3 II were detected in cardiomyocytes harboring Tat. These results suggest that, by targeting mitochondria and protein quality control, Tat significantly impacts bioenergetics and autophagy resulting in dysregulation of cardiomyocyte health and homeostasis.

Menin mediates Tat-induced neuronal apoptosis in brain frontal cortex of SIV-infected macaques and in Tat-treated cells.

The molecular mechanisms involved in human immunodeficiency virus (HIV)-associated neurocognitive disorder (HAND) remain poorly understood. It has been recently reported that HIV-1 Tat transactivation requires menin, suggesting that menin may be involved in HAND pathogenesis. But the role of menin is not clear. Here, we found that protein level of menin was increased in simian-human immunodeficiency chimeric virus (SHIV)-SF162.P4 and simian immunodeficiency virus (SIV) sm543-3-infected rhesus macaques compared with the controls by immunohistochemistry (IHC) and western blot. Menin mainly expressed in the frontal cortex neurons of the brain, more importantly, the number of menin-staining cells was positively correlated with cleaved-caspase-3-positive cells while it was negatively correlated with a neuron-specific nuclear protein NeuN-positive cells, suggesting that expression of menin may induce neuronal apoptosis. Further studies showed that menin level was significantly increased during Tat-induced apoptosis, while downregulation of menin by pll3.7-MEN1-shRNA attenuated the Tat-induced cleavage of caspase-3 and caspase-8 in SY5Y cells and primary neuron cultures. Together, our findings reveal a pro-apoptotic role of menin in the brains of the SIV-infected macaques and the cultured neurons, indicating that targeting menin may be potential to block the HIV-1 Tat induced neuronal damage in HAND.

Probing interaction of a fluorescent ligand with HIV TAR RNA.

Trans-activator of Transcription (Tat) antagonists could block the interaction between Tat protein and its target, trans-activation responsive region (TAR) RNA, to inhibit Tat function and prevent human immunodeficiency virus type 1 (HIV-1) replication. For the first time, a small fluorescence ligand, ICR 191, was found to interact with TAR RNA at the Tat binding site and compete with Tat. It was also observed that the fluorescence of ICR 191 could be quenched when binding to TAR RNA and recovered when discharged via competition with Tat peptide or a well-known Tat inhibitor, neomycin B. The binding parameters of ICR 191 to TAR RNA were determined through theoretical calculations. Mass spectrometry, circular dichroism and molecular docking were used to further confirm the interaction of ICR 191 with TAR RNA. Inspired by these discoveries, a primary fluorescence model for the discovery of Tat antagonists was built using ICR 191 as a fluorescence indicator and the feasibility of this model was evaluated. This ligand-RNA interaction could provide a new strategy for research aimed at discovering Tat antagonists.

HIV-1 Tat Protein Activates both the MyD88 and TRIF Pathways To Induce Tumor Necrosis Factor Alpha and Interleukin-10 in Human Monocytes.

