The trajectory patterns of single HIV-1 virus-like particle in live CD4 cells: A real time three-dimensional multi-resolution microscopy study using encapsulated nonblinking giant quantum dot.

BACKGROUND: The exploration of virology knowledge was limited by the optical technology for the observation of virus. Previously, a three-dimensional multi-resolution real-time microscope system (3D-MRM) was developed to observe the uptake of HIV-1-tat peptide-modified nanoparticles in cell membrane. In this study, we labeled HIV-1 virus-like particles (VLPs) with passivated giant quantum dots (gQDs) and recorded their interactive trajectories with human Jurkat CD4 cells through 3D-MRM. METHODS: The labeled of gQDs of the HIV-1 VLPs in sucrose-gradient purified viral lysates was first confirmed by Cryo-electronic microscopy and Western blot assay. After the infection with CD4 cells, the gQD-labeled VLPs were visualized and their extracellular and intracellular trajectories were recorded by 3D-MRM. RESULTS: A total of 208 prime trajectories was identified and classified into three distinct patterns: cell-free random diffusion pattern, directional movement pattern and cell-associated movement pattern, with distributions and mean durations were 72.6%/87.6 s, 9.1%/402.7 s and 18.3%/68.7 s, respectively. Further analysis of the spatial-temporal relationship between VLP trajectories and CD4 cells revealed the three stages of interactions: (1) cell-associated (extracellular) diffusion stage, (2) cell membrane surfing stage and (3) intracellular directional movement stage. CONCLUSION: A complete trajectory of HIV-1 VLP interacting with CD4 cells was presented in animation. This encapsulating method could increase the accuracy for the observation of HIV-1-CD4 cell interaction in real time and three dimensions.

New insights into pathogenesis point to HIV-1 Tat as a key vaccine target.

Despite over 30 years of enormous effort and progress in the field, no preventative and/or therapeutic vaccines against human immunodeficiency virus (HIV) are available. Here, we briefly summarize the vaccine strategies and vaccine candidates that in recent years advanced to efficacy trials with mostly unsatisfactory results. Next, we discuss a novel and somewhat contrarian approach based on biological and epidemiological evidence, which led us to choose the HIV protein Tat for the development of preventive and therapeutic HIV vaccines. Toward this goal, we review here the role of Tat in the virus life cycle as well as experimental and epidemiological evidence supporting its key role in the natural history of HIV infection and comorbidities. We then discuss the preclinical and clinical development of a Tat therapeutic vaccine, which, by improving the functionality and homeostasis of the immune system and by reducing the viral reservoir in virologically suppressed vaccinees, helps to establish key determinants for intensification of combination antiretroviral therapy (cART) and a functional cure. Future developments and potential applications of the Tat therapeutic vaccine are also discussed, as well as the rationale for its use in preventative strategies. We hope this contribution will lead to a reconsideration of the current paradigms for the development of HIV/AIDS vaccines, with a focus on targeting of viral proteins with key roles in HIV pathogenesis.

HIV-1 Tat: An update on transcriptional and non-transcriptional functions.

Over the past decades, much have been learned about HIV-1 virus and its molecular strategies for pathogenesis. However, HIV-1 still remains an enigmatic virus, particularly because of its unique proteins. Establishment of latency and reactivation is still a puzzling question and various temporal and spatial dynamics between HIV-1 proteins itself have given us new way of thinking about its pathogenesis. HIV-1 replication depends on Tat which is a small unstructured protein and subjected to various post-translational modifications for its myriad of functions. HIV-1 Tat protein modulates the functions of various strategic cellular pathways like proteasomal machinery and inflammatory pathways to aid in HIV-1 pathogenesis. Many of the recent findings have shown that Tat is associated with exosomes, cleared from HIV-1 infected cells through its degradation by diverse routes ranging from lysosomal to proteasomal pathways. HIV-1 Tat was also found to be associated with other HIV-1 proteins including Vpr, Nef, Nucleocapsid (NC) and Rev. Interaction of Tat with Vpr and Nef increases its transactivation function, whereas, interaction of Tat with NC or Rev leads to Tat protein degradation and hence suppression of Tat functions. Research in the recent years has established that Tat is not only important for HIV-1 promoter transactivation and virus replication but also modulating multiple cellular and molecular functions leading to HIV-1 pathogenicity. In this review we discussed various transcriptional and non-transcriptional HIV-1 Tat functions which modulate host cell metabolism during HIV-1 pathogenesis.

