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Sample records for hiv-1 tat protein

  1. In vitro nuclear interactome of the HIV-1 Tat protein.

    LENUS (Irish Health Repository)

    Gautier, Virginie W

    2009-01-01

    BACKGROUND: One facet of the complexity underlying the biology of HIV-1 resides not only in its limited number of viral proteins, but in the extensive repertoire of cellular proteins they interact with and their higher-order assembly. HIV-1 encodes the regulatory protein Tat (86-101aa), which is essential for HIV-1 replication and primarily orchestrates HIV-1 provirus transcriptional regulation. Previous studies have demonstrated that Tat function is highly dependent on specific interactions with a range of cellular proteins. However they can only partially account for the intricate molecular mechanisms underlying the dynamics of proviral gene expression. To obtain a comprehensive nuclear interaction map of Tat in T-cells, we have designed a proteomic strategy based on affinity chromatography coupled with mass spectrometry. RESULTS: Our approach resulted in the identification of a total of 183 candidates as Tat nuclear partners, 90% of which have not been previously characterised. Subsequently we applied in silico analysis, to validate and characterise our dataset which revealed that the Tat nuclear interactome exhibits unique signature(s). First, motif composition analysis highlighted that our dataset is enriched for domains mediating protein, RNA and DNA interactions, and helicase and ATPase activities. Secondly, functional classification and network reconstruction clearly depicted Tat as a polyvalent protein adaptor and positioned Tat at the nexus of a densely interconnected interaction network involved in a range of biological processes which included gene expression regulation, RNA biogenesis, chromatin structure, chromosome organisation, DNA replication and nuclear architecture. CONCLUSION: We have completed the in vitro Tat nuclear interactome and have highlighted its modular network properties and particularly those involved in the coordination of gene expression by Tat. Ultimately, the highly specialised set of molecular interactions identified will

  2. Nucleolar protein trafficking in response to HIV-1 Tat: rewiring the nucleolus.

    Science.gov (United States)

    Jarboui, Mohamed Ali; Bidoia, Carlo; Woods, Elena; Roe, Barbara; Wynne, Kieran; Elia, Giuliano; Hall, William W; Gautier, Virginie W

    2012-01-01

    The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these

  3. HIV-1 Tat protein induces glial cell autophagy through enhancement of BAG3 protein levels.

    Science.gov (United States)

    Bruno, Anna Paola; De Simone, Francesca Isabella; Iorio, Vittoria; De Marco, Margot; Khalili, Kamel; Sariyer, Ilker Kudret; Capunzo, Mario; Nori, Stefania Lucia; Rosati, Alessandra

    2014-01-01

    BAG3 protein has been described as an anti-apoptotic and pro-autophagic factor in several neoplastic and normal cells. We previously demonstrated that BAG3 expression is elevated upon HIV-1 infection of glial and T lymphocyte cells. Among HIV-1 proteins, Tat is highly involved in regulating host cell response to viral infection. Therefore, we investigated the possible role of Tat protein in modulating BAG3 protein levels and the autophagic process itself. In this report, we show that transfection with Tat raises BAG3 levels in glioblastoma cells. Moreover, BAG3 silencing results in highly reducing Tat- induced levels of LC3-II and increasing the appearance of sub G0/G1 apoptotic cells, in keeping with the reported role of BAG3 in modulating the autophagy/apoptosis balance. These results demonstrate for the first time that Tat protein is able to stimulate autophagy through increasing BAG3 levels in human glial cells.

  4. Modulation of microtubule assembly by the HIV-1 Tat protein is strongly dependent on zinc binding to Tat

    Directory of Open Access Journals (Sweden)

    Muller Sylviane

    2008-07-01

    Full Text Available Abstract Background During HIV-1 infection, the Tat protein plays a key role by transactivating the transcription of the HIV-1 proviral DNA. In addition, Tat induces apoptosis of non-infected T lymphocytes, leading to a massive loss of immune competence. This apoptosis is notably mediated by the interaction of Tat with microtubules, which are dynamic components essential for cell structure and division. Tat binds two Zn2+ ions through its conserved cysteine-rich region in vitro, but the role of zinc in the structure and properties of Tat is still controversial. Results To investigate the role of zinc, we first characterized Tat apo- and holo-forms by fluorescence correlation spectroscopy and time-resolved fluorescence spectroscopy. Both of the Tat forms are monomeric and poorly folded but differ by local conformational changes in the vicinity of the cysteine-rich region. The interaction of the two Tat forms with tubulin dimers and microtubules was monitored by analytical ultracentrifugation, turbidity measurements and electron microscopy. At 20°C, both of the Tat forms bind tubulin dimers, but only the holo-Tat was found to form discrete complexes. At 37°C, both forms promoted the nucleation and increased the elongation rates of tubulin assembly. However, only the holo-Tat increased the amount of microtubules, decreased the tubulin critical concentration, and stabilized the microtubules. In contrast, apo-Tat induced a large amount of tubulin aggregates. Conclusion Our data suggest that holo-Tat corresponds to the active form, responsible for the Tat-mediated apoptosis.

  5. Improved intracellular delivery of glucocerebrosidase mediated by the HIV-1 TAT protein transduction domain

    International Nuclear Information System (INIS)

    Lee, Kyun Oh; Luu, Nga; Kaneski, Christine R.; Schiffmann, Raphael; Brady, Roscoe O.; Murray, Gary J.

    2005-01-01

    Enzyme replacement therapy (ERT) for Gaucher disease designed to target glucocerebrosidase (GC) to macrophages via mannose-specific endocytosis is very effective in reversing hepatosplenomegaly, and normalizing hematologic parameters but is less effective in improving bone and lung involvement and ineffective in brain. Recombinant GCs containing an in-frame fusion to the HIV-1 trans-activator protein transduction domain (TAT) were expressed in eukaryotic cells in order to obtain active, normally glycosylated GC fusion proteins for enzyme uptake studies. Despite the absence of mannose-specific endocytic receptors on the plasma membranes of various fibroblasts, the recombinant GCs with C-terminal TAT fusions were readily internalized by these cells. Immunofluorescent confocal microscopy demonstrated the recombinant TAT-fusion proteins with a mixed endosomal and lysosomal localization. Thus, TAT-modified GCs represent a novel strategy for a new generation of therapeutic enzymes for ERT for Gaucher disease

  6. Antibodies to the HIV-1 Tat protein correlated with nonprogression to AIDS: a rationale for the use of Tat toxoid as an HIV-1 vaccine.

    Science.gov (United States)

    Zagury, J F; Sill, A; Blattner, W; Lachgar, A; Le Buanec, H; Richardson, M; Rappaport, J; Hendel, H; Bizzini, B; Gringeri, A; Carcagno, M; Criscuolo, M; Burny, A; Gallo, R C; Zagury, D

    1998-01-01

    To investigate which immune parameters, such as antibodies against HIV-1 specificities, or viral parameters, such as p24 antigenemia, are predictive of disease progression. We performed studies on serum collected from individuals exhibiting two extremes of disease evolution--67 fast progressors (FP) and 182 nonprogressors (NP)--at their enrollment. After a 1- to 2-year clinical follow-up of 104 nonprogressors after their enrollment, we could determine the best serologic predictors for disease progression. We investigated levels of antibodies to tetanus toxoid and to HIV antigens including Env, Gag, Nef, and Tat proteins, as well as p24 antigenemia, viremia, CD4 cell count, and interferon-alpha (IFN-alpha) titers in FPs and NPs, and we correlated these data with clinical and biologic signs of progression. p24 Antigenemia, a marker of viral replication, and anti-Tat antibodies were highly and inversely correlated in both groups (P < .001). Furthermore, anti-p24 antibodies and low serum IFN-alpha levels were correlated to the NP versus the FP cohort. Finally, among NPs, only antibodies to Tat and not to the other HIV specificities (Env, Nef, Gag) were significantly predictive of clinical stability during their follow-up. Antibodies toward HIV-1 Tat, which are inversely correlated to p24 antigenemia, appear as a critical marker for a lack of disease progression. This study strongly suggests that rising anti-Tat antibodies through active immunization may be beneficial in AIDS vaccine development to control viral replication.

  7. LRRK2 kinase inhibition prevents pathological microglial phagocytosis in response to HIV-1 Tat protein

    Directory of Open Access Journals (Sweden)

    Marker Daniel F

    2012-11-01

    Full Text Available Abstract Background Human Immunodeficiency Virus-1 (HIV-1 associated neurocognitive disorders (HANDs are accompanied by significant morbidity, which persists despite the use of combined antiretroviral therapy (cART. While activated microglia play a role in pathogenesis, changes in their immune effector functions, including phagocytosis and proinflammatory signaling pathways, are not well understood. We have identified leucine-rich repeat kinase 2 (LRRK2 as a novel regulator of microglial phagocytosis and activation in an in vitro model of HANDs, and hypothesize that LRRK2 kinase inhibition will attenuate microglial activation during HANDs. Methods We treated BV-2 immortalized mouse microglia cells with the HIV-1 trans activator of transcription (Tat protein in the absence or presence of LRRK2 kinase inhibitor (LRRK2i. We used Western blot, qRT-PCR, immunocytochemistry and latex bead engulfment assays to analyze LRRK2 protein levels, proinflammatory cytokine and phagocytosis receptor expression, LRRK2 cellular distribution and phagocytosis, respectively. Finally, we utilized ex vivo microfluidic chambers containing primary hippocampal neurons and BV-2 microglia cells to investigate microglial phagocytosis of neuronal axons. Results We found that Tat-treatment of BV-2 cells induced kinase activity associated phosphorylation of serine 935 on LRRK2 and caused the formation of cytoplasmic LRRK2 inclusions. LRRK2i decreased Tat-induced phosphorylation of serine 935 on LRRK2 and inhibited the formation of Tat-induced cytoplasmic LRRK2 inclusions. LRRK2i also decreased Tat-induced process extension in BV-2 cells. Furthermore, LRRK2i attenuated Tat-induced cytokine expression and latex bead engulfment. We examined relevant cellular targets in microfluidic chambers and found that Tat-treated BV-2 microglia cells cleared axonal arbor and engulfed neuronal elements, whereas saline treated controls did not. LRRK2i was found to protect axons in the presence

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

    Science.gov (United States)

    Kesby, James P; Najera, Julia A; Romoli, Benedetto; Fang, Yiding; Basova, Liana; Birmingham, Amanda; Marcondes, Maria Cecilia G; Dulcis, Davide; Semenova, Svetlana