UNLABELLED: In this study, we show that the HIV-1 Tat protein interacts with rapid kinetics to engage the Toll-like receptor 4 (TLR4) pathway, leading to the production of proinflammatory and anti-inflammatory cytokines. The pretreatment of human monocytes with Tat protein for 10 to 30 min suffices to irreversibly engage the activation of the TLR4 pathway, leading to the production of tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10), two cytokines strongly implicated in the chronic activation and dysregulation of the immune system during HIV-1 infection. Therefore, this study analyzed whether the HIV-1 Tat protein is able to activate these two pathways separately or simultaneously. Using three complementary approaches, including mice deficient in the MyD88, TIRAP/MAL, or TRIF adaptor, biochemical analysis, and the use of specific small interfering RNAs (siRNAs), we demonstrated (i) that Tat was able to activate both the MyD88 and TRIF pathways, (ii) the capacity of Tat to induce TIRAP/MAL degradation, (iii) the crucial role of the MyD88 pathway in the production of Tat-induced TNF-α and IL-10, (iv) a reduction but not abrogation of IL-10 and TNF-α by Tat-stimulated macrophages from mice deficient in TIRAP/MAL, and (v) the crucial role of the TRIF pathway in Tat-induced IL-10 production. Further, we showed that downstream of the MyD88 and TRIF pathways, the Tat protein activated the protein kinase C (PKC) ßII isoform, the mitogen-activated protein (MAP) kinases p38 and extracellular signal-regulated kinase 1/2 (ERK1/2), and NF-κB in a TLR4-dependent manner. Collectively, our data show that by recruiting the TLR4 pathway with rapid kinetics, the HIV-1 Tat protein leads to the engagement of both the MyD88 and TRIF pathways and to the activation of PKC, MAP kinase, and NF-κB signaling to induce the production of TNF-α and IL-10. IMPORTANCE: In this study, we demonstrate that by recruiting the TLR4 pathway with rapid kinetics, the HIV-1 Tat protein leads to the engagement of both the MyD88 and TRIF pathways and to the activation of PKC-ßII, MAP kinase, and NF-κB signaling to induce the production of TNF-α and IL-10, two cytokines strongly implicated in the chronic activation and dysregulation of the immune system during HIV-1 infection. Thus, it may be interesting to target Tat as a pathogenic factor early after HIV-1 infection. This could be achieved either by vaccination approaches including Tat as an immunogen in potential candidate vaccines or by developing molecules capable of neutralizing the effect of the Tat protein.

A Natural Product from Polygonum cuspidatum Sieb. Et Zucc. Promotes Tat-Dependent HIV Latency Reversal through Triggering P-TEFb's Release from 7SK snRNP.

The latent reservoirs of HIV represent a major impediment to eradication of HIV/AIDS. To overcome this problem, agents that can activate latent HIV proviruses have been actively sought after, as they can potentially be used in combination with the highly active antiretroviral therapy (HAART) to eliminate the latent reservoirs. Although several chemical compounds have been shown to activate latency, they are of limited use due to high toxicity and poor clinical outcomes. In an attempt to identify natural products as effective latency activators from traditional Chinese medicinal herbs that have long been widely used in human population, we have isolated procyanidin C-13,3',3"-tri-O-gallate (named as REJ-C1G3) from Polygonum cuspidatum Sieb. et Zucc., that can activate HIV in latently infected Jurkat T cells. REJ-C1G3 preferentially stimulates HIV transcription in a process that depends on the viral encoded Tat protein and acts synergistically with prostratin (an activator of the NF-κB pathway) or JQ1 (an inhibitor of Brd4) to activate HIV latency. Our mechanistic analyses further show that REJ-C1G3 accomplishes these tasks by inducing the release of P-TEFb, a host cofactor essential for Tat-activation of HIV transcription, from the cellular P-TEFb reservoir 7SK snRNP.

HIV-Tat Protein Forms Phosphoinositide-dependent Membrane Pores Implicated in Unconventional Protein Secretion.

HIV-Tat has been demonstrated to be secreted from cells in a phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-dependent manner. Here we show that HIV-Tat forms membrane-inserted oligomers, a process that is accompanied by changes in secondary structure with a strong increase in antiparallel ß sheet content. Intriguingly, oligomerization of HIV-Tat on membrane surfaces leads to the formation of membrane pores, as demonstrated by physical membrane passage of small fluorescent tracer molecules. Although membrane binding of HIV-Tat did not strictly depend on PI(4,5)P2 but, rather, was mediated by a range of acidic membrane lipids, a functional interaction between PI(4,5)P2 and HIV-Tat was critically required for efficient membrane pore formation by HIV-Tat oligomers. These properties are strikingly similar to what has been reported previously for fibroblast growth factor 2 (FGF2), providing strong evidence of a common core mechanism of unconventional secretion shared by HIV-Tat and fibroblast growth factor 2.

Determining the Functions of HIV-1 Tat and a Second Magnesium Ion in the CDK9/Cyclin T1 Complex: A Molecular Dynamics Simulation Study.