HIV-1 Tat Dysregulates the Hypothalamic-Pituitary-Adrenal Stress Axis and Potentiates Oxycodone-Mediated Psychomotor and Anxiety-Like Behavior of Male Mice.

Human immunodeficiency virus (HIV) is associated with co-morbid affective and stress-sensitive neuropsychiatric disorders that may be related to dysfunction of the hypothalamic-pituitary-adrenal (HPA) stress axis. The HPA axis is perturbed in up to 46% of HIV patients, but the mechanisms are not known. The neurotoxic HIV-1 regulatory protein, trans-activator of transcription (Tat), may contribute. We hypothesized that HPA dysregulation may contribute to Tat-mediated interactions with oxycodone, a clinically-used opioid often prescribed to HIV patients. In transgenic male mice, Tat expression produced significantly higher basal corticosterone levels with adrenal insufficiency in response to a natural stressor or pharmacological blockade of HPA feedback, recapitulating the clinical phenotype. On acute exposure, HIV-1 Tat interacted with oxycodone to potentiate psychomotor and anxiety like-behavior in an open field and light-dark transition tasks, whereas repeated exposure sensitized stress-related psychomotor behavior and the HPA stress response. Pharmacological blockade of glucocorticoid receptors (GR) partially-restored the stress response and decreased oxycodone-mediated psychomotor behavior in Tat-expressing mice, implicating GR in these effects. Blocking corticotrophin-releasing factor (CRF) receptors reduced anxiety-like behavior in mice that were exposed to oxycodone. Together, these effects support the notion that Tat exposure can dysregulate the HPA axis, potentially raising vulnerability to stress-related substance use and affective disorders.

[Viral latency of HIV-1]. / La latence virale du VIH-1.

Human immunodeficiency virus (HIV-1) latency is clinically highlighting via the persistence of a residual viral load in cART-treated patients due to the reactivation of cellular reservoirs. Two forms of latency coexist but the contribution of the pre-integrationnal latency clearly plays a minor role in viral persistence. In contrast, the post-integrationnal latency significantly contributes to the evasion of the immune system by the HIV-1 cellular reservoir and consequently to HIV-1 pathogenesis. Although post-transcriptional mechanisms can contribute to the maintenance of viral latency, HIV-1 transcriptional inhibition is critical for the establishment and maintenance of post-integrational latency. This inhibition is a multifactorial phenomenon, making the development of anti-latency therapeutic strategies complex. These different notions will be described throughout this review.

A Review of Angiogenic Imbalance in HIV-Infected Hypertensive Disorders of Pregnancy.

PURPOSE OF REVIEW: This review provides a comprehensive insight into the angiogenic profile of hypertensive and normotensive pregnancies compromised by HIV infection. Furthermore, we evaluate the economic implementation of the sFlt-1/PlGF ratio and review the reports on therapeutic apheresis in limiting sFlt-1 production. RECENT FINDINGS: In preeclampsia, an increased expression of sFlt-1 triggers angiogenic imbalance. Women of African ancestry have high levels of angiogenic factors than other racial groups. The sFlt-1/PlGF ratio shows promise in the early assessment of preeclampsia, while sFlt-1 apheresis restores angiogenic imbalance. Studies suggest antiretroviral therapy does not impact the angiogenic shift in preeclampsia development. The angiogenic profile in pregnant women of different races influences preeclampsia development. Despite the opposing immune response in HIV infection and preeclampsia, the HIV tat protein strongly mimics vascular endothelial growth factor (VEGF); hence, it is plausible to assume that HIV infection may ameliorate the angiogenic imbalance in preeclampsia.

HIV Tat excites D1 receptor-like expressing neurons from rat nucleus accumbens.

