HIV-1 Viral Protein Research Articles

Presence of p24-Antigen Associated to Erythrocyte in HIV-Positive Individuals Even in Patients with Undetectable Plasma Viral Load

BackgroundHIV adherence to erythrocytes has been demonstrated in vitro, and it has been suggested that erythrocytes may be carriers of the virus. However, the association between HIV particles or viral proteins and erythrocytes in HIV-infected individuals is still to be elucidated.Methodology/Principal FindingsHIV-positive participants (n = 112) were classified into two groups according to values of three plasma viral loads (pVL) determined during the 12-month period prior to the study. The first group included 71 individuals with detectable pVL, whereas the second group included 41 individuals with undetectable pVL. Plasma viral load, erythrocyte-associated p24-antigen and p24-antigen in plasma were determined at the moment of the study. A total of 51 out of the 71 patients with detectable pVL showed erythrocyte-associated p24-antigen whereas 13 showed p24-antigen in plasma. Twenty-two out of the 51 patients with erythrocyte-associated p24-antigen showed pVL<10,000 copies/ml and undetectable p24-antigen in plasma. The data indicates that the amount of erythrocyte-associated p24-antigen was not related to p24-antigen in plasma or pVL levels in this group. Among the 41 patients with prior undetectable pVL, eight presented detectable pVL and erythrocyte-associated p24-antigen at the moment of the study. The other 33 showed undetectable pVL and five of these presented erythrocyte-associated p24-antigen. A positive relationship was found between the presence of erythrocyte-associated p24-antigen and the detectable pVL at the moment of the study (p<0.00001). Even more, in another series of assays, a detectable viral load associated to erythrocytes was determined and it was always accompanied by erythrocyte-associated p24-antigen detection.Conclusions/SignificanceThis study demonstrates the presence of erythrocyte-associated p24-antigen in HIV-infected individuals. Since erythrocyte-associated p24-antigen is not always related to pVL or p24-antigen in plasma, erythrocyte-associated p24-antigen showed viral expression not represented in plasma. Therefore, the determination of erythrocyte-associated p24-antigen may contribute to better understand the kinetics and/or evolution of HIV infection.

Identification of Amino Acids in the Human Tetherin Transmembrane Domain Responsible for HIV-1 Vpu Interaction and Susceptibility

Tetherin, also known as BST-2/CD317/HM1.24, is an antiviral cellular protein that inhibits the release of HIV-1 particles from infected cells. HIV-1 viral protein U (Vpu) is a specific antagonist of human tetherin that might contribute to the high virulence of HIV-1. In this study, we show that three amino acid residues (I34, L37, and L41) in the transmembrane (TM) domain of human tetherin are critical for the interaction with Vpu by using a live cell-based assay. We also found that the conservation of an additional amino acid at position 45 and two residues downstream of position 22, which are absent from monkey tetherins, are required for the antagonism by Vpu. Moreover, computer-assisted structural modeling and mutagenesis studies suggest that an alignment of these four amino acid residues (I34, L37, L41, and T45) on the same helical face in the TM domain is crucial for the Vpu-mediated antagonism of human tetherin. These results contribute to the molecular understanding of human tetherin-specific antagonism by HIV-1 Vpu.

PP2A1 Binding, Cell Transducing and Apoptotic Properties of Vpr77–92: A New Functional Domain of HIV-1 Vpr Proteins