    2017-10-01

    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

  9. HIV-1 tat protein recruits CIS to the cytoplasmic tail of CD127 to induce receptor ubiquitination and proteasomal degradation

    Energy Technology Data Exchange (ETDEWEB)

    Sugden, Scott, E-mail: scott.sugden@ircm.qc.ca [The Ottawa Hospital Research Institute, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6 (Canada); Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5 (Canada); Ghazawi, Feras [The Ottawa Hospital Research Institute, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6 (Canada); Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5 (Canada); MacPherson, Paul, E-mail: pmacpherson@toh.on.ca [The Ottawa Hospital Research Institute, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6 (Canada); Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5 (Canada); Division of Infectious Diseases, The Ottawa Hospital General Campus, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6 (Canada)

    2016-11-15

    HIV-1 Tat protein down regulates expression of the IL-7 receptor alpha-chain (CD127) from the surface of CD8 T cells resulting in impaired T cell proliferation and cytolytic capacity. We have previously shown that soluble Tat protein is taken up by CD8 T cells and interacts with the cytoplasmic tail of CD127 to induce receptor degradation. The N-terminal domain of Tat interacts with CD127 while the basic domain directs CD127 to the proteasome. We have also shown that upon IL-7 binding to its receptor, CD127 is phosphorylated resulting in CIS-mediated proteasomal degradation. Here, we show that Tat mimics this process by recruiting CIS to CD127 in the absence of IL-7 and receptor phosphorylation, leading to CD127 ubiquitination and degradation. Tat therefore acts as an adapter to induce cellular responses under conditions where they may not otherwise occur. Thusly, Tat reduces IL-7 signaling and impairs CD8 T cell survival and function. -- Highlights: •Soluble HIV-1 Tat decreases CD127 expression on CD8 T cells, causing dysfunction. •Tat induces CD127 ubiquitination without activating IL-7 signaling. •Tat binds CD127 and recruits the E3 ubiquitin ligase CIS via its basic domain. •Tat hijacks a normal cellular mechanism to degrade CD127 without IL-7 signaling.

  10. HIV-1 tat protein recruits CIS to the cytoplasmic tail of CD127 to induce receptor ubiquitination and proteasomal degradation

    International Nuclear Information System (INIS)

    Sugden, Scott; Ghazawi, Feras; MacPherson, Paul

    2016-01-01

    HIV-1 Tat protein down regulates expression of the IL-7 receptor alpha-chain (CD127) from the surface of CD8 T cells resulting in impaired T cell proliferation and cytolytic capacity. We have previously shown that soluble Tat protein is taken up by CD8 T cells and interacts with the cytoplasmic tail of CD127 to induce receptor degradation. The N-terminal domain of Tat interacts with CD127 while the basic domain directs CD127 to the proteasome. We have also shown that upon IL-7 binding to its receptor, CD127 is phosphorylated resulting in CIS-mediated proteasomal degradation. Here, we show that Tat mimics this process by recruiting CIS to CD127 in the absence of IL-7 and receptor phosphorylation, leading to CD127 ubiquitination and degradation. Tat therefore acts as an adapter to induce cellular responses under conditions where they may not otherwise occur. Thusly, Tat reduces IL-7 signaling and impairs CD8 T cell survival and function. -- Highlights: •Soluble HIV-1 Tat decreases CD127 expression on CD8 T cells, causing dysfunction. •Tat induces CD127 ubiquitination without activating IL-7 signaling. •Tat binds CD127 and recruits the E3 ubiquitin ligase CIS via its basic domain. •Tat hijacks a normal cellular mechanism to degrade CD127 without IL-7 signaling.

  11. Cloning, Expression and Purification of the Recombinant HIV-1 Tat-Nef Fusion Protein in Prokaryotic Expression System

    OpenAIRE

    Somayeh Kadkhodayan; Shiva Irani; Seyed Mehdi Sadat; Fatemeh Fotouhi; Azam Bolhassani

    2016-01-01

    Abstract Background: Nef is one of the HIV-1 critical proteins, because it is essential for viral replication and AIDS disease progression and induction of immune response against it can partially inhibit viral infection. Moreover, a domain of the HIV-1 Trans-Activator of Transcription (Tat, 48-60 aa) could act as a cell penetrating peptide (CPP). In current study, cloning and expression of Tat-Nef fusion protein was performed in E. coli for the first time. The protein expression was confi...

  12. Cloning, Expression and Purification of the Recombinant HIV-1 Tat-Nef Fusion Protein in Prokaryotic Expression System

    Directory of Open Access Journals (Sweden)

    Somayeh Kadkhodayan

    2016-07-01

    Full Text Available Abstract Background: Nef is one of the HIV-1 critical proteins, because it is essential for viral replication and AIDS disease progression and induction of immune response against it can partially inhibit viral infection. Moreover, a domain of the HIV-1 Trans-Activator of Transcription (Tat, 48-60 aa could act as a cell penetrating peptide (CPP. In current study, cloning and expression of Tat-Nef fusion protein was performed in E. coli for the first time. The protein expression was confirmed by western blot analysis and was purified using reverse staining method. Materials and Methods: In this experimental study, primarily, cloning of Tat-Nef fusion gene was done in pGEX6p2 expression vector. Then, the expression of Tat-Nef recombinat protein in E.coli BL21 (DE3 strain was performed by using IPTG inducer. The protein expression was confirmed by SDS-PAGE and western blotting using anti-Nef monoclonal antibody. Then, the recombinant fusion protein was purified from gel using reverse staining method. Results: The results of PCR analysis and enzyme digestion showed a clear band of ~ 726 bp in agarose gel indicating the correct Tat-Nef fusion cloning in pGEX6p2 prokaryotic expression vector. In addition, a 54 kDa band of Tat-Nef on SDS-PAGE revealed Tat-Nef protein expression that western blot analysis using anti-Nef monoclonal antibody confirmed it. Conclusion: The purified Tat-Nef recombinant fusion protein will be used as an antigen for protein vaccine design against HIV infection.

  13. Interaction of the protein transduction domain of HIV-1 TAT with heparan sulfate: binding mechanism and thermodynamic parameters.

    Science.gov (United States)

    Ziegler, André; Seelig, Joachim

    2004-01-01

    The positively charged protein transduction domain of the HIV-1 TAT protein (TAT-PTD; residues 47-57 of TAT) rapidly translocates across the plasma membrane of living cells. This property is exploited for the delivery of proteins, drugs, and genes into cells. The mechanism of this translocation is, however, not yet understood. Recent theories for translocation suggest binding of the protein transduction domain (PTD) to extracellular glycosaminoglycans as a possible mechanism. We have studied the binding equilibrium between TAT-PTD and three different glycosaminoglycans with high sensitivity isothermal titration calorimetry and provide the first quantitative thermodynamic description. The polysulfonated macromolecules were found to exhibit multiple identical binding sites for TAT-PTD with only small differences between the three species as far as the thermodynamic parameters are concerned. Heparan sulfate (HS, molecular weight, 14.2 +/- 2 kDa) has 6.3 +/- 1.0 independent binding sites for TAT-PTD which are characterized by a binding constant K0 = (6.0 +/- 0.6) x 10(5) M(-1) and a reaction enthalpy deltaHpep0 = -4.6 +/- 1.0 kcal/mol at 28 degrees C. The binding affinity, deltaGpep0, is determined to equal extent by enthalpic and entropic contributions. The HS-TAT-PTD complex formation entails a positive heat capacity change of deltaCp0 = +135 cal/mol peptide, which is characteristic of a charge neutralization reaction. This is in contrast to hydrophobic binding reactions which display a large negative heat capacity change. The stoichiometry of 6-7 TAT-PTD molecules per HS corresponds to an electric charge neutralization. Light scattering data demonstrate a maximum scattering intensity at this stoichiometric ratio, the intensity of which depends on the order of mixing of the two components. The data suggest cross-linking and/or aggregation of HS-TAT-PTD complexes. Two other glycosaminoglycans, namely heparin and chondroitin sulfate B, were also studied with isothermal

  14. On the role of the second coding exon of the HIV-1 Tat protein in virus replication and MHC class I downregulation

    NARCIS (Netherlands)

    Verhoef, K.; Bauer, M.; Meyerhans, A.; Berkhout, B.

    1998-01-01

    Tat is an essential protein of human immunodeficiency virus type 1 (HIV-1) and activates transcription from the viral long terminal repeat (LTR) promoter. The tat gene is composed of two coding exons of which the first, corresponding to the N-terminal 72 amino acid residues, has been reported to be

  15. Structure-based design of ligands for protein basic domains: Application to the HIV-1 Tat protein

    Science.gov (United States)

    Filikov, Anton V.; James, Thomas L.

    1998-05-01

    A methodology has been developed for designing ligands to bind a flexible basic protein domain where the structure of the domain is essentially known. It is based on an empirical binding free energy function developed for highly charged complexes and on Monte Carlo simulations in internal coordinates with both the ligand and the receptor being flexible. HIV-1 encodes a transactivating regulatory protein called Tat. Binding of the basic domain of Tat to TAR RNA is required for efficient transcription of the viral genome. The structure of a biologically active peptide containing the Tat basic RNA-binding domain is available from NMR studies. The goal of the current project is to design a ligand which will bind to that basic domain and potentially inhibit the TAR-Tat interaction. The basic domain contains six arginine and two lysine residues. Our strategy was to design a ligand for arginine first and then a superligand for the basic domain by joining arginine ligands with a linker. Several possible arginine ligands were obtained by searching the Available Chemicals Directory with DOCK 3.5 software. Phytic acid, which can potentially bind multiple arginines, was chosen as a building block for the superligand. Calorimetric binding studies of several compounds to methylguanidine and Arg-/Lys-containing peptides were performed. The data were used to develop an empirical binding free energy function for prediction of affinity of the ligands for the Tat basic domain. Modeling of the conformations of the complexes with both the superligand and the basic domain being flexible has been carried out via Biased Probability Monte Carlo (BPMC) simulations in internal coordinates (ICM 2.6 suite of programs). The simulations used parameters to ensure correct folding, i.e., consistent with the experimental NMR structure of a 25-residue Tat peptide, from a random starting conformation. Superligands for the basic domain were designed by joining together two molecules of phytic acid with

  16. Combined metabonomic and quantitative real-time PCR analyses reveal systems metabolic changes in Jurkat T-cells treated with HIV-1 Tat protein.