BACKGROUND: HIV-1 infection and drug abuse are frequently co-morbid and their association greatly increases the severity of HIV-1-induced neuropathology. While nucleus accumbens (NAcc) function is severely perturbed by drugs of abuse, little is known about how HIV-1 infection affects NAcc. METHODS: We used calcium and voltage imaging to investigate the effect of HIV-1 trans-activator of transcription (Tat) on rat NAcc. Based on previous neuronal studies, we hypothesized that Tat modulates intracellular Ca2+ homeostasis of NAcc neurons. RESULTS: We provide evidence that Tat triggers a Ca2+ signaling cascade in NAcc medium spiny neurons (MSN) expressing D1-like dopamine receptors leading to neuronal depolarization. Firstly, Tat induced inositol 1,4,5-trisphsophate (IP3) receptor-mediated Ca2+ release from endoplasmic reticulum, followed by Ca2+ and Na+ influx via transient receptor potential canonical channels. The influx of cations depolarizes the membrane promoting additional Ca2+ entry through voltage-gated P/Q-type Ca2+ channels and opening of tetrodotoxin-sensitive Na+ channels. By activating this mechanism, Tat elicits a feed-forward depolarization increasing the excitability of D1-phosphatidylinositol-linked NAcc MSN. We previously found that cocaine targets NAcc neurons directly (independent of the inhibition of dopamine transporter) only when IP3-generating mechanisms are concomitantly initiated. When tested here, cocaine produced a dose-dependent potentiation of the effect of Tat on cytosolic Ca2+. CONCLUSION: We describe for the first time a HIV-1 Tat-triggered Ca2+ signaling in MSN of NAcc involving TRPC and depolarization and a potentiation of the effect of Tat by cocaine, which may be relevant for the reward axis in cocaine-abusing HIV-1-positive patients.

HIV-1 TAT protein enhances sensitization to methamphetamine by affecting dopaminergic function.

Methamphetamine abuse is common among humans with immunodeficiency virus (HIV). The HIV-1 regulatory protein TAT induces dysfunction of mesolimbic dopaminergic systems which may result in impaired reward processes and contribute to methamphetamine abuse. These studies investigated the impact of TAT expression on methamphetamine-induced locomotor sensitization, underlying changes in dopamine function and adenosine receptors in mesolimbic brain areas and neuroinflammation (microgliosis). Transgenic mice with doxycycline-induced TAT protein expression in the brain were tested for locomotor activity in response to repeated methamphetamine injections and methamphetamine challenge after a 7-day abstinence period. Dopamine function in the nucleus accumbens (Acb) was determined using high performance liquid chromatography. Expression of dopamine and/or adenosine A receptors (ADORA) in the Acb and caudate putamen (CPu) was assessed using RT-PCR and immunohistochemistry analyses. Microarrays with pathway analyses assessed dopamine and adenosine signaling in the CPu. Activity-dependent neurotransmitter switching of a reserve pool of non-dopaminergic neurons to a dopaminergic phenotype in the ventral tegmental area (VTA) was determined by immunohistochemistry and quantified with stereology. TAT expression enhanced methamphetamine-induced sensitization. TAT expression alone decreased striatal dopamine (D1, D2, D4, D5) and ADORA1A receptor expression, while increasing ADORA2A receptors expression. Moreover, TAT expression combined with methamphetamine exposure was associated with increased adenosine A receptors (ADORA1A) expression and increased recruitment of dopamine neurons in the VTA. TAT expression and methamphetamine exposure induced microglia activation with the largest effect after combined exposure. Our findings suggest that dopamine-adenosine receptor interactions and reserve pool neuronal recruitment may represent potential targets to develop new treatments for methamphetamine abuse in individuals with HIV.

Cellular protein GLTSCR2: A valuable target for the development of broad-spectrum antivirals.

Viral infection induces translocation of the nucleolar protein GLTSCR2 from the nucleus to the cytoplasm, resulting in attenuation of the type I interferon IFN-ß. Addressing the role of GLTSCR2 in viral replication, we detect that knocking down GLTSCR2 by shRNAs results in significant suppression of viral replication in mammalian and chicken cells. Injection of chicken embryo with the GLTSCR2-specific shRNA-1370 simultaneously or 24 h prior to infection with Newcastle disease virus (NDV) substantially reduces viral replication in chicken embryo fibroblasts. Injection of shRNA-1370 into chicken embryo also reduces the replication of avian influenza virus (AIV). In contrast, GLTSCR2-derived protein G4-T, forming α-helical dimers, increases replication of seven various DNA and RNA viruses in cells. Our studies reveal that alteration of the function of cellular GLTSCR2 plays a role in supporting viral replication. GLTSCR2 should be seriously considered as a therapeutic target for developing broad spectrum antiviral agents to effectively control viral infection.

Alarmins and central nervous system inflammation in HIV-associated neurological disorders.