BackgroundThe hallmark of HIV-1 pathogenesis is the progressive CD4+ T cell depletion and high propensity of CD4+ T cells to apoptosis. HIV-1 viral protein R (Vpr) is a major pro-apoptotic gene product. A first Vpr-mediated apoptotic mechanism that requires a physical interaction of HIV-1 Vpr71-82 mitochondriotoxic domain containing the conserved sequence 71-HFRIGCRHSRIG-82 with the Adenine Nucleotide Translocator (ANT) has been characterized. The family of Ser/Thr protein phosphatase PP2A interacts with several viral proteins to regulate cell growth and apoptotic pathways. Previous studies based on yeast two hybrid assays and mutational experiments indicated that PP2A1 is involved in the induction of G2 arrest by HIV-1 Vpr.Principal FindingsExperiments combining pull-down, cell penetration and apoptosis analyses in distinct human cells indicate that the PP2A1 binding sequence from Vpr77–92 is a new cell penetrating apoptotic sequence. We also found that the I84P mutation or the IIQ/VTR83–85 and T89A substitutions in the Vpr77–92 sequence prevent PP2A1 binding, cell penetration and apoptosis. In addition the double R77A and R80A mutation known to inactivate the mitochondriotoxic Vpr71–82 domain, has no effect on the biological properties of the Vpr77–92 domain.ConclusionTogether our data provide evidence for the first time that the Vpr77–92 sequence delineates a biological active domain of Vpr with PP2A1 binding and pro-apopototic capacities and, it is conceivable that this cell penetrating sequence may account for the Vpr internalization in uninfected cells. Finally, our data also implicate the existence of two partially overlapping pro-apoptotic domains in the Vpr C-terminal part, a redundancy that represents a new approach to address the question of biological relevance of HIV-1 Vpr. In this context, future studies will be required to determine the functional relevance of the Vpr77–92 domain in full length Vpr protein and also in entire HIV provirus.

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.

Hepatitis C Virus Core Protein Induces Neuroimmune Activation and Potentiates Human Immunodeficiency Virus-1 Neurotoxicity

BackgroundHepatitis C virus (HCV) genomes and proteins are present in human brain tissues although the impact of HIV/HCV co-infection on neuropathogenesis remains unclear. Herein, we investigate HCV infectivity and effects on neuronal survival and neuroinflammation in conjunction with HIV infection.MethodologyHuman microglia, astrocyte and neuron cultures were infected with cell culture-derived HCV or exposed to HCV core protein with or without HIV-1 infection or HIV-1 Viral Protein R (Vpr) exposure. Host immune gene expression and cell viability were measured. Patch-clamp studies of human neurons were performed in the presence or absence of HCV core protein. Neurobehavioral performance and neuropathology were examined in HIV-1 Vpr-transgenic mice in which stereotaxic intrastriatal implants of HCV core protein were performed.Principal FindingsHCV-encoded RNA as well as HCV core and non-structural 3 (NS3) proteins were detectable in human microglia and astrocytes infected with HCV. HCV core protein exposure induced expression of pro-inflammatory cytokines including interleukin-1β, interleukin-6 and tumor necrosis factor-α in microglia (p<0.05) but not in astrocytes while increased chemokine (e.g. CXCL10 and interleukin-8) expression was observed in both microglia and astrocytes (p<0.05). HCV core protein modulated neuronal membrane currents and reduced both β-III-tubulin and lipidated LC3-II expression (p<0.05). Neurons exposed to supernatants from HCV core-activated microglia exhibited reduced β-III-tubulin expression (p<0.05). HCV core protein neurotoxicity and interleukin-6 induction were potentiated by HIV-1 Vpr protein (p<0.05). HIV-1 Vpr transgenic mice implanted with HCV core protein showed gliosis, reduced neuronal counts together with diminished LC3 immunoreactivity. HCV core-implanted animals displayed neurobehavioral deficits at days 7 and 14 post-implantation (p<0.05).ConclusionsHCV core protein exposure caused neuronal injury through suppression of neuronal autophagy in addition to neuroimmune activation. The additive neurotoxic effects of HCV- and HIV-encoded proteins highlight extrahepatic mechanisms by which HCV infection worsens the disease course of HIV infection.

Solid-phase synthesis and screening of N-acylated polyamine (NAPA) combinatorial libraries for protein binding

Inhibitors for protein–protein interactions are challenging to design, in part due to the unique and complex architectures of each protein’s interaction domain. Most approaches to develop inhibitors for these interactions rely on rational design, which requires prior structural knowledge of the target and its ligands. In the absence of structural information, a combinatorial approach may be the best alternative to finding inhibitors of a protein–protein interaction. Current chemical libraries, however, consist mostly of molecules designed to inhibit enzymes. In this manuscript, we report the synthesis and screening of a library based on an N-acylated polyamine (NAPA) scaffold that we designed to have specific molecular features necessary to inhibit protein–protein interactions. Screens of the library identified a member with favorable binding properties to the HIV viral protein R (Vpr), a regulatory protein from HIV, that is involved in numerous interactions with other proteins critical for viral replication.