    Science.gov (United States)

    Liao, Wenting; Tan, Guangguo; Zhu, Zhenyu; Chen, Qiuli; Lou, Ziyang; Dong, Xin; Zhang, Wei; Pan, Wei; Chai, Yifeng

    2012-11-02

    HIV-1 Tat protein is released by infected cells and can affect bystander uninfected T cells and induce numerous biological responses which contribute to its pathogenesis. To elucidate the complex pathogenic mechanism, we conducted a comprehensive investigation on Tat protein-related extracellular and intracellular metabolic changes in Jurkat T-cells using combined gas chromatography-mass spectrometry (GC-MS), reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) and a hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS)-based metabonomics approach. Quantitative real-time PCR (qRT-PCR) analyses were further employed to measure expressions of several relevant enzymes together with perturbed metabolic pathways. Combined metabonomic and qRT-PCR analyses revealed that HIV-1 Tat caused significant and comprehensive metabolic changes, as represented by significant changes of 37 metabolites and 10 relevant enzymes in HIV-1 Tat-treated cells. Using MetaboAnalyst 2.0, it was found that 11 pathways (Impact-value >0.10) among the regulated pathways were acutely perturbed, including sphingolipid metabolism, glycine, serine and threonine metabolism, pyruvate metabolism, inositol phosphate metabolism, arginine and proline metabolism, citrate cycle, phenylalanine metabolism, tryptophan metabolism, pentose phosphate pathway, glycerophospholipid metabolism, glycolysis or gluconeogenesis. These results provide metabolic evidence of the complex pathogenic mechanism of HIV-1 Tat protein as a "viral toxin", and would help obligate Tat protein as "an important target" for therapeutic intervention and vaccine development.

  17. The HIV-1 Tat protein modulates CD4 expression in human T cells through the induction of miR-222.

    Science.gov (United States)

    Orecchini, Elisa; Doria, Margherita; Michienzi, Alessandro; Giuliani, Erica; Vassena, Lia; Ciafrè, Silvia Anna; Farace, Maria Giulia; Galardi, Silvia

    2014-01-01

    Several cellular microRNAs show substantial changes in expression during HIV-1 infection and their active role in the viral life cycle is progressively emerging. In the present study, we found that HIV-1 infection of Jurkat T cells significantly induces the expression of miR-222. We show that this induction depends on HIV-1 Tat protein, which is able to increase the transcriptional activity of NFkB on miR-222 promoter. Moreover, we demonstrate that miR-222 directly targets CD4, a key receptor for HIV-1, thus reducing its expression. We propose that Tat, by inducing miR-222 expression, complements the CD4 downregulation activity exerted by other viral proteins (i.e., Nef, Vpu, and Env), and we suggest that this represents a novel mechanism through which HIV-1 efficiently represses CD4 expression in infected cells.

  18. The HIV-1 Tat protein modulates CD4 expression in human T cells through the induction of miR-222

    Science.gov (United States)

    Orecchini, Elisa; Doria, Margherita; Michienzi, Alessandro; Giuliani, Erica; Vassena, Lia; Ciafrè, Silvia Anna; Farace, Maria Giulia; Galardi, Silvia

    2014-01-01

    Several cellular microRNAs show substantial changes in expression during HIV-1 infection and their active role in the viral life cycle is progressively emerging. In the present study, we found that HIV-1 infection of Jurkat T cells significantly induces the expression of miR-222. We show that this induction depends on HIV-1 Tat protein, which is able to increase the transcriptional activity of NFkB on miR-222 promoter. Moreover, we demonstrate that miR-222 directly targets CD4, a key receptor for HIV-1, thus reducing its expression. We propose that Tat, by inducing miR-222 expression, complements the CD4 downregulation activity exerted by other viral proteins (i.e., Nef, Vpu, and Env), and we suggest that this represents a novel mechanism through which HIV-1 efficiently represses CD4 expression in infected cells. PMID:24717285

  19. Progesterone protects normative anxiety-like responding among ovariectomized female mice that conditionally express the HIV-1 regulatory protein, Tat, in the CNS.

    Science.gov (United States)

    Paris, Jason J; Fenwick, Jason; McLaughlin, Jay P

    2014-05-01

    Increased anxiety is co-morbid with human immunodeficiency virus (HIV) infection. Actions of the neurotoxic HIV-1 regulatory protein, Tat, may contribute to affective dysfunction. We hypothesized that Tat expression would increase anxiety-like behavior of female GT-tg bigenic mice that express HIV-1 Tat protein in the brain in a doxycycline-dependent manner. Furthermore, given reports that HIV-induced anxiety may occur at lower rates among women, and that the neurotoxic effects of Tat are ameliorated by sex steroids in vitro, we hypothesized that 17β-estradiol and/or progesterone would ameliorate Tat-induced anxiety-like effects. Among naturally-cycling proestrous and diestrous mice, Tat-induction via 7days of doxycycline treatment significantly increased anxiety-like responding in an open field, elevated plus maze and a marble-burying task, compared to treatment with saline. Proestrous mice demonstrated less anxiety-like behavior than diestrous mice in the open field and elevated plus maze, but these effects did not significantly interact with Tat-induction. Among ovariectomized mice, doxycycline-induced Tat protein significantly increased anxiety-like behavior in an elevated plus maze and a marble burying task compared to saline-treated mice, but not an open field (where anxiety-like responding was already maximal). Co-administration of progesterone (4mg/kg), but not 17β-estradiol (0.09mg/kg), with doxycycline significantly ameliorated anxiety-like responding in the elevated plus maze and marble burying tasks. When administered together, 17β-estradiol partially antagonized the protective effects of progesterone on Tat-induced anxiety-like behavior. These findings support evidence of steroid-protection over HIV-1 proteins, and extend them by demonstrating the protective capacity of progesterone on Tat-induced anxiety-like behavior of ovariectomized female mice. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. The HIV-1 viral protein Tat increases glutamate and decreases GABA exocytosis from human and mouse neocortical nerve endings.

    Science.gov (United States)

    Musante, Veronica; Summa, Maria; Neri, Elisa; Puliti, Aldamaria; Godowicz, Tomasz T; Severi, Paolo; Battaglia, Giuseppe; Raiteri, Maurizio; Pittaluga, Anna

    2010-08-01

    Human immunodeficiency virus-1 (HIV-1)-encoded transactivator of transcription (Tat) potentiated the depolarization-evoked exocytosis of [(3)H]D-aspartate ([(3)H]D-ASP) from human neocortical terminals. The metabotropic glutamate (mGlu) 1 receptor antagonist 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) prevented this effect, whereas the mGlu5 receptor antagonist 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP) was ineffective. Western blot analysis showed that human neocortex synaptosomes possess mGlu1 and mGlu5 receptors. Tat potentiated the K(+)-evoked release of [(3)H]D-ASP or of endogenous glutamate from mouse neocortical synaptosomes in a CPCCOEt-sensitive and MPEP-insensitive manner. Deletion of mGlu1 receptors (crv4/crv4 mice) or mGlu5 receptors (mGlu5(-/-)mouse) silenced Tat effects. Tat enhanced inositol 1,4,5-trisphosphate production in human and mouse neocortical synaptosomes, consistent with the involvement of group I mGlu receptors. Tat inhibited the K(+)-evoked release of [(3)H]gamma-aminobutyric acid ([(3)H]GABA) from human synaptosomes and that of endogenous GABA or [(3)H]GABA from mouse nerve terminals; the inhibition was insensitive to CPCCOEt or MPEP. Tat-induced effects were retained by Tat(37-72) but not by Tat(48-85). In mouse neocortical slices, Tat facilitated the K(+)- and the veratridine-induced release of [(3)H]D-ASP in a CPCCOEt-sensitive manner and was ineffective in crv4/crv4 mouse slices. These observations are relevant to the comprehension of the pathophysiological effects of Tat in central nervous system and may suggest new potential therapeutic approaches to the cure of HIV-1-associated dementia.

  1. Potent inhibition of HIV-1 replication by a Tat mutant.

    Directory of Open Access Journals (Sweden)

    Luke W Meredith

    Full Text Available Herein we describe a mutant of the two-exon HIV-1 Tat protein, termed Nullbasic, that potently inhibits multiple steps of the HIV-1 replication cycle. Nullbasic was created by replacing the entire arginine-rich basic domain of wild type Tat with glycine/alanine residues. Like similarly mutated one-exon Tat mutants, Nullbasic exhibited transdominant negative effects on Tat-dependent transactivation. However, unlike previously reported mutants, we discovered that Nullbasic also strongly suppressed the expression of unspliced and singly-spliced viral mRNA, an activity likely caused by redistribution and thus functional inhibition of HIV-1 Rev. Furthermore, HIV-1 virion particles produced by cells expressing Nullbasic had severely reduced infectivity, a defect attributable to a reduced ability of the virions to undergo reverse transcription. Combination of these inhibitory effects on transactivation, Rev-dependent mRNA transport and reverse transcription meant that permissive cells constitutively expressing Nullbasic were highly resistant to a spreading infection by HIV-1. Nullbasic and its activities thus provide potential insights into the development of potent antiviral therapeutics that target multiple stages of HIV-1 infection.

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

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    Wei, Jinsong; Zhang, Yumin; Knapp, Pamela E; Zhao, Tianyong

    2016-01-01

    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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Chemical Composition of Essential Oils from Thymus vulgaris, Cymbopogon citratus, and Rosmarinus officinalis, and Their Effects on the HIV-1 Tat Protein Function.

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    Feriotto, Giordana; Marchetti, Nicola; Costa, Valentina; Beninati, Simone; Tagliati, Federico; Mischiati, Carlo

    2018-02-01

    New drugs would be beneficial to fight resistant HIV strains, in particular those capable of interfering with essential viral functions other than those targeted by highly active antiretroviral therapy drugs. Despite the central role played by Tat protein in HIV transcription, a search for vegetable extracts able to hamper this important viral function was never carried out. In this work, we evaluated the chemical composition and possible interference of essential oil from Thymus vulgaris, Cananga odorata, Cymbopogon citratus, and Rosmarinus officinalis with the Tat/TAR-RNA interaction and with Tat-induced HIV-1 LTR transcription. GC/MS Analysis demonstrated the biodiversity of herbal species translated into essential oils composed of different blends of terpenes. In all of them, 4 - 6 constituents represent from 81.63% to 95.19% of the total terpenes. Essential oils of Thymus vulgaris, Cymbopogon citratus, and Rosmarinus officinalis were active in interfering with Tat functions, encouraging further studies to identify single terpenes responsible for the antiviral activity. In view of the quite different composition of these essential oils, we concluded that their interference on Tat function depends on specific terpene or a characteristic blend. © 2018 Wiley-VHCA AG, Zurich, Switzerland.