The current paradigm of cyclin-dependent kinase (CDK) regulation based on the well-established CDK2 has been recently expanded. The determination of CDK9 crystal structures suggests the requirement of an additional regulatory protein, such as human immunodeficiency virus type 1 (HIV-1) Tat, to exert its physiological functions. In most kinases, the exact number and roles of the cofactor metal ions remain unappreciated, and the repertoire has thus gained increasing attention recently. Here, molecular dynamics (MD) simulations were implemented on CDK9 to explore the functional roles of HIV-1 Tat and the second Mg2+ ion at site 1 (Mg12+). The simulations unveiled that binding of HIV-1 Tat to CDK9 not only stabilized hydrogen bonds (H-bonds) between ATP and hinge residues Asp104 and Cys106, as well as between ATP and invariant Lys48, but also facilitated the salt bridge network pertaining to the phosphorylated Thr186 at the activation loop. By contrast, these H-bonds cannot be formed in CDK9 owing to the absence of HIV-1 Tat. MD simulations further revealed that the Mg12+ ion, coupled with the Mg22+ ion, anchored to the triphosphate moiety of ATP in its catalytic competent conformation. This observation indicates the requirement of the Mg12+ ion for CDK9 to realize its function. Overall, the introduction of HIV-1 Tat and Mg12+ ion resulted in the active site architectural characteristics of phosphorylated CDK9. These data highlighted the functional roles of HIV-1 Tat and Mg12+ ion in the regulation of CDK9 activity, which contributes an important complementary understanding of CDK molecular underpinnings.

Levofloxacin and indolicidin for combination antimicrobial therapy.

Despite the increasing need for antibiotics to fight infectious diseases, fewer new antibiotics are available on the market. Unfortunately, developing a new class of antibiotics is associated with high commercial risk. Therefore, modification or combination of existing antibiotics to improve their efficacy is a promising strategy. Herein, we conjugated the antibiotic, levofloxacin, with two peptides, i.e. an antimicrobial peptide indolicidin and a cell penetrating peptide (TAT). Glycolic acid and glycine linkers were used between levofloxacin and peptides. We developed an optimized condition for coupling of levofloxacin via its carboxylic group to glycolic acid using solid phase peptide synthesis (SPPS). Antibacterial and haemolytic assays were carried out on the conjugates and only the levofloxacin-indolicidin conjugate demonstrated moderate antibacterial activity. Interestingly, physical mixture of levofloxacin and indolicidin showed improvement in the activity against Gram-positive bacteria.

Astrocyte elevated gene-1 is a novel modulator of HIV-1-associated neuroinflammation via regulation of nuclear factor-κB signaling and excitatory amino acid transporter-2 repression.

Astrocyte elevated gene-1 (AEG-1), a novel human immunodeficiency virus (HIV)-1 and tumor necrosis factor (TNF)-α-inducible oncogene, has generated significant interest in the field of cancer research as a therapeutic target for many metastatic aggressive tumors. However, little is known about its role in astrocyte responses during HIV-1 central nervous system (CNS) infection and whether it contributes toward the development of HIV-associated neurocognitive disorders (HAND). Therefore, in this study, we investigated changes in AEG-1 CNS expression in HIV-1-infected brain tissues and elucidated a potential mechanism of AEG-1-mediated regulation of HAND. Immunoblotting and immunohistochemical analyses of HIV-1 seropositive and HIV-1 encephalitic human brain tissues revealed significantly elevated levels of AEG-1 protein. Immunohistochemical analyses of HIV-1 Tat transgenic mouse brain tissues also showed a marked increase in AEG-1 staining. Similar to in vivo observations, cultured astrocytes expressing HIV-1 Tat also revealed AEG-1 and cytokine up-regulation. Astrocytes treated with HAND-relevant stimuli, TNF-α, interleukin (IL)-1ß, and HIV-1, also significantly induced AEG-1 expression and nuclear translocation via activation of the nuclear factor (NF)-κB pathway. Co-immunoprecipitation studies demonstrated IL-1ß- or TNF-α-induced AEG-1 interaction with NF-κB p65 subunit. AEG-1 knockdown decreased NF-κB activation, nuclear translocation, and transcriptional output in TNF-α-treated astrocytes. Moreover, IL-1ß treatment of AEG-1-overexpressing astrocytes significantly lowered expression of excitatory amino acid transporter 2, increased expression of excitatory amino acid transporter 2 repressor ying yang 1, and reduced glutamate clearance, a major transducer of excitotoxic neuronal damage. Findings from this study identify a novel transcriptional co-factor function of AEG-1 and further implicate AEG-1 in HAND-associated neuroinflammation.