Formation of Mobile Chromatin-Associated Nuclear Foci Containing HIV-1 Vpr and VPRBP Is Critical for the Induction of G2 Cell Cycle Arrest

HIV-1 Viral protein R (Vpr) induces a cell cycle arrest at the G2/M phase by activating the ATR DNA damage/stress checkpoint. Recently, we and several other groups showed that Vpr performs this activity by recruiting the DDB1-CUL4A (VPRBP) E3 ubiquitin ligase. While recruitment of this E3 ubiquitin ligase complex has been shown to be required for G2 arrest, the subcellular compartment where this complex forms and functionally acts is unknown. Herein, using immunofluorescence and confocal microscopy, we show that Vpr forms nuclear foci in several cell types including HeLa cells and primary CD4+ T-lymphocytes. These nuclear foci contain VPRBP and partially overlap with DNA repair foci components such as γ-H2AX, 53BP1 and RPA32. While treatment with the non-specific ATR inhibitor caffeine or depletion of VPRBP by siRNA did not inhibit formation of Vpr nuclear foci, mutations in the C-terminal domain of Vpr and cytoplasmic sequestration of Vpr by overexpression of Gag-Pol resulted in impaired formation of these nuclear structures and defective G2 arrest. Consistently, we observed that G2 arrest-competent sooty mangabey Vpr could form these foci but not its G2 arrest-defective paralog Vpx, suggesting that formation of Vpr nuclear foci represents a critical early event in the induction of G2 arrest. Indeed, we found that Vpr could associate to chromatin via its C-terminal domain and that it could form a complex with VPRBP on chromatin. Finally, analysis of Vpr nuclear foci by time-lapse microscopy showed that they were highly mobile and stable structures. Overall, our results suggest that Vpr recruits the DDB1-CUL4A (VPRBP) E3 ligase to these nuclear foci and uses these mobile structures to target a chromatin-bound cellular substrate for ubiquitination in order to induce DNA damage/replication stress, ultimately leading to ATR activation and G2 cell cycle arrest.

HIV-1 Viral Protein R: From Structure to Function

The viral protein r (Vpr) of HIV-1 binds several host proteins leading to pleiotropic functions, such as G2/M cell cycle arrest, apoptosis induction and gene transactivation. Vpr is encapsidated through the Gag C-terminus into the nascent viral particles, suggesting that Vpr plays several important functions in the early stages of the viral lifecycle. In this regard, Vpr interacts with nucleic acids and membranes to facilitate the preintegration complex migration and incorporation into the nucleus of nondividing cells. Thus, Vpr has to recruit several host and viral factors to promote its functions during HIV-1 pathogenesis. This article focuses on its interacting partners by giving an overview of the functional outcome of the different Vpr complexes, as well as the structural determinants of Vpr required for its binding properties.

Fission yeast as a HTS platform for molecular probes of HIV-1 Vpr-induced cell death