  4. Inhibition of Non Canonical HIV-1 Tat Secretion Through the Cellular Na+,K+-ATPase Blocks HIV-1 Infection

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    Silvia Agostini

    2017-07-01

    Full Text Available Besides its essential role in the activation of HIV-1 gene expression, the viral Tat protein has the unusual property of trafficking in and out of cells. In contrast to Tat internalization, the mechanism involved in extracellular Tat release has so far remained elusive. Here we show that Tat secretion occurs through a Golgi-independent pathway requiring binding of Tat with three short, non-consecutive intracytoplasmic loops at the C-terminus of the cellular Na+,K+-ATPase pump alpha subunit. Ouabain, a pump inhibitor, blocked this interaction and prevented Tat secretion; virions produced in the presence of this drug were less infectious, consistent the capacity of virion-associated Tat to increase HIV-1 infectivity. Treatment of CD4+ T-cells with short peptides corresponding to the Tat-binding regions of the pump alpha subunit impaired extracellular Tat release and blocked HIV-1 replication. Thus, non canonical, extracellular Tat secretion is essential for viral infectivity.

  5. Safety and immunogenicity of HIV-1 Tat toxoid in immunocompromised HIV-1-infected patients.

    Science.gov (United States)

    Gringeri, A; Santagostino, E; Muça-Perja, M; Mannucci, P M; Zagury, J F; Bizzini, B; Lachgar, A; Carcagno, M; Rappaport, J; Criscuolo, M; Blattner, W; Burny, A; Gallo, R C; Zagury, D

    1998-01-01

    To antagonize the deleterious effects of the HIV-1 toxin extracellular Tat on uninfected immune cells, we developed a new strategy of anti-HIV-1 vaccine using an inactivated but immunogenic Tat (Tat toxoid). Tat toxoid has been assayed for safety and immunogenicity in seropositive patients. The phase I vaccine clinical trial testing Tat toxoid preparation in Seppic Isa 51 oil adjuvant was performed on 14 HIV-1-infected asymptomatic although biologically immunocompromised individuals (500-200 CD4+ cells/mm3). Following as many as 8 injections, no clinical defects were observed. All patients exhibited an antibody (Ab) response to Tat, and some had cell-mediated immunity (CMI) as evaluated by skin test in vivo and T-cell proliferation in vitro. These results provide initial evidence of safety and potency of Tat toxoid vaccination in HIV-1-infected individuals.

  6. An attenuated herpes simplex virus type 1 (HSV1) encoding the HIV-1 Tat protein protects mice from a deadly mucosal HSV1 challenge.

    Science.gov (United States)

    Sicurella, Mariaconcetta; Nicoli, Francesco; Gallerani, Eleonora; Volpi, Ilaria; Berto, Elena; Finessi, Valentina; Destro, Federica; Manservigi, Roberto; Cafaro, Aurelio; Ensoli, Barbara; Caputo, Antonella; Gavioli, Riccardo; Marconi, Peggy C

    2014-01-01

    Herpes simplex virus types 1 and 2 (HSV1 and HSV2) are common infectious agents in both industrialized and developing countries. They cause recurrent asymptomatic and/or symptomatic infections, and life-threatening diseases and death in newborns and immunocompromised patients. Current treatment for HSV relies on antiviral medications, which can halt the symptomatic diseases but cannot prevent the shedding that occurs in asymptomatic patients or, consequently, the spread of the viruses. Therefore, prevention rather than treatment of HSV infections has long been an area of intense research, but thus far effective anti-HSV vaccines still remain elusive. One of the key hurdles to overcome in anti-HSV vaccine development is the identification and effective use of strategies that promote the emergence of Th1-type immune responses against a wide range of epitopes involved in the control of viral replication. Since the HIV1 Tat protein has several immunomodulatory activities and increases CTL recognition of dominant and subdominant epitopes of heterologous antigens, we generated and assayed a recombinant attenuated replication-competent HSV1 vector containing the tat gene (HSV1-Tat). In this proof-of-concept study we show that immunization with this vector conferred protection in 100% of mice challenged intravaginally with a lethal dose of wild-type HSV1. We demonstrate that the presence of Tat within the recombinant virus increased and broadened Th1-like and CTL responses against HSV-derived T-cell epitopes and elicited in most immunized mice detectable IgG responses. In sharp contrast, a similarly attenuated HSV1 recombinant vector without Tat (HSV1-LacZ), induced low and different T cell responses, no measurable antibody responses and did not protect mice against the wild-type HSV1 challenge. These findings strongly suggest that recombinant HSV1 vectors expressing Tat merit further investigation for their potential to prevent and/or contain HSV1 infection and

  7. An attenuated herpes simplex virus type 1 (HSV1 encoding the HIV-1 Tat protein protects mice from a deadly mucosal HSV1 challenge.

    Directory of Open Access Journals (Sweden)

    Mariaconcetta Sicurella

    Full Text Available Herpes simplex virus types 1 and 2 (HSV1 and HSV2 are common infectious agents in both industrialized and developing countries. They cause recurrent asymptomatic and/or symptomatic infections, and life-threatening diseases and death in newborns and immunocompromised patients. Current treatment for HSV relies on antiviral medications, which can halt the symptomatic diseases but cannot prevent the shedding that occurs in asymptomatic patients or, consequently, the spread of the viruses. Therefore, prevention rather than treatment of HSV infections has long been an area of intense research, but thus far effective anti-HSV vaccines still remain elusive. One of the key hurdles to overcome in anti-HSV vaccine development is the identification and effective use of strategies that promote the emergence of Th1-type immune responses against a wide range of epitopes involved in the control of viral replication. Since the HIV1 Tat protein has several immunomodulatory activities and increases CTL recognition of dominant and subdominant epitopes of heterologous antigens, we generated and assayed a recombinant attenuated replication-competent HSV1 vector containing the tat gene (HSV1-Tat. In this proof-of-concept study we show that immunization with this vector conferred protection in 100% of mice challenged intravaginally with a lethal dose of wild-type HSV1. We demonstrate that the presence of Tat within the recombinant virus increased and broadened Th1-like and CTL responses against HSV-derived T-cell epitopes and elicited in most immunized mice detectable IgG responses. In sharp contrast, a similarly attenuated HSV1 recombinant vector without Tat (HSV1-LacZ, induced low and different T cell responses, no measurable antibody responses and did not protect mice against the wild-type HSV1 challenge. These findings strongly suggest that recombinant HSV1 vectors expressing Tat merit further investigation for their potential to prevent and/or contain HSV1

  8. The effects of HIV-1 regulatory TAT protein expression on brain reward function, response to psychostimulants and delay-dependent memory in mice.

    Science.gov (United States)

    Kesby, James P; Markou, Athina; Semenova, Svetlana

    2016-10-01

    Depression and psychostimulant abuse are common comorbidities among humans with immunodeficiency virus (HIV) disease. The HIV regulatory protein TAT is one of multiple HIV-related proteins associated with HIV-induced neurotoxicity. TAT-induced dysfunction of dopamine and serotonin systems in corticolimbic brain areas may result in impaired reward function, thus, contributing to depressive symptoms and psychostimulant abuse. Transgenic mice with doxycycline-induced TAT protein expression in the brain (TAT+, TAT- control) show neuropathology resembling brain abnormalities in HIV+ humans. We evaluated brain reward function in response to TAT expression, nicotine and methamphetamine administration in TAT+ and TAT- mice using the intracranial self-stimulation procedure. We evaluated the brain dopamine and serotonin systems with high-performance liquid chromatography. The effects of TAT expression on delay-dependent working memory in TAT+ and TAT- mice using the operant delayed nonmatch-to-position task were also assessed. During doxycycline administration, reward thresholds were elevated by 20% in TAT+ mice compared with TAT- mice. After the termination of doxycycline treatment, thresholds of TAT+ mice remained significantly higher than those of TAT- mice and this was associated with changes in mesolimbic serotonin and dopamine levels. TAT+ mice showed a greater methamphetamine-induced threshold lowering compared with TAT- mice. TAT expression did not alter delay-dependent working memory. These results indicate that TAT expression in mice leads to reward deficits, a core symptom of depression, and a greater sensitivity to methamphetamine-induced reward enhancement. Our findings suggest that the TAT protein may contribute to increased depressive-like symptoms and continued methamphetamine use in HIV-positive individuals. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. RNA glycosidase and other agents target Tat to inhibit HIV-1 transcription.

    Science.gov (United States)

    Harrich, David; Jin, Hongping

    2018-03-20

    The HIV-1 tat gene encodes a small 86-104 amino acid protein depending on the HIV-1 strain. Tat is essential for HIV-1 replication through interactions with numerous cellular transcription factors. The interaction between Tat and P-TEFb, which is a cellular protein complex composed of cyclin T1 and CDK9, delivers P-TEFb to the newly transcribed viral mRNAs where phosphorylation of RNA polymerase II by CDK9 leads to highly efficient mRNA transcription. It has long been recognized that Tat is a potential anti-HIV-1 target and possibly a viral Achilles' heel. However, specifically targeting Tat without affecting normal host cell functions has been challenging. Means to inactivate Tat have been reported that includes small compounds, transdominant negative Tat proteins, and by plant-derived antivirals. Investigations of these agents have reported encouraging outcomes that inform and may hopefully affect strategies for a functional HIV-1 cure. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  10. Interaction between HIV-1 Tat and DNA-PKcs modulates HIV transcription and class switch recombination.

    Science.gov (United States)

    Zhang, Shi-Meng; Zhang, He; Yang, Tian-Yi; Ying, Tian-Yi; Yang, Pei-Xiang; Liu, Xiao-Dan; Tang, Sheng-Jian; Zhou, Ping-Kun

    2014-01-01

    HIV-1 tat targets a variety of host cell proteins to facilitate viral transcription and disrupts host cellular immunity by inducing lymphocyte apoptosis, but whether it influences humoral immunity remains unclear. Previously, our group demonstrated that tat depresses expression of DNA-PKcs, a critical component of the non-homologous end joining pathway (NHEJ) of DNA double-strand breaks repair, immunoglobulin class switch recombination (CSR) and V(D)J recombination, and sensitizes cells to ionizing radiation. In this study, we demonstrated that HIV-1 Tat down-regulates DNA-PKcs expression by directly binding to the core promoter sequence. In addition, Tat interacts with and activates the kinase activity of DNA-PKcs in a dose-dependent and DNA independent manner. Furthermore, Tat inhibits class switch recombination (CSR) at low concentrations (≤ 4 µg/ml) and stimulates CSR at high concentrations (≥ 8 µg/ml). On the other hand, low protein level and high kinase activity of DNA-PKcs promotes HIV-1 transcription, while high protein level and low kinase activity inhibit HIV-1 transcription. Co-immunoprecipitation results revealed that DNA-PKcs forms a large complex comprised of Cyclin T1, CDK9 and Tat via direct interacting with CDK9 and Tat but not Cyclin T1. Taken together, our results provide new clues that Tat regulates host humoral immunity via both transcriptional depression and kinase activation of DNA-PKcs. We also raise the possibility that inhibitors and interventions directed towards DNA-PKcs may inhibit HIV-1 transcription in AIDS patients.