In the era of highly active antiretroviral therapy (HAART), HIV-1-associated neurocognitive disorders (HAND) persist in infected individuals with adequate immunological and virological status. Risk factors for cognitive impairment include hepatitis C virus co-infection, host genetic factors predisposing to HAND, the early establishment of the virus in the CNS and its persistence under HAART; thus, the CNS is an important reservoir for HIV. Microglial cells are permissive to HIV-1, and NLRP3 inflammasome-associated genes were found expressed in brains of HIV-1-infected persons, contributing to brain disease. Inflammasomes can be triggered by alarmins or danger-associated molecular patterns (DAMPs), which directly stimulate the production of proinflammatory mediators by glial cells, contribute to blood-brain barrier injury through induction of release of various proteases and allow the passage of infected macrophages, and trigger IL-1ß release from primed cells. Amongst alarmins involved in HIV-1-induced neuropathogenesis, IL-33 and high-mobility group box 1 (HMGB1) are of particular interest. Neurocognitive alterations were recently associated with dysregulation of the IL-33/ST2 axis in the CNS, leading to the induction of neuronal apoptosis, decrease in synaptic function and neuroinflammation. Specific biomarkers, including HMGB1 and anti-HMGB1 antibodies, have been identified in cerebrospinal fluid from patients with HAND, correlated with immune activation and identifying a very early stage of neurocognitive impairment that precedes changes in metabolites detected by magnetic resonance spectroscopy. Moreover, HMGB1 plays a crucial role in HIV-1 persistence in dendritic cells and in the constitution of viral reservoirs. In this review, the mechanisms whereby alarmins contribute to HIV-1-induced CNS inflammation and neuropathogenesis will be discussed.

HIV-1 Tat exacerbates lipopolysaccharide-induced cytokine release via TLR4 signaling in the enteric nervous system.

The loss of gut epithelium integrity leads to translocation of microbes and microbial products resulting in immune activation and drives systemic inflammation in acquired immunodeficiency syndrome (AIDS) patients. Although viral loads in HIV patients are significantly reduced in the post-cART era, inflammation and immune activation persist and can lead to morbidity. Here, we determined the interactive effects of the viral protein HIV-1 Tat and lipopolysaccharide (LPS) on enteric neurons and glia. Bacterial translocation was significantly enhanced in Tat-expressing (Tat+) mice. Exposure to HIV-1 Tat in combination with LPS enhanced the expression and release of the pro-inflammatory cytokines IL-6, IL-1ß and TNF-α in the ilea of Tat+ mice and by enteric glia. This coincided with enhanced NF-κB activation in enteric glia that was abrogated in glia from TLR4 knockout mice and by knockdown (siRNA) of MyD88 siRNA in wild type glia. The synergistic effects of Tat and LPS resulted in a reduced rate of colonic propulsion in Tat+ mice treated with LPS. These results show that HIV-1 Tat interacts with the TLR4 receptor to enhance the pro-inflammatory effects of LPS leading to gastrointestinal dysmotility and enhanced immune activation.

Tat-dependent production of an HIV-1 TAR-encoded miRNA-like small RNA.

Evidence is accumulating that retroviruses can produce microRNAs (miRNAs). To prevent cleavage of their RNA genome, retroviruses have to use an alternative RNA source as miRNA precursor. The transacting responsive (TAR) hairpin structure in HIV-1 RNA has been suggested as source for miRNAs, but how these small RNAs are produced without impeding virus replication remained unclear. We used deep sequencing analysis of AGO2-bound HIV-1 RNAs to demonstrate that the 3' side of the TAR hairpin is processed into a miRNA-like small RNA. This ∼21 nt RNA product is able to repress the expression of mRNAs bearing a complementary target sequence. Analysis of the small RNAs produced by wild-type and mutant HIV-1 variants revealed that non-processive transcription from the HIV-1 LTR promoter results in the production of short TAR RNAs that serve as precursor. These TAR RNAs are cleaved by Dicer and processing is stimulated by the viral Tat protein. This biogenesis pathway differs from the canonical miRNA pathway and allows HIV-1 to produce the TAR-encoded miRNA-like molecule without cleavage of the RNA genome.

The Dynamic Landscape of the Full-Length HIV-1 Transactivator of Transcription.