Fission yeast as a HTS platform for molecular probes of HIV-1 Vpr-induced cell death Zsigmond Benko1, Robert T Elder2, Dong Liang1, Richard Y Zhao11Department of Pathology, Department of Microbiology-Immunology, Institute of Human Virology, University of Maryland Medical School, Baltimore, MD, USA; 2Children&rsquo;s Memorial Research Center, Northwestern University Feinberg School of Medicine, Chicago, IL, USAAbstract: HIV-1 viral protein R (Vpr) plays an important role in the viral life cycle and pathogenesis of HIV-1. Its activities associate with activation of viral replication, suppression of host immune responses and depletion of CD4+ T-lymphocytes. In particular, Vpr induces cell death through apoptosis that may contribute to the depletion of CD4 T cells, which is a hallmark of HIV-1 infection. Currently, there are no anti-Vpr drugs or adequate assays for high throughput screening to identify selective and potent inhibitors of Vpr activities. In this report, we developed a simple fission yeast-based High Throughput Screening (HTS) system that allows us to screen small molecule compounds that specifically inhibit HIV-1 Vpr. This HTS system includes an absorbance-based primary growth assay, a secondary assay using a semi-quantitative colony-forming dot test, and a fluorescence-based LIVE/DEAD yeast viability assay, which is used as the counter screen tests. We further present here results of a pilot study using a Microsource Spectrum Collection Library, which contains 2,000 biologically active and structurally diverse compounds of known drugs, experimental bioactives, and pure natural products. One compound Benfotiamine showed &gt;50% inhibitory concentration (IC50) and 100% inhibition at 20 and 100 &micro;M, respectively. Interestingly, Benfotiamine is in the same family as thiamine, which was used in this system to control Vpr production through transcriptional inhibition. Even though transcriptional suppressor is not what we are looking for, finding Benfotiamine through the described HTS system from a drug library nevertheless demonstrated the feasibility of the screening assay.Keywords: HIV-1 viral protein R (Vpr), fission yeast (Schizosaccharomyces pombe), high throughput screening (HTS), small molecules, drug library, Benfotiamine

CD4 dimerization requires two cysteines in the cytoplasmic domain of the molecule and occurs in microdomains distinct from lipid rafts

CD4 is engaged in APC-mediated T-cell activation and serves as the primary receptor for HIV. CD4 dimerization and location in specific microdomains has been previously suggested to control its pathophysiological activity. In this study, we investigated (i) whether the CD4 cytoplasmic domain contributes to its dimerization by evaluating the dimerization of mutants, bearing deletions or point mutations in their cytoplasmic tail, (ii) whether CD4 monomers and dimers segregate in distinct microdomains by subcellular fractionation, and (iii) how CD4 dimerization is affected by T-cell activation or HIV-1 viral proteins. Our results indicated that within the cytoplasmic tail of CD4, two cysteines played a crucial role in the dimers formation, since point mutations or truncation upstream of these residues prevented dimerization. The solubility of CD4 dimers and monomers in various detergents was different and CD4 dimers were poorly associated with lipid rafts, but strongly interacted with the tetraspanin CD81. Neither cytoskeleton-disrupting drugs nor cholesterol-sequestering agents had an effect on the CD4 dimerization indicating that dimers formation was independent of CD4 association with the cytoskeleton or lipid rafts. Finally, whereas T-cell activation poorly impact on CD4 dimerization, HIV-1 gp120 and Nef drastically reduced the ratio of CD4 dimers/monomers. Together, these findings demonstrate that two cysteines within the CD4 cytoplasmic tail are critical for dimerization, that CD4 dimers locate preferentially in microdomains distinct than classical lipid rafts, likely tetraspanin-enriched microdomains, and that CD4 dimers are implicated in the process of HIV infection.

Identification of host proteins associated with HIV-1 preintegration complexes isolated from infected CD4+ cells

An integrated HIV-1 genomic DNA leads to an infected cell becoming either an active or a latent virus-producing cell. Upon appropriate activation, a latently infected cell can result in production of progeny viruses that spread the infection to uninfected cells. The host proteins influence several steps of HIV-1 infection including formation of the preintegration complex (PIC), a key nucleoprotein intermediate essential for integration of reverse transcribed viral DNA into the chromosome. Much effort has gone into the identification of host proteins contributing to the assembly of functional PICs. Experimental approaches included the use of yeast two-hybrid system, co-immunoprecipitation, affinity tagged HIV-1 viral proteins and in vitro reconstitution of salt-stripped PIC activity. Several host proteins identified using these approaches have been shown to affect HIV-1 replication in cells and influence catalytic activities of recombinant IN in vitro. However, the comprehensive identification and characterization of host proteins associated with HIV-1 PICs of infected cells have been hindered in part by the technical limitation in acquiring sufficient amount of catalytically active PICs. To efficiently identify additional host factors associated with PICs in infected cells, we have developed the following novel approach. The catalytically active PICs from HIV-1-infected CD4+ cells were isolated using biotinylated target DNA, and the proteins selectively co-purifying with PICs have been analyzed by mass spectrometry. This technology enabled us to reveal at least 19 host proteins that are associated with HIV-1 PICs, of which 18 proteins have not been described previously with respect to HIV-1 integration. Physiological functions of the identified proteins range from chromatin organization to protein transport. A detailed characterization of these host proteins could provide new insights into the mechanism of HIV-1 integration and uncover new antiviral targets to block HIV-1 integration.