  11. The HIV-1 transcriptional activator Tat has potent nucleic acid chaperoning activities in vitro.

    Science.gov (United States)

    Kuciak, Monika; Gabus, Caroline; Ivanyi-Nagy, Roland; Semrad, Katharina; Storchak, Roman; Chaloin, Olivier; Muller, Sylviane; Mély, Yves; Darlix, Jean-Luc

    2008-06-01

    The human immunodeficiency virus type 1 (HIV-1) is a primate lentivirus that causes the acquired immunodeficiency syndrome (AIDS). In addition to the virion structural proteins and enzyme precursors, that are Gag, Env and Pol, HIV-1 encodes several regulatory proteins, notably a small nuclear transcriptional activator named Tat. The Tat protein is absolutely required for virus replication since it controls proviral DNA transcription to generate the full-length viral mRNA. Tat can also regulate mRNA capping and splicing and was recently found to interfere with the cellular mi- and siRNA machinery. Because of its extensive interplay with nucleic acids, and its basic and disordered nature we speculated that Tat had nucleic acid-chaperoning properties. This prompted us to examine in vitro the nucleic acid-chaperoning activities of Tat and Tat peptides made by chemical synthesis. Here we report that Tat has potent nucleic acid-chaperoning activities according to the standard DNA annealing, DNA and RNA strand exchange, RNA ribozyme cleavage and trans-splicing assays. The active Tat(44-61) peptide identified here corresponds to the smallest known sequence with DNA/RNA chaperoning properties.

  12. HIV-1 Tat and AIDS-associated cancer: targeting the cellular anti-cancer barrier?

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    Daniel René

    2008-05-01

    Full Text Available Abstract The acquired immunodeficiency syndrome (AIDS is accompanied by a significant increase in the incidence of neoplasms. Several causative agents have been proposed for this phenomenon. These include immunodeficiency and oncogenic DNA viruses and the HIV-1 protein Tat. Cancer in general is closely linked to genomic instability and DNA repair mechanisms. The latter maintains genomic stability and serves as a cellular anti-cancer barrier. Defects in DNA repair pathway are associated with carcinogenesis. This review focuses on newly discovered connections of the HIV-1 protein Tat, as well as cellular co-factors of Tat, to double-strand break DNA repair. We propose that the Tat-induced DNA repair deficiencies may play a significant role in the development of AIDS-associated cancer.

  13. Direct effects of HIV-1 Tat on excitability and survival of primary dorsal root ganglion neurons: possible contribution to HIV-1-associated pain.

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    Xianxun Chi

    Full Text Available The vast majority of people living with human immunodeficiency virus type 1 (HIV-1 have pain syndrome, which has a significant impact on their quality of life. The underlying causes of HIV-1-associated pain are not likely attributable to direct viral infection of the nervous system due to the lack of evidence of neuronal infection by HIV-1. However, HIV-1 proteins are possibly involved as they have been implicated in neuronal damage and death. The current study assesses the direct effects of HIV-1 Tat, one of potent neurotoxic viral proteins released from HIV-1-infected cells, on the excitability and survival of rat primary dorsal root ganglion (DRG neurons. We demonstrated that HIV-1 Tat triggered rapid and sustained enhancement of the excitability of small-diameter rat primary DRG neurons, which was accompanied by marked reductions in the rheobase and resting membrane potential (RMP, and an increase in the resistance at threshold (R(Th. Such Tat-induced DRG hyperexcitability may be a consequence of the inhibition of cyclin-dependent kinase 5 (Cdk5 activity. Tat rapidly inhibited Cdk5 kinase activity and mRNA production, and roscovitine, a well-known Cdk5 inhibitor, induced a very similar pattern of DRG hyperexcitability. Indeed, pre-application of Tat prevented roscovitine from having additional effects on the RMP and action potentials (APs of DRGs. However, Tat-mediated actions on the rheobase and R(Th were accelerated by roscovitine. These results suggest that Tat-mediated changes in DRG excitability are partly facilitated by Cdk5 inhibition. In addition, Cdk5 is most abundant in DRG neurons and participates in the regulation of pain signaling. We also demonstrated that HIV-1 Tat markedly induced apoptosis of primary DRG neurons after exposure for longer than 48 h. Together, this work indicates that HIV-1 proteins are capable of producing pain signaling through direct actions on excitability and survival of sensory neurons.

  14. A real-time view of the TAR:Tat:P-TEFb complex at HIV-1 transcription sites

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    Knezevich Anna

    2007-05-01

    Full Text Available Abstract HIV-1 transcription is tightly regulated: silent in long-term latency and highly active in acutely-infected cells. Transcription is activated by the viral protein Tat, which recruits the elongation factor P-TEFb by binding the TAR sequence present in nascent HIV-1 RNAs. In this study, we analyzed the dynamic of the TAR:Tat:P-TEFb complex in living cells, by performing FRAP experiments at HIV-1 transcription sites. Our results indicate that a large fraction of Tat present at these sites is recruited by Cyclin T1. We found that in the presence of Tat, Cdk9 remained bound to nascent HIV-1 RNAs for 71s. In contrast, when transcription was activated by PMA/ionomycin, in the absence of Tat, Cdk9 turned-over rapidly and resided on the HIV-1 promoter for only 11s. Thus, the mechanism of trans-activation determines the residency time of P-TEFb at the HIV-1 gene, possibly explaining why Tat is such a potent transcriptional activator. In addition, we observed that Tat occupied HIV-1 transcription sites for 55s, suggesting that the TAR:Tat:P-TEFb complex dissociates from the polymerase following transcription initiation, and undergoes subsequent cycles of association/dissociation.

  15. Interplay between Structure and Charge as a Key to Allosteric Modulation of Human 20S Proteasome by the Basic Fragment of HIV-1 Tat Protein.

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    Przemysław Karpowicz

    Full Text Available The proteasome is a giant protease responsible for degradation of the majority of cytosolic proteins. Competitive inhibitors of the proteasome are used against aggressive blood cancers. However, broadening the use of proteasome-targeting drugs requires new mechanistic approaches to the enzyme's inhibition. In our previous studies we described Tat1 peptide, an allosteric inhibitor of the proteasome derived from a fragment of the basic domain of HIV-Tat1 protein. Here, we attempted to dissect the structural determinants of the proteasome inhibition by Tat1. Single- and multiple- alanine walking scans were performed. Tat1 analogs with stabilized beta-turn conformation at positions 4-5 and 8-9, pointed out by the molecular dynamics modeling and the alanine scan, were synthesized. Structure of Tat1 analogs were analyzed by circular dichroism, Fourier transform infrared and nuclear magnetic resonance spectroscopy studies, supplemented by molecular dynamics simulations. Biological activity tests and structural studies revealed that high flexibility and exposed positive charge are hallmarks of Tat1 peptide. Interestingly, stabilization of a beta-turn at the 8-9 position was necessary to significantly improve the inhibitory potency.

  16. Creatine protects against mitochondrial dysfunction associated with HIV-1 Tat-induced neuronal injury

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    Stevens, Patrick R.; Gawryluk, Jeremy W.; Hui, Liang; Chen, Xuesong; Geiger, Jonathan D.

    2015-01-01

    HIV-1 infected individuals are living longer but experiencing a prevalence rate of over 50% for HIV-1 associated neurocognitive disorders (HAND) for which no effective treatment is available. Viral and cellular factors secreted by HIV-1 infected cells leads to neuronal injury and HIV-1 Tat continues to be implicated in the pathogenesis of HAND. Here we tested the hypothesis that creatine protected against HIV-1 Tat-induced neuronal injury by preventing mitochondrial bioenergetic crisis and/or redox catastrophe. Creatine blocked HIV-1 Tat1-72-induced increases in neuron cell death and synaptic area loss. Creatine protected against HIV-1 Tat-induced decreases in ATP. Creatine and creatine plus HIV-1 Tat increased cellular levels of creatine, and creatine plus HIV-1 Tat further decreased ratios of phosphocreatine to creatine observed with creatine or HIV-1 Tat treatments alone. Additionally, creatine protected against HIV-1 Tat-induced mitochondrial hypopolarization and HIV-1 Tat-induced mitochondrial permeability transition pore opening. Thus, creatine may be a useful adjunctive therapy against HAND. PMID:25613139

  17. The cross-talk of HIV-1 Tat and methamphetamine in HIV-associated neurocognitive disorders

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    Susana T Valente

    2015-10-01

    Full Text Available Antiretroviral therapy (ART has dramatically improved the lives of HIV-1 infected individuals. Nonetheless, HIV associated neurocognitive disorders (HAND, which range from undetectable neurocognitive impairments to severe dementia, still affect approximately 50% of the infected population, hampering their quality of life.The persistence of HAND is promoted by several factors, including longer life expectancies, the residual levels of virus in the central nervous system and the continued presence of HIV-1 regulatory proteins such as the transactivator of transcription (Tat in the brain. Tat is a secreted viral protein that crosses the blood brain barrier into the central nervous system, where it has the ability to directly act on neurons and non-neuronal cells alike. These actions result in the release of soluble factors involved in inflammation, oxidative stress and excitotoxicity, ultimately resulting in neuronal damage. The percentage of methamphetamine abusers is high among the HIV-1-positive population compared to the general population. On the other hand, methamphetamine abuse is correlated with increased viral replication, enhanced Tat-mediated neurotoxicity and neurocognitive impairments. Although several strategies have been investigated to reduce HAND and methamphetamine use, no clinically approved treatment is currently available. Here, we review the latest findings of the effects of Tat and methamphetamine in HAND and discuss a few promising potential therapeutic developments.