The type 1 human immunodeficiency virus (HIV-1) transactivator of transcription (Tat) is a small RNA-binding protein essential for viral gene expression and replication. It has also been shown to bind to a large number of human proteins and to modulate many different cellular activities. We have used nuclear magnetic resonance (NMR) spectroscopy and hydrogen exchange chemistry to measure backbone dynamics over the millisecond to picosecond time scales. Sequential backbone assignment was facilitated by several isotope labeling schemes, including uniform labeling, site-specific labeling, and unlabeling. (15)N NMR relaxation parameters were measured and analyzed by reduced spectral density mapping and the Lipari-Szabo Model-Free approach to characterize the backbone dynamics on the picosecond to nanosecond time scale. The results indicate that the protein exists in an extended disordered conformational ensemble. NMR relaxation dispersion profiles show that on the millisecond time scale no conformational exchange is detected for any of the residues, supporting the model of a disordered backbone. NMR chemical shift differences from random coil values suggest that some segments of the protein have a modest propensity to fold; comparison to X-ray diffraction structures of Tat complexes indicates that some segments of the protein function through an induced-fit mechanism whereas other segments likely operate by conformational selection. Surprisingly, measured hydrogen exchange rates are higher than predicted for a disordered polymer, but this is explained as being caused by the high net charge on the protein that enhances base-catalyzed hydrogen exchange. The dynamics results provide a deeper understanding of the protein conformational ensemble and form a foundation for future studies of the conformational changes that accompany the formation of the superelongation complex that activates viral transcription.

HIV-Tat Induces the Nrf2/ARE Pathway through NMDA Receptor-Elicited Spermine Oxidase Activation in Human Neuroblastoma Cells.

Previously, we reported that HIV-Tat elicits spermine oxidase (SMO) activity upregulation through NMDA receptor (NMDAR) stimulation in human SH-SY5Y neuroblastoma cells, thus increasing ROS generation, which in turn leads to GSH depletion, oxidative stress, and reduced cell viability. In several cell types, ROS can trigger an antioxidant cell response through the transcriptional induction of oxidative stress-responsive genes regulated by the nuclear factor erythroid 2-related factor 2 (Nrf2). Here, we demonstrate that Tat induces both antioxidant gene expression and Nrf2 activation in SH-SY5Y cells, mediated by SMO activity. Furthermore, NMDAR is involved in Tat-induced Nrf2 activation. These findings suggest that the NMDAR/SMO/Nrf2 pathway is an important target for protection against HIV-associated neurocognitive disorders.

Functional roles of HIV-1 Tat protein in the nucleus.

Human immunodeficiency virus-1 (HIV-1) Tat protein is one of the most important regulatory proteins for viral gene expression in the host cell and can modulate different cellular processes. In addition, Tat is secreted by the infected cell and can be internalized by neighboring cells; therefore, it affects both infected and uninfected cells. Tat can modulate cellular processes by interacting with different cellular structures and signaling pathways. In the nucleus, Tat might be localized either in the nucleoplasm or the nucleolus depending on its concentration. Here we review the distinct functions of Tat in the nucleoplasm and the nucleolus in connection with viral infection and HIV-induced oncogenesis.

HIV-1 Tat regulates the expression of the dcw operon and stimulates the proliferation of bacteria.

Infections of pathogenic bacteria are very common in acquired immunodeficiency syndrome (AIDS) patients. However, the biological effects of HIV-1 Tat on bacteria are incompletely understood. In this study, HIV-1 Tat was expressed in Escherichia coli and Pseudomonas aeruginosa (PA01) to investigate its biological effects on bacteria. Bacterial cells expressing either HIV-1 Tat1-86 (Tat1-86) or HIV-1 Tat1-72 (Tat1-72) grow significantly faster than those with either only an empty vector or an unrelated control (GFP or Rluc). Supplementation of purified HIV-1 Tat1-86 or Tat1-101 protein into bacterial culture medium stimulated the growth of both E. coli and PA01. The expression profile of certain cell division-associated genes, such as those in the division cell wall (dcw) operon (ftsA, ftsQ, ftsW and ftsZ), yafO and zipA, was altered in HIV-1 Tat1-86 expressing E. coli BL21(DE3). Furthermore, the expression of firefly luciferase (Fluc) reporter gene, when engineered for control by the dcw promoter and terminator, was enhanced by HIV-1 Tat in E. coli, confirming that HIV-1 Tat transcriptionally regulates the expression of the dcw operon. The finding that HIV-1 Tat stimulates bacterial growth whether it is produced intracellularly or applied extracellularly may have relevance for HIV patients who are highly susceptible to opportunistic bacterial infections. Contents category: Viruses -Retroviruses. The GenBank accession number for the sequence of HIV-1 Tat1-86 is AF324439.1.