Molecular Evolution of the Primate Antiviral Restriction Factor Tetherin

BackgroundTetherin is a recently identified antiviral restriction factor that restricts HIV-1 particle release in the absence of the HIV-1 viral protein U (Vpu). It is reminiscent of APOBEC3G and TRIM5a that also antagonize HIV. APOBEC3G and TRIM5a have been demonstrated to evolve under pervasive positive selection throughout primate evolution, supporting the red-queen hypothesis. Therefore, one naturally presumes that Tetherin also evolves under pervasive positive selection throughout primate evolution and supports the red-queen hypothesis. Here, we performed a detailed evolutionary analysis to address this presumption.Methodology/Principal FindingsResults of non-synonymous and synonymous substitution rates reveal that Tetherin as a whole experiences neutral evolution rather than pervasive positive selection throughout primate evolution, as well as in non-primate mammal evolution. Sliding-window analyses show that the regions of the primate Tetherin that interact with viral proteins are under positive selection or relaxed purifying selection. In particular, the sites identified under positive selection generally focus on these regions, indicating that the main selective pressure acting on the primate Tetherin comes from virus infection. The branch-site model detected positive selection acting on the ancestral branch of the New World Monkey lineage, suggesting an episodic adaptive evolution. The positive selection was also found in duplicated Tetherins in ruminants. Moreover, there is no bias in the alterations of amino acids in the evolution of the primate Tetherin, implying that the primate Tetherin may retain broad spectrum of antiviral activity by maintaining structure stability.Conclusions/SignificanceThese results conclude that the molecular evolution of Tetherin may be attributed to the host–virus arms race, supporting the Red Queen hypothesis, and Tetherin may be in an intermediate stage in transition from neutral to pervasive adaptive evolution.

Cell cycle G2/M arrest through an S phase-dependent mechanism by HIV-1 viral protein R

BackgroundCell cycle G2 arrest induced by HIV-1 Vpr is thought to benefit viral proliferation by providing an optimized cellular environment for viral replication and by skipping host immune responses. Even though Vpr-induced G2 arrest has been studied extensively, how Vpr triggers G2 arrest remains elusive.ResultsTo examine this initiation event, we measured the Vpr effect over a single cell cycle. We found that even though Vpr stops the cell cycle at the G2/M phase, but the initiation event actually occurs in the S phase of the cell cycle. Specifically, Vpr triggers activation of Chk1 through Ser345 phosphorylation in an S phase-dependent manner. The S phase-dependent requirement of Chk1-Ser345 phosphorylation by Vpr was confirmed by siRNA gene silencing and site-directed mutagenesis. Moreover, downregulation of DNA replication licensing factors Cdt1 by siRNA significantly reduced Vpr-induced Chk1-Ser345 phosphorylation and G2 arrest. Even though hydroxyurea (HU) and ultraviolet light (UV) also induce Chk1-Ser345 phosphorylation in S phase under the same conditions, neither HU nor UV-treated cells were able to pass through S phase, whereas vpr-expressing cells completed S phase and stopped at the G2/M boundary. Furthermore, unlike HU/UV, Vpr promotes Chk1- and proteasome-mediated protein degradations of Cdc25B/C for G2 induction; in contrast, Vpr had little or no effect on Cdc25A protein degradation normally mediated by HU/UV.ConclusionsThese data suggest that Vpr induces cell cycle G2 arrest through a unique molecular mechanism that regulates host cell cycle regulation in an S-phase dependent fashion.