  18. Interaction of the phospholipid scramblase 1 with HIV-1 Tat results in the repression of Tat-dependent transcription

    International Nuclear Information System (INIS)

    Kusano, Shuichi; Eizuru, Yoshito

    2013-01-01

    Highlights: •PLSCR1 specifically interacted with HIV-1 Tat in vitro and in vivo. •PLSCR1 repressed Tat-dependent transactivation of the HIV-1 LTR. •Suppression of PLSCR1 expression enhanced the levels of HIV-1 transcripts. •PLSCR1 reduced the nuclear localization of Tat. -- Abstract: Human phospholipid scramblase 1 (PLSCR1) is an interferon (IFN)-stimulated gene and possesses an IFN-mediated antiviral function. We show here that PLSCR1 directly interacts with human immunodeficiency virus type-1 (HIV-1) Tat. This interaction occurs both in vitro and in vivo through amino acids 160–250 of PLSCR1. Overexpression of PLSCR1 efficiently represses the Tat-dependent transactivation of the HIV-1 long terminal repeat (LTR) and reduces the nuclear translocation of Tat. In addition, shRNA-mediated suppression of endogenous PLSCR1 expression enhances the levels of gag mRNA in an HIV-1-infected T-cell line. These findings indicate that PLSCR1 negatively regulates the Tat-dependent transactivation of the HIV-1 LTR during HIV-1 infection

  19. Controlling cellular P-TEFb activity by the HIV-1 transcriptional transactivator Tat.

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    Lisa Muniz

    Full Text Available The human immunodeficiency virus 1 (HIV-1 transcriptional transactivator (Tat is essential for synthesis of full-length transcripts from the integrated viral genome by RNA polymerase II (Pol II. Tat recruits the host positive transcription elongation factor b (P-TEFb to the HIV-1 promoter through binding to the transactivator RNA (TAR at the 5'-end of the nascent HIV transcript. P-TEFb is a general Pol II transcription factor; its cellular activity is controlled by the 7SK small nuclear RNA (snRNA and the HEXIM1 protein, which sequester P-TEFb into transcriptionally inactive 7SK/HEXIM/P-TEFb snRNP. Besides targeting P-TEFb to HIV transcription, Tat also increases the nuclear level of active P-TEFb through promoting its dissociation from the 7SK/HEXIM/P-TEFb RNP by an unclear mechanism. In this study, by using in vitro and in vivo RNA-protein binding assays, we demonstrate that HIV-1 Tat binds with high specificity and efficiency to an evolutionarily highly conserved stem-bulge-stem motif of the 5'-hairpin of human 7SK snRNA. The newly discovered Tat-binding motif of 7SK is structurally and functionally indistinguishable from the extensively characterized Tat-binding site of HIV TAR and importantly, it is imbedded in the HEXIM-binding elements of 7SK snRNA. We show that Tat efficiently replaces HEXIM1 on the 7SK snRNA in vivo and therefore, it promotes the disassembly of the 7SK/HEXIM/P-TEFb negative transcriptional regulatory snRNP to augment the nuclear level of active P-TEFb. This is the first demonstration that HIV-1 specifically targets an important cellular regulatory RNA, most probably to promote viral transcription and replication. Demonstration that the human 7SK snRNA carries a TAR RNA-like Tat-binding element that is essential for the normal transcriptional regulatory function of 7SK questions the viability of HIV therapeutic approaches based on small drugs blocking the Tat-binding site of HIV TAR.

  20. Celastrol ameliorates HIV-1 Tat-induced inflammatory responses via NF-kappaB and AP-1 inhibition and heme oxygenase-1 induction in astrocytes

    International Nuclear Information System (INIS)

    Youn, Gi Soo; Kwon, Dong-Joo; Ju, Sung Mi; Rhim, Hyangshuk; Bae, Yong Soo; Choi, Soo Young; Park, Jinseu

    2014-01-01

    HIV-1 Tat causes extensive neuroinflammation that may progress to AIDS-related encephalitis and dementia. Celastrol possesses various biological activities such as anti-oxidant, anti-tumor, and anti-inflammatory activities. In this study, we investigated the modulatory effects of celastrol on HIV-1 Tat-induced inflammatory responses and the molecular mechanisms underlying its action in astrocytes. Pre-treatment of CRT-MG human astroglioma cells with celastrol significantly inhibited HIV-1 Tat-induced expression of ICAM-1/VCAM-1 and subsequent monocyte adhesiveness in CRT-MG cells. In addition, celastrol suppressed HIV-1 Tat-induced expression of pro-inflammatory chemokines, such as CXCL10, IL-8, and MCP-1. Celastrol decreased HIV-1 Tat-induced activation of JNK MAPK, AP-1, and NF-κB. Furthermore, celastrol induced mRNA and protein expression of HO-1 as well as Nrf2 activation. Blockage of HO-1 expression using siRNA reversed the inhibitory effect of celastrol on HIV-1 Tat-induced inflammatory responses. These results suggest that celastrol has regulatory effects on HIV-1 Tat-induced inflammatory responses by blocking the JNK MAPK-AP-1/NF-κB signaling pathways and inducing HO-1 expression in astrocytes. - Highlights: • Celastrol suppressed HIV-1 Tat-induced expression of pro-inflammatory genes. • Celastrol inhibited HIV-1 Tat -induced activation of JNK MAPK. • Celastrol inhibited HIV-1 Tat-induced activation of both NF-κB and AP-1. • Celastrol inhibited HIV-1 Tat-induced inflammatory responses via HO-1 induction

  1. Celastrol ameliorates HIV-1 Tat-induced inflammatory responses via NF-kappaB and AP-1 inhibition and heme oxygenase-1 induction in astrocytes

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    Youn, Gi Soo; Kwon, Dong-Joo; Ju, Sung Mi [Department of Biomedical Science and Research Institute for Bioscience and Biotechnology, Hallym University, Chunchon 200-702 (Korea, Republic of); Rhim, Hyangshuk [Department of Biomedical Sciences, Department of Medical Life Sciences, College of Medicine, the Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Bae, Yong Soo [Department of Biological Science, College of Natural Sciences, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Choi, Soo Young [Department of Biomedical Science and Research Institute for Bioscience and Biotechnology, Hallym University, Chunchon 200-702 (Korea, Republic of); Park, Jinseu, E-mail: jinpark@hallym.ac.kr [Department of Biomedical Science and Research Institute for Bioscience and Biotechnology, Hallym University, Chunchon 200-702 (Korea, Republic of)

    2014-10-01

    HIV-1 Tat causes extensive neuroinflammation that may progress to AIDS-related encephalitis and dementia. Celastrol possesses various biological activities such as anti-oxidant, anti-tumor, and anti-inflammatory activities. In this study, we investigated the modulatory effects of celastrol on HIV-1 Tat-induced inflammatory responses and the molecular mechanisms underlying its action in astrocytes. Pre-treatment of CRT-MG human astroglioma cells with celastrol significantly inhibited HIV-1 Tat-induced expression of ICAM-1/VCAM-1 and subsequent monocyte adhesiveness in CRT-MG cells. In addition, celastrol suppressed HIV-1 Tat-induced expression of pro-inflammatory chemokines, such as CXCL10, IL-8, and MCP-1. Celastrol decreased HIV-1 Tat-induced activation of JNK MAPK, AP-1, and NF-κB. Furthermore, celastrol induced mRNA and protein expression of HO-1 as well as Nrf2 activation. Blockage of HO-1 expression using siRNA reversed the inhibitory effect of celastrol on HIV-1 Tat-induced inflammatory responses. These results suggest that celastrol has regulatory effects on HIV-1 Tat-induced inflammatory responses by blocking the JNK MAPK-AP-1/NF-κB signaling pathways and inducing HO-1 expression in astrocytes. - Highlights: • Celastrol suppressed HIV-1 Tat-induced expression of pro-inflammatory genes. • Celastrol inhibited HIV-1 Tat -induced activation of JNK MAPK. • Celastrol inhibited HIV-1 Tat-induced activation of both NF-κB and AP-1. • Celastrol inhibited HIV-1 Tat-induced inflammatory responses via HO-1 induction.

  2. HIV-1 Tat depresses DNA-PKCS expression and DNA repair, and sensitizes cells to ionizing radiation

    International Nuclear Information System (INIS)

    Sun Yi; Huang Yuechen; Xu Qinzhi; Wang Huiping; Bai Bei; Sui Jianli; Zhou Pingkun

    2006-01-01

    Purpose There is accumulating evidence that cancer patients with human immmunodeficiency virus-1/acquired immunodeficency syndrome (HIV-1/AIDS) have more severe tissue reactions and often develop cutaneous toxic effects when subjected to radiotherapy. Here we explored the effects of the HIV-1 Tat protein on cellular responses to ionizing radiation. Methods and Materials Two Tat-expressing cell lines, TT2 and TE671-Tat, were derived from human rhabdomyosarcoma cells by transfecting with the HIV-1 tat gene. Radiosensitivity was determined using colony-forming ability. Gene expression was assessed by cDNA microarray and immunohybridization. The Comet assay and γ-H2AX foci were use to detect DNA double-strand breaks (DSBs) and repair. Radiation-induced cell cycle changes were detected by flow cytometry. Results The radiosensitivity of TT2 and TE671-Tat cells was significantly increased as compared with parental TE671 cells or the control TE671-pCI cells. Tat also increased proliferation activity. The comet assay and γH2AX foci detection revealed a decreased capacity to repair radiation-induced DNA DSBs in Tat-expressing cells. Microarray assay demonstrated that the DNA repair gene DNA-PKcs, and cell cycle-related genes Cdc20, Cdc25C, KIF2C and CTS1 were downregulated in Tat-expressing cells. Depression of DNA-PKcs in Tat-expressing cells was further confirmed by RT-PCR and immuno-hybridization analysis. Tat-expressing cells exhibited a prolonged S phase arrest after 4 Gy γ-irradiation, and a noticeable delay in the initiation and elimination of radiation-induced G 2 /M arrest as compared with parental cells. In addition, the G 2 /M arrest was incomplete in TT2 cells. Moreover, HIV-1 Tat resulted in a constitutive overexpression of cyclin B1 protein. Conclusion HIV-1 Tat protein sensitizes cells to ionizing radiation via depressing DNA repair and dysregulating cell cycle checkpoints. These observations provide new insight into the increased tissue reactions of AIDS

  3. Morphine Tolerance and Physical Dependence Are Altered in Conditional HIV-1 Tat Transgenic Mice.

    Science.gov (United States)

    Fitting, Sylvia; Stevens, David L; Khan, Fayez A; Scoggins, Krista L; Enga, Rachel M; Beardsley, Patrick M; Knapp, Pamela E; Dewey, William L; Hauser, Kurt F