HIV-1 Tat disrupts CX3CL1-CX3CR1 axis in microglia via the NF-κBYY1 pathway.

Microglia are critical for the pathogenesis of HIV-associated dementia not only by acting as conduits of viral entry but also as reservoirs for productive and latent virus infection, and as producers of neurotoxins. Interaction between CX3CL1 (fractalkine) and FKN receptor (CX3CR1) is highly functional in the brain, and is known to regulate a complex network of paracrine and autocrine interactions between neurons and microglia. The aim of the present study was to determine which extent of HIV-1 Tat protein causes the alteration of CX3CR1 expression and to investigate the regulatory mechanism for CX3CR1 expression. Here we showed that exposure of primary microglia and BV2 cells to exogenous Tat protein resulted in down-regulation of CX3CR1 mRNA and protein expression, with a concomitant induction of proinflammatory responses. Next, we further showed that NF-κB activation by Tat treatment negatively regulated CX3CR1 expression. Since a YY1 binding site ~10kb upstream of CX3CR1 promoter was predicted in rats, mice and humans, the classical NF-κB-YY1 regulatory pathway was considered. Our findings indicated that Tat repressed CX3CR1 expression via NF-κB-YY1 regulatory pathway. To gain insight into the effect of Tat on CX3CL1-CX3CR1 communication, calcium mobilization, MAPK activation and microglial migration, respectively, were tested in microglial cells after successive treatment with Tat and CX3CL1. The results suggested that Tat disrupted the responses of microglia to CX3CL1. Taken together, these results demonstrate that HIV-1 Tat protein suppresses CX3CR1 expression in microglia via NF-κB-YY1 pathway and attenuates CX3CL1-induced functional response of microglia.

Poligapolide, a PI3K/Akt inhibitor in immunodeficiency virus type 1 TAT-transduced CHME5 cells, isolated from the rhizome of Polygala tenuifolia.

The rhizome of Polygala tenuifolia WILLD (PT, family Polygalaceae) has been used in traditional Chinese medicine for inflammation, dementia, amnesia, neurasthenia and cancer. The phosphoinositide 3-kinase (PI3K)/Akt inhibitor(s) was isolated from PT by using the cytoprotective phenotype of human immunodeficiency virus type 1 (HIV-1) Tat-transduced CHME5 cells against lipopolysaccharide/cycloheximide. We isolated 9 constituents (1)-(9) from ethyl acetate fraction of PT, which potently showed anti-cytoprotective effect against HIV-1 TAT-transduced cells. Of them, (9R)-(-)-9-peptandecanolide (2), a new compound named poligapolide, most potently abolished the cytoprotective effect of HIV-1 Tat-transduced CHME5 cells. The compound (2) inhibited the phosphorylation of Akt and its downstream molecule, glycogen synthase kinase-3 beta (GSK3ß) in PI3K/Akt cell survival signaling pathway, but did not suppress the phosphorylation of PI3K and pyruvate dehydrogenase lipoamide kinase isozyme 1. Based on these finding, poligapolide may abolish the cytoprotective phenotype of HIV-1 Tat-transduced CHME5 cells by inhibiting Akt phosphorylation in PI3K/Akt pathway.

A staggered decameric assembly of human C-reactive protein stabilized by zinc ions revealed by X-ray crystallography.

Human C-reactive protein (CRP) is an acute phase protein, which harbours both host defence and scavenging properties. In this study, we obtained two new crystal forms of CRP, where CRP forms a symmetric, staggered dimer of pentamers. In one of these structures, obtained in the presence of HIV-1 Tat protein, this dimer of pentamers is stabilized by two zinc ions trapped within a cleft of the effector face of CRP. These two decameric interfaces involve complementary surfaces of CRP pentamers and bury a large area of ~2000 Å(2) per pentamer, suggesting a biological role of this interface. These two novel decameric interfaces and the involvement of zinc might have important consequences in the understanding of CRP biological functions.