HIV-1 viral protein r induces ERK and caspase-8-dependent apoptosis in renal tubular epithelial cells

HIV-associated nephropathy (HIVAN) is the most common cause of end-stage renal disease in persons with HIV/AIDS and is characterized by focal glomerulosclerosis and dysregulated renal tubular epithelial cell (RTEC) proliferation and apoptosis. HIV-1 viral protein r (Vpr) has been implicated in HIV-induced RTEC apoptosis but the mechanisms of Vpr-induced RTEC apoptosis are unknown. The aim of this study was therefore to determine the mechanisms of Vpr-induced apoptosis in RTEC. Apoptosis and caspase activation were analyzed in human RTEC (HK2) after transduction with Vpr-expressing and control lentiviral vectors. Bax and BID were inhibited with lentiviral shRNA, and ERK activation was blocked with the MEK1,2 inhibitor, U0126. Vpr induced apoptosis as indicated by caspase 3/7 activation, PARP-1 cleavage and mitochondrial injury. Vpr activated both caspases-8 and 9. Inhibition of Bax reduced Vpr-induced apoptosis, as reported in other cell types. Additionally, Vpr-induced cleavage of BID to tBID and suppression of BID expression prevented Vpr-induced apoptosis. Since sustained ERK activation can activate caspase-8 in some cell types, we studied the role of ERK in Vpr-induced caspase-8 activation. Vpr induced sustained ERK activation in HK2 cells and incubation with U0126 reduced Vpr-induced caspase-8 activation, BID cleavage and apoptosis. We detected phosphorylated ERK in RTEC in HIVAN biopsy specimens by immunohistochemistry. These studies delineate a novel pathway of Vpr-induced apoptosis in RTEC, which is mediated by sustained ERK activation, resulting in caspase 8-mediated cleavage of BID to tBID, thereby facilitating Bax-mediated mitochondrial injury and apoptosis.

The HIV-1 viral protein Tat modulates glutamate and GABA exocytosis from human and mouse neocortical nerve endings by acting at different binding sites

Background Central Nervous System (CNS) disorders often accompany the acquired immunodeficiency syndrome (AIDS) and are typified by neuropsychiatric symptoms, such as cognitive and motor impairments, sometimes paralleled by neuropathological hallmarks. Collectively, these events are referred to as HIV-1 associated dementia (HAD). Before the advent of the Highly Active Antiretroviral Therapy (HAART), about 20% of adult patients, but as many as 40% of children/adolescent infected subjects developed HAD. Nowadays, in the era of HAART, the prevalence of HAD has decreased, but a more subtle form of disorder, referred to as Minor Cognitive Motor Disorder (MCMD), has emerged in about 20% of symptomatic HIV-1 seropositive patients, including those receiving HAART. This work was aimed at investigating the impact of the HIV-1 viral protein Tat on central neurotransmission since this protein has been proposed as one of the viral component involved in the onset of central neuropsychiatric symptoms. Methods The approach used was the up-down superfusion of purified synaptosomes isolated from human neocortical specimens removed during neurosurgery from consenting patients suffering of brain tumours. Experiments were also carried out by using mouse purified synaptosomes or slices in an attempt to propose an animal model suitable to investigate Tat-induced modification to central nervous system. Functional studies were paralleled by biochemical investigation on the existence of receptor protein potential involved in the effects observed and changes to second messenger production. Results

Absence of Mitochondrial Toxicity in Hearts of Transgenic Mice Treated with Abacavir