    2016-01-01

    Despite considerable evidence that chronic opiate use selectively affects the pathophysiologic consequences of human immunodeficiency virus type 1 (HIV-1) infection in the nervous system, few studies have examined whether neuro-acquired immune deficiency syndrome (neuroAIDS) might intrinsically alter the pharmacologic responses to chronic opiate exposure. This is an important matter because HIV-1 and opiate abuse are interrelated epidemics, and HIV-1 patients are often prescribed opiates as a treatment of HIV-1-related neuropathic pain. Tolerance and physical dependence are inevitable consequences of frequent and repeated administration of morphine. In the present study, mice expressing HIV-1 Tat in a doxycycline (DOX)-inducible manner [Tat(+)], their Tat(-) controls, and control C57BL/6 mice were chronically exposed to placebo or 75-mg morphine pellets to explore the effects of Tat induction on morphine tolerance and dependence. Antinociceptive tolerance and locomotor activity tolerance were assessed using tail-flick and locomotor activity assays, respectively, and physical dependence was measured with the platform-jumping assay and recording of other withdrawal signs. We found that Tat(+) mice treated with DOX [Tat(+)/DOX] developed an increased tolerance in the tail-flick assay compared with control Tat(-)/DOX and/or C57/DOX mice. Equivalent tolerance was developed in all mice when assessed by locomotor activity. Further, Tat(+)/DOX mice expressed reduced levels of physical dependence to chronic morphine exposure after a 1-mg/kg naloxone challenge compared with control Tat(-)/DOX and/or C57/DOX mice. Assuming the results seen in Tat transgenic mice can be generalized to neuroAIDS, our findings suggest that HIV-1-infected individuals may display heightened analgesic tolerance to similar doses of opiates compared with uninfected individuals and show fewer symptoms of physical dependence. Copyright © 2015 by The American Society for Pharmacology and Experimental

  4. HIV-1 Tat reduces nephrin in human podocytes: a potential mechanism for enhanced glomerular permeability in HIV-associated nephropathy.

    Science.gov (United States)

    Doublier, Sophie; Zennaro, Cristina; Spatola, Tiziana; Lupia, Enrico; Bottelli, Antonella; Deregibus, Maria Chiara; Carraro, Michele; Conaldi, Pier Giulio; Camussi, Giovanni

    2007-02-19

    To determine whether HIV-1 Tat may directly alter glomerular permeability in HIV-associated nephropathy (HIVAN). Heavy proteinuria is a hallmark of HIVAN. The slit diaphragm is the ultimate glomerular filtration barrier critical for maintaining the efficiency of the ultrafiltration unit of the kidney. In this study, we evaluated the direct effect of Tat protein on the permeability of isolated glomeruli and on the expression of nephrin, the main slit diaphragm component, by human cultured podocytes. Permeability was studied by measuring the permeability to albumin in isolated rat glomeruli. We also evaluated the expression of nephrin in human cultured podocytes by using immunofluorescence and Western blot. We found that Tat increased albumin permeability in isolated glomeruli, and rapidly induced the redistribution and loss of nephrin in cultured podocytes. Pretreatment of glomeruli and podocytes with blocking antibodies showed that Tat reduced nephrin expression by engaging vascular endothelial growth factor receptors types 2 and 3 and the integrin alphavbeta3. Pre-incubation of podocytes with two platelet-activating factor (PAF) receptor antagonists prevented the loss and redistribution of nephrin induced by Tat, suggesting that PAF is an intracellular mediator of Tat action. Tat induced a rapid PAF synthesis by podocytes. When podocytes transfected to overexpress PAF-acetylhydrolase, the main catabolic enzyme of PAF, were stimulated with Tat, the redistribution and loss of nephrin was abrogated. The present results define a mechanism by which Tat may reduce nephrin expression in podocytes, thus increasing glomerular permeability. This provides new insights in the understanding of HIVAN pathogenesis.

  5. The Tat protein of human immunodeficiency virus-1 enhances hepatitis C virus replication through interferon gamma-inducible protein-10

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    Qu Jing

    2012-04-01

    Full Text Available Abstract Background Co-infection with human immunodeficiency virus-1 (HIV-1 and hepatitis C virus (HCV is associated with faster progression of liver disease and an increase in HCV persistence. However, the mechanism by which HIV-1 accelerates the progression of HCV liver disease remains unknown. Results HIV-1/HCV co-infection is associated with increased expression of interferon gamma-induced protein-10 (IP-10 mRNA in peripheral blood mononuclear cells (PBMCs. HCV RNA levels were higher in PBMCs of patients with HIV-1/HCV co-infection than in patients with HCV mono-infection. HIV-1 Tat and IP-10 activated HCV replication in a time-dependent manner, and HIV-1 Tat induced IP-10 production. In addition, the effect of HIV-1 Tat on HCV replication was blocked by anti-IP-10 monoclonal antibody, demonstrating that the effect of HIV-1 Tat on HCV replication depends on IP-10. Taken together, these results suggest that HIV-1 Tat protein activates HCV replication by upregulating IP-10 production. Conclusions HIV-1/HCV co-infection is associated with increased expression of IP-10 mRNA and replication of HCV RNA. Furthermore, both HIV-1 Tat and IP-10 activate HCV replication. HIV-1 Tat activates HCV replication by upregulating IP-10 production. These results expand our understanding of HIV-1 in HCV replication and the mechanism involved in the regulation of HCV replication mediated by HIV-1 during co-infection.

  6. HIV-1 Tat C-mediated regulation of tumor necrosis factor receptor-associated factor-3 by microRNA 32 in human microglia

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    Mishra Ritu

    2012-06-01

    Full Text Available Abstract Background HIV-1 Tat protein is known to be associated with neuroinflammation, a condition that develops in almost half of patients infected with HIV-1. HIV-1 Tat can alter glial neuroprotective functions, leading to neurotoxicity within the CNS. HIV-1 Tat is known to be secreted from productively infected cells and can affect neighboring uninfected cells by modulating cellular gene expression in a bystander fashion. Methods We were interested to study whether exogenous exposure to HIV-1 Tat-C protein perturbs the microRNA (miRNA expression profile of human microglial cells, leading to altered protein expression. We used protein expression and purification, miRNA overexpression, miRNA knockdown, transfection, site-directed mutagenesis, real-time PCR, luciferase assay and western blotting techniques to perform our study. Results HIV-1 Tat-C treatment of human microglial cells resulted in a dose-dependent increase in miR-32 expression. We found that tumor necrosis factor-receptor–associated factor 3 TRAF3 is a direct target for miR-32, and overexpression of miR-32 in CHME3 cells decreased TRAF3 both at the mRNA and the protein level. Recovery of TRAF3 protein expression after transfection of anti-miR-32 and the results of the luciferase reporter assay provided direct evidence of TRAF3 regulation by miR-32. We found that the regulation of interferon regulatory factor 3 (IRF3 and IRF7 is controlled by cellular levels of TRAF3 protein in microglial cells, as after overexpression of miR-32 and application of anti-miR-32, expression levels of IRF3 and IRF7 were inversely regulated by expression levels of TRAF3. Thus, our results suggest a novel miRNA mediated mechanism for regulation of TRAF3 in human microglial cells exposed to HIV-1 Tat C protein. These results may help to elucidate the detrimental neuroinflammatory consequences of HIV-1 Tat C protein in bystander fashion. Conclusion HIV-1 Tat protein can modulate TRAF3 expression through

  7. Epitopes of human immunodeficiency virus regulatory proteins tat, nef, and rev are expressed in normal human tissue

    NARCIS (Netherlands)

    Parmentier, H. K.; van Wichen, D. F.; Meyling, F. H.; Goudsmit, J.; Schuurman, H. J.

    1992-01-01

    The expression of regulatory proteins tat, rev, and nef of human immunodeficiency virus type-1 (HIV-1) and tat of HIV-2 was studied in frozen sections of lymph nodes from HIV-1-infected individuals, and various tissues from uninfected persons. In HIV-1-positive lymph nodes, monoclonal antibodies to

  8. Inhibition of GABAergic Neurotransmission by HIV-1 Tat and Opioid Treatment in the Striatum Involves ?-Opioid Receptors

    OpenAIRE

    Xu, Changqing; Fitting, Sylvia

    2016-01-01

    Due to combined antiretroviral therapy (cART), human immunodeficiency virus type 1 (HIV-1) is considered a chronic disease with high prevalence of mild forms of neurocognitive impairments, also referred to as HIV-associated neurocognitive disorders (HAND). Although opiate drug use can exacerbate HIV-1 Tat-induced neuronal damage, it remains unknown how and to what extent opioids interact with Tat on the GABAergic system. We conducted whole-cell recordings in mouse striatal slices and examined...

  9. Targeted PEG-based bioconjugates enhance the cellular uptake and transport of a HIV-1 TAT nonapeptide.

    Science.gov (United States)

    Ramanathan, S; Qiu, B; Pooyan, S; Zhang, G; Stein, S; Leibowitz, M J; Sinko, P J

    2001-12-13

    We previously described the enhanced cell uptake and transport of R.I-K(biotin)-Tat9, a large ( approximately 1500 Da) peptidic inhibitor of HIV-1 Tat protein, via SMVT, the intestinal biotin transporter. The aim of the present study was to investigate the feasibility of targeting biotinylated PEG-based conjugates to SMVT in order to enhance cell uptake and transport of Tat9. The 29 kDa peptide-loaded bioconjugate (PEG:(R.I-Cys-K(biotin)-Tat9)8) used in these studies contained eight copies of R.I-K(biotin)-Tat9 appended to PEG by means of a cysteine linkage. The absorptive transport of biotin-PEG-3400 (0.6-100 microM) and the bioconjugate (0.1-30 microM) was studied using Caco-2 cell monolayers. Inhibition of biotin-PEG-3400 by positive controls (biotin, biocytin, and desthiobiotin) was also determined. Uptake of these two compounds was also determined in CHO cells transfected with human SMVT (CHO/hSMVT) and control cells (CHO/pSPORT) over the concentration ranges of 0.05-12.5 microM and 0.003-30 microM, respectively. Nonbiotinylated forms of these two compounds, PEG-3350 and PEG:(R.I-Cys-K-Tat9)8, were used in the control studies. Biotin-PEG-3400 transport was found to be concentration-dependent and saturable in Caco-2 cells (K(m)=6.61 microM) and CHO/hSMVT cells (K(m)=1.26 microM). Transport/uptake was significantly inhibited by positive control substrates of SMVT. PEG:(R.I-Cys-K(biotin)Tat9)8 also showed saturable transport kinetics in Caco-2 cells (K(m)=6.13 microM) and CHO/hSMVT cells (K(m)=8.19 microM). Maximal uptake in molar equivalents of R.I-Cys-K(biotin)Tat9 was 5.7 times greater using the conjugate versus the biotinylated peptide alone. Transport of the nonbiotinylated forms was significantly lower (PPEG-3400 and PEG:(R.I-Cys-K(biotin)Tat9)8 interact with human SMVT to enhance the cellular uptake and transport of these larger molecules and that targeted bioconjugates may have potential for enhancing the cellular uptake and transport of small peptide

  10. Inhibition of both HIV-1 reverse transcription and gene expression by a cyclic peptide that binds the Tat-transactivating response element (TAR RNA.