Comparative analysis of RNA/protein dynamics for the arginine-rich-binding motif and zinc-finger-binding motif proteins encoded by HIV-1.

We report a comparative study in which a single-molecule fluorescence resonance energy transfer approach was used to examine how the binding of two families of HIV-1 viral proteins to viral RNA hairpins locally changes the RNA secondary structures. The single-molecule fluorescence resonance energy transfer results indicate that the zinc finger protein (nucleocapsid) locally melts the TAR RNA and RRE-IIB RNA hairpins, whereas arginine-rich motif proteins (Tat and Rev) may strengthen the hairpin structures through specific binding interactions. Competition experiments show that Tat and Rev can effectively inhibit the nucleocapsid-chaperoned annealing of complementary DNA oligonucleotides to the TAR and RRE-IIB RNA hairpins, respectively. The competition binding data presented here suggest that the specific nucleic acid binding interactions of Tat and Rev can effectively compete with the general nucleic acid binding/chaperone functions of the nucleocapsid protein, and thus may in principle help regulate critical events during the HIV life cycle.

Crystal structure of HIV-1 Tat complexed with human P-TEFb.

Regulation of the expression of the human immunodeficiency virus (HIV) genome is accomplished in large part by controlling transcription elongation. The viral protein Tat hijacks the host cell's RNA polymerase II elongation control machinery through interaction with the positive transcription elongation factor, P-TEFb, and directs the factor to promote productive elongation of HIV mRNA. Here we describe the crystal structure of the Tat.P-TEFb complex containing HIV-1 Tat, human Cdk9 (also known as CDK9), and human cyclin T1 (also known as CCNT1). Tat adopts a structure complementary to the surface of P-TEFb and makes extensive contacts, mainly with the cyclin T1 subunit of P-TEFb, but also with the T-loop of the Cdk9 subunit. The structure provides a plausible explanation for the tolerance of Tat to sequence variations at certain sites. Importantly, Tat induces significant conformational changes in P-TEFb. This finding lays a foundation for the design of compounds that would specifically inhibit the Tat.P-TEFb complex and block HIV replication.

Synthesis and biological evaluation of 2-phenylquinolones targeted at Tat/TAR recognition.

Tat (transactivator of transcription) is a small HIV protein rich in arginines that interacts with a viral RNA structure called TAR (trans-activation responsive region). Tat-TAR interaction is essential for viral gene expression, replication and pathogenesis. Small molecules able to interfere with TAR and to compete for Tat binding possess antiviral activity due to inhibition of viral transcription and expression, thus impairing formation of infectious virions. We report here, the synthesis and biological evaluation of a new series of quinolone derivatives, namely 2-phenylquinolones, designed with the aim of interfering with the protein/RNA complex. These new derivatives are able to efficiently interfere with Tat/TAR complex in vitro depending on precise structural requirements as demonstrated by fluorescence quenching assay analysis.

Thioredoxin reductase-1 negatively regulates HIV-1 transactivating protein Tat-dependent transcription in human macrophages.

Epidemiological studies suggest a correlation between severity of acquired immunodeficiency syndrome (AIDS) and selenium deficiency, indicating a protective role for this anti-oxidant during HIV infection. Here we demonstrate that thioredoxin reductase-1 (TR1), a selenium-containing pyridine nucleotide-disulfide oxidoreductase that reduces protein disulfides to free thiols, negatively regulates the activity of the HIV-1 encoded transcriptional activator, Tat, in human macrophages. We used a small interfering RNA approach as well as a high affinity substrate of TR1, ebselen, to demonstrate that Tat-dependent transcription and HIV-1 replication were significantly increased in human macrophages when TR1 activity was reduced. The increase in HIV-1 replication in TR1 small interfering RNA-treated cells was independent of the redox-sensitive transcription factor, NF-kappaB. These studies indicate that TR-1 acts as a negative regulator of Tat-dependent transcription. Furthermore, in vitro biochemical assays with recombinant Tat protein confirmed that TR1 targets two disulfide bonds within the Cys-rich motif required for efficient HIV-1 transactivation. Increasing TR1 expression along with other selenoproteins by supplementing with selenium suggests a potential inexpensive adjuvant therapy for HIV/AIDS patients.