Abacavir (ABC) is a guanosine nucleoside reverse transcriptase inhibitor (NRTI) with potent antiretroviral activity. Since NRTIs exhibit tissue-specific inhibition of mitochondrial DNA (mtDNA) synthesis, the ability of ABC to inhibit mtDNA synthesis in vivo was evaluated. Inbred wild-type (WT) and transgenic mice (TG) treated with ABC (3.125 mg/d p. o., 35 days) were used to define mitochondrial oxidative stress and cardiac function. Chosen TGs exhibited overexpression of HIV-1 viral proteins (NL4-3Deltagag/pol, non-replication competent), hemizygous depletion or overexpression of mitochondrial superoxide dismutase (SOD2(+/-) knock-out (KO) or MnSOD OX, respectively), overexpression of mitochondrially targeted catalase (MCAT), or double "knockout" deletion of aldehyde dehydrogenase activity (ALDH2 KO). Impact on mtDNA synthesis was assessed by comparing changes in mtDNA abundance between ABC-treated and vehicle-treated WTs and TGs. No changes in mtDNA abundance occurred from ABC treatment in any mice, suggesting no inhibition of mtDNA synthesis. Left ventricle (LV) mass and LV end-diastolic dimension (LVEDD) were defined echocardiographically and remained unchanged as well. These results indicate that treatment with ABC has no visible cardiotoxicity in these adult mice exposed for 5 weeks compared to findings with other antiretroviral NRTI studies and support some claims for its relative safety.

Characterization of Antibodies to Human Immunodeficiency Viral Proteins in the Sera of HIV Infected and Non HIV Infected HBsAg Seropositive Patients

This work was designed to characterize the HIV viral antibodies in HIV infected and non-HIV infected HBsAgseropositive patients. Fifty non HIV infected (male = 25; female = 25)and 50 HIV infected HBsAg seropositivepatients (male – n = 25; female – n = 25) aged 6 – 64 years recruited from the medical outpatient department ofBaptist Medical Centre, Saki – Oyo State – Nigeria were investigated as test subjects. Fifty apparently healthyHIV and HBsAg seronegative individuals (male = 25; female = 25) aged 4 – 72years were recruited as controlsubjects. All subjects were counseled and were subjected to HBsAg and HIV immunoassays by Enzyme LinkedImmunosorbent Assay and Western blot assay. All subjects were monitored for twelve months. The subjectswere investigated on recruitment and 12months after recruitment. The result obtained indicated higher frequencyof occurrence of each of the HIV antibodies to each of the viral proteins in HIV infected HBsAg seropositivepatients than in non HIV infected HBsAg seropositive patients (94% vs 0% (gp 160,), 84% vs 0% (gp 120), 94%vs 4% (p66), 94% vs 4% (p51), 84% vs 0% (gp 41), 94% vs 0% (p31), 100% vs 24% (p24) and 84% vs 6% (p17)during the first bleeding. The result obtained after 12 months showed a slight difference with the expression ofantibody to gp41 by 4% of the non HIV infected HBsAg seropositive patients in addition to antibody to p24 orp17 which confirms HIV infection. Some of the non HIV infected HBsAg seropositive patients expressedantibodies to the following proteins p66, p51, p24, p17 during the initial investigation and after 12 months. Thefrequency of occurrence of antibody to p24 obtained in all HIV – HBsAg and some of the non HIV infectedHBsAg seropositive patients was higher compared antibodies to other HIV proteins. This recent work hastherefore been used to suggest the possibilities of antibodies to HIV viral proteins (p66, p51, p24, p17) in HBsAgseropositive sera. It also confirms an encouraging degree of specificity of antibodies to HIV envelopeglycoproteins (gp160, gp120, gp41) in the diagnosis of HIV infection.

HIV proteins (gp120 and Tat) and methamphetamine in oxidative stress-induced damage in the brain: Potential role of the thiol antioxidant N-acetylcysteine amide