Cortical consequences of HIV-1 Tat exposure in rats are enhanced by chronic cocaine.

The life span of individuals that are sero-positive for human immunodeficiency virus (HIV) has greatly improved; however, complications involving the central nervous system (CNS) remain a concern. While HIV does not directly infect neurons, the proteins produced by the virus, including HIV transactivator of transcription (Tat), are released from infected glia; these proteins can be neurotoxic. This neurotoxicity is thought to mediate the pathology underlying HIVassociated neurological impairments. Cocaine abuse is common among HIV infected individuals, and this abuse augments HIV-associated neurological deficits. The brain regions and pathophysiological mechanisms that are dysregulated by both chronic cocaine and Tat are the focus of the current review.

Mechanisms of HIV-1 Tat neurotoxicity via CDK5 translocation and hyper-activation: role in HIV-associated neurocognitive disorders.

The advent of more effective antiretroviral therapies has reduced the frequency of HIV dementia, however the prevalence of milder HIV associated neurocognitive disorders [HAND] is actually rising. Neurodegenerative mechanisms in HAND might include toxicity by secreted HIV-1 proteins such as Tat, gp120 and Nef that could activate neuro-inflammatory pathways, block autophagy, promote excitotoxicity, oxidative stress, mitochondrial dysfunction and dysregulation of signaling pathways. Recent studies have shown that Tat could interfere with several signal transduction mechanisms involved in cytoskeletal regulation, cell survival and cell cycle re-entry. Among them, Tat has been shown to hyper-activate cyclin-dependent kinase [CDK] 5, a member of the Ser/Thr CDKs involved in cell migration, angiogenesis, neurogenesis and synaptic plasticity. CDK5 is activated by binding to its regulatory subunit, p35 or p39. For this manuscript we review evidence showing that Tat, via calcium dysregulation, promotes calpain-1 cleavage of p35 to p25, which in turn hyper-activates CDK5 resulting in abnormal phosphorylation of downstream targets such as Tau, collapsin response mediator protein-2 [CRMP2], doublecortin [DCX] and MEF2. We also present new data showing that Tat interferes with the trafficking of CDK5 between the nucleus and cytoplasm. This results in prolonged presence of CDK5 in the cytoplasm leading to accumulation of aberrantly phosphorylated cytoplasmic targets [e.g.: Tau, CRMP2, DCX] that impair neuronal function and eventually lead to cell death. Novel therapeutic approaches with compounds that block Tat mediated hyper-activation of CDK5 might be of value in the management of HAND.

Chronic HIV-1 Tat and HIV reduce Rbfox3/NeuN: evidence for sex-related effects.

The NeuN antibody has been widely used to identify and quantify neurons in normal and disease situations based on binding to a nuclear epitope in most types of neurons. This epitope was recently identified as the RNA-binding, feminizing locus on X-3 (Rbfox3), a member of the larger, mammalian Fox1 family of RNA binding proteins. Fox1 proteins recognize a unique UGCAUG mRNA motif and regulate alternative splicing of precursor mRNA to control post-transcriptional events important in neuronal differentiation and central nervous system development. Recent clinical findings show that Rbfox3/NeuN gene dosage is altered in certain human neurodevelopmental disorders, and redistribution has been noted in HIV(+) tissue. We hypothesized that HIV-1 Tat might affect Rbfox3/NeuN expression, and examined this question in vivo using inducible transgenic mice, and in vitro using human mesencephalic-derived neurons. Rbfox3/NeuN expression and localization in HIV+ basal ganglia and hippocampus was also examined. Chronic Tat exposure reduced Rbfox3/NeuN protein levels and increased cytoplasmic localization, similar to the effect of HIV exposure. Cytoplasmic Rbfox3/NeuN signal has occasionally been reported, although the meaning or function of cytoplasmic versus nuclear localization remains speculative. Importantly, Rbfox3/NeuN reductions were more significant in male mice. Although Rbfox3/NeuN-expressing cells were significantly decreased by Tat exposure, stereology showed that Nissl(+) neuron numbers remained normal. Thus, loss of Rbfox3/NeuN may relate more to functional change than to neuron loss. The effects of Tat by itself are highly relevant to HIV(+) individuals maintained on antiretroviral therapy, since Tat is released from infected cells even when viral replication is inhibited.