An increased risk of HIV-1 associated dementia (HAD) has been observed in patients abusing methamphetamine (METH). Since both HIV viral proteins (gp120, Tat) and METH induce oxidative stress, drug abusing patients are at a greater risk of oxidative stress-induced damage. The objective of this study was to determine if N-acetylcysteine amide (NACA) protects the blood brain barrier (BBB) from oxidative stress-induced damage in animals exposed to gp120, Tat and METH. To study this, CD-1 mice pre-treated with NACA/saline, received injections of gp120, Tat, gp120 + Tat or saline for 5 days, followed by three injections of METH/saline on the fifth day, and sacrificed 24 h after the final injection. Various oxidative stress parameters were measured, and animals treated with gp120 + Tat + Meth were found to be the most challenged group, as indicated by their GSH and MDA levels. Treatment with NACA significantly rescued the animals from oxidative stress. Further, NACA-treated animals had significantly higher expression of TJ proteins and BBB permeability as compared to the group treated with gp120 + Tat + METH alone, indicating that NACA can protect the BBB from oxidative stress-induced damage in gp120, Tat and METH exposed animals, and thus could be a viable therapeutic option for patients with HAD.

Niemann-Pick C1 Functions Independently of Niemann-Pick C2 in the Initial Stage of Retrograde Transport of Membrane-impermeable Lysosomal Cargo

The rare neurodegenerative disease Niemann-Pick Type C (NPC) results from mutations in either NPC1 or NPC2, which are membrane-bound and soluble lysosomal proteins, respectively. Previous studies have shown that mutations in either protein result in biochemically indistinguishable phenotypes, most notably the hyper-accumulation of cholesterol and other cargo in lysosomes. We comparatively evaluated the kinetics of [(3)H]dextran release from lysosomes of wild type, NPC1, NPC2, and NPC1/NPC2 pseudo-double mutant cells and found significant differences between all cell types examined. Specifically, NPC1 or NPC2 mutant fibroblasts treated with NPC1 or NPC2 siRNA (to create NPC1/NPC2 pseudo-double mutants) secreted dextran less efficiently than did either NPC1 or NPC2 single mutant cell lines, suggesting that the two proteins may work independently of one another in the egress of membrane-impermeable lysosomal cargo. To investigate the basis for these differences, we examined the role of NPC1 and NPC2 in the retrograde fusion of lysosomes with late endosomes to create so-called hybrid organelles, which is believed to be the initial step in the egress of cargo from lysosomes. We show here that cells with mutated NPC1 have significantly reduced rates of late endosome/lysosome fusion relative to wild type cells, whereas cells with mutations in NPC2 have rates that are similar to those observed in wild type cells. Instead of being involved in hybrid organelle formation, we show that NPC2 is required for efficient membrane fission events from nascent hybrid organelles, which is thought to be required for the reformation of lysosomes and the release of lysosomal cargo-containing membrane vesicles. Collectively, these results suggest that NPC1 and NPC2 can function independently of one another in the egress of certain membrane-impermeable lysosomal cargo.

MicroRNA profiling reveals new aspects of HIV neurodegeneration: caspase‐6 regulates astrocyte survival

MicroRNAs (miRNAs) are small noncoding RNA molecules, which are known to regulate gene expression in physiological and pathological conditions. miRNA profiling was performed using brain tissue from patients with HIV encephalitis (HIVE), a neuroinflammatory/degenerative disorder caused by HIV infection of the brain. Microarray analysis showed differential expression of multiple miRNAs in HIVE compared to control brains. Target prediction and gene ontology enrichment analysis disclosed targeting of several gene families/biological processes by differentially expressed miRNAs (DEMs), with cell death-related genes, including caspase-6, showing a bias toward down-regulated DEMs. Consistent with the miRNA data, HIVE brains exhibited higher levels of caspase-6 transcripts compared with control patients. Immunohistochemical analysis showed localization of the cleaved form of caspase-6 in astrocytes in HIVE brain sections. Exposure of cultured human primary astrocytes to HIV viral protein R (Vpr) induced p53 up-regulation, loss of mitochondrial membrane potential, and caspase-6 activation followed by cell injury. Transgenic mice, expressing Vpr in microglial cells, demonstrated astrocyte apoptosis in brain, which was associated with caspase-6 activation and neurobehavioral abnormalities. Overall, these data point to previously unrecognized alterations in miRNA profile in the brain during HIV infection, which contribute to cell death through dysregulation of cell death machinery.