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    Matthew S Lalonde

    2011-05-01

    Full Text Available The RNA response element TAR plays a critical role in HIV replication by providing a binding site for the recruitment of the viral transactivator protein Tat. Using a structure-guided approach, we have developed a series of conformationally-constrained cyclic peptides that act as structural mimics of the Tat RNA binding region and block Tat-TAR interactions at nanomolar concentrations in vitro. Here we show that these compounds block Tat-dependent transcription in cell-free systems and in cell-based reporter assays. The compounds are also cell permeable, have low toxicity, and inhibit replication of diverse HIV-1 strains, including both CXCR4-tropic and CCR5-tropic primary HIV-1 isolates of the divergent subtypes A, B, C, D and CRF01_AE. In human peripheral blood mononuclear cells, the cyclic peptidomimetic L50 exhibited an IC(50 ∼250 nM. Surprisingly, inhibition of LTR-driven HIV-1 transcription could not account for the full antiviral activity. Timed drug-addition experiments revealed that L-50 has a bi-phasic inhibition curve with the first phase occurring after HIV-1 entry into the host cell and during the initiation of HIV-1 reverse transcription. The second phase coincides with inhibition of HIV-1 transcription. Reconstituted reverse transcription assays confirm that HIV-1 (- strand strong stop DNA synthesis is blocked by L50-TAR RNA interactions in-vitro. These findings are consistent with genetic evidence that TAR plays critical roles both during reverse transcription and during HIV gene expression. Our results suggest that antiviral drugs targeting TAR RNA might be highly effective due to a dual inhibitory mechanism.

  11. Tat-mediated protein delivery in living Caenorhabditis elegans

    International Nuclear Information System (INIS)

    Delom, Frederic; Fessart, Delphine; Caruso, Marie-Elaine; Chevet, Eric

    2007-01-01

    The Tat protein from HIV-1 fused with heterologous proteins traverses biological membranes in a transcellular process called: protein transduction. This has already been successfully exploited in various biological models, but never in the nematode worm Caenorhabditis elegans. TAT-eGFP or GST-eGFP proteins were fed to C. elegans worms, which resulted in the specific localization of Tat-eGFP to epithelial intestinal cells. This system represents an efficient tool for transcellular transduction in C. elegans intestinal cells. Indeed, this approach avoids the use of tedious purification steps to purify the TAT fusion proteins and allows for rapid analyses of the transduced proteins. In addition, it may represent an efficient tool to functionally analyze the mechanisms of protein transduction as well as to complement RNAi/KO in the epithelial intestinal system. To sum up, the advantage of this technology is to combine the potential of bacterial expression system and the Tat-mediated transduction technique in living worm

  12. Inhibition of GABAergic Neurotransmission by HIV-1 Tat and Opioid Treatment in the Striatum Involves μ-opioid Receptors

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    Changqing Xu

    2016-11-01

    Full Text Available Due to combined antiretroviral therapy (cART, human immunodeficiency virus type 1 (HIV-1 is considered a chronic disease with high prevalence of mild forms of neurocognitive impairments, also referred to as HIV-associated neurocognitive disorders (HAND. Although opiate drug use can exacerbate HIV-1 Tat-induced neuronal damage, it remains unknown how and to what extent opioids interact with Tat on the GABAergic system. We conducted whole-cell recordings in mouse striatal slices and examined the effects of HIV-1 Tat in the presence and absence of morphine (1 μM and damgo (1 μM on GABAergic neurotransmission. Results indicated a decrease in the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs and miniature IPSCs (mIPSCs by Tat (5 – 50 nM in a concentration-dependent manner. The significant Tat-induced decrease in IPSCs was abolished when removing extracellular and/or intracellular calcium. Treatment with morphine or damgo alone significantly decreased the frequency, but not amplitude of IPSCs. Interestingly, morphine but not damgo indicated an additional downregulation of the mean frequency of mIPSCs in combination with Tat. Pretreatment with naloxone (1 μM and CTAP (1 μM prevented the Tat-induced decrease in sIPSCs frequency but only naloxone prevented the combined Tat and morphine effect on mIPSCs frequency. Results indicate a Tat- or opioid-induced decrease in GABAergic neurotransmission via µ-opioid receptors with combined Tat and morphine effects involving additional opioid receptor-related mechanisms. Exploring the interactions between Tat and opioids on the GABAergic system may help to guide future research on HAND in the context of opiate drug use.

  13. Crosstalk between HDAC6 and Nox2-based NADPH oxidase mediates HIV-1 Tat-induced pro-inflammatory responses in astrocytes

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    Gi Soo Youn

    2017-08-01

    Full Text Available Histone deacetylase 6 (HDAC6 likely is important in inflammatory diseases. However, how HDAC6 exerts its effect on inflammatory processes remains unclear. HIV-1 transactivator of transcription (Tat activates NADPH oxidase resulting in generation of reactive oxygen species (ROS, leading to extensive neuro-inflammation in the central nervous system. We investigated the correlation of HDAC6 and NADPH oxidase in HIV-1 Tat-stimulated astrocytes. HDAC6 knockdown attenuated HIV-1 Tat-induced ROS generation and NADPH oxidase activation. HDAC6 knockdown suppressed HIV-1 Tat-induced expression of NADPH oxidase subunits, such as Nox2, p47phox, and p22phox. Specific inhibition of HDAC6 using tubastatin A suppressed HIV-1 Tat-induced ROS generation and activation of NADPH oxidase. N-acetyl cysteine, diphenyl iodonium, and apocynin suppressed HIV-1 Tat-induced expression of HDAC6 and the pro-inflammatory chemokines CCL2, CXCL8, and CXCL10. Nox2 knockdown attenuated HIV-1 Tat-induced HDAC6 expression and subsequent expression of chemokines. The collective results point to the potential crosstalk between HDAC6 and NADPH oxidase, which could be a combined therapeutic target for relief of HIV-1 Tat-mediated neuro-inflammation. Keywords: HIV-1 Tat, HDAC6, NADPH oxidase, ROS, Inflammation, Astrocytes

  14. MAS NMR of HIV-1 protein assemblies

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    Suiter, Christopher L.; Quinn, Caitlin M.; Lu, Manman; Hou, Guangjin; Zhang, Huilan; Polenova, Tatyana

    2015-04-01

    The negative global impact of the AIDS pandemic is well known. In this perspective article, the utility of magic angle spinning (MAS) NMR spectroscopy to answer pressing questions related to the structure and dynamics of HIV-1 protein assemblies is examined. In recent years, MAS NMR has undergone major technological developments enabling studies of large viral assemblies. We discuss some of these evolving methods and technologies and provide a perspective on the current state of MAS NMR as applied to the investigations into structure and dynamics of HIV-1 assemblies of CA capsid protein and of Gag maturation intermediates.

  15. Fractalkine/CX3CL1 protects striatal neurons from synergistic morphine and HIV-1 Tat-induced dendritic losses and death

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    Suzuki Masami

    2011-11-01

    Full Text Available Abstract Background Fractalkine/CX3CL1 and its cognate receptor CX3CR1 are abundantly expressed in the CNS. Fractalkine is an unusual C-X3-C motif chemokine that is important in neuron-microglial communication, a co-receptor for HIV infection, and can be neuroprotective. To assess the effects of fractalkine on opiate-HIV interactive neurotoxicity, wild-type murine striatal neurons were co-cultured with mixed glia from the striata of wild-type or Cx3cr1 knockout mice ± HIV-1 Tat and/or morphine. Time-lapse digital images were continuously recorded at 20 min intervals for up to 72 h using computer-aided microscopy to track the same cells repeatedly. Results Co-exposure to Tat and morphine caused synergistic increases in neuron death, dendritic pruning, and microglial motility as previously reported. Exogenous fractalkine prevented synergistic Tat and morphine-induced dendritic losses and neuron death even though the inflammatory mediator TNF-α remained significantly elevated. Antibody blockade of CX3CR1 mimicked the toxic effects of morphine plus Tat, but did not add to their toxicity; while fractalkine failed to protect wild-type neurons co-cultured with Cx3cr1-/--null glia against morphine and Tat toxicity. Exogenous fractalkine also normalized microglial motility, which is elevated by Tat and morphine co-exposure, presumably limiting microglial surveillance that may lead to toxic effects on neurons. Fractalkine immunofluorescence was expressed in neurons and to a lesser extent by other cell types, whereas CX3CR1 immunoreactivity or GFP fluorescence in cells cultured from the striatum of Cx3cr1-/- (Cx3cr1GFP/GFP mice were associated with microglia. Immunoblotting shows that fractalkine levels were unchanged following Tat and/or morphine exposure and there was no increase in released fractalkine as determined by ELISA. By contrast, CX3CR1 protein levels were markedly downregulated. Conclusions The results suggest that deficits in fractalkine

  16. HIV-1 Tat affects the programming and functionality of human CD8⁺ T cells by modulating the expression of T-box transcription factors.

    Science.gov (United States)

    Sforza, Fabio; Nicoli, Francesco; Gallerani, Eleonora; Finessi, Valentina; Reali, Eva; Cafaro, Aurelio; Caputo, Antonella; Ensoli, Barbara; Gavioli, Riccardo

    2014-07-31

    HIV infection is characterized by several immune dysfunctions of both CD8⁺ and CD4⁺ T cells as hyperactivation, impairment of functionality and expansion of memory T cells. CD8⁺ T-cell dysfunctions have been associated with increased expression of T-bet, Eomesdermin and pro-inflammatory cytokines, and with down-regulation of CD127. The HIV-1 trans-activator of transcription (Tat) protein, which is released by infected cells and detected in tissues of HIV-positive individuals, is known to contribute to the dysregulation of CD4⁺ T cells; however, its effects on CD8⁺ T cells have not been investigated. Thus, in this study, we sought to address whether Tat may affect CD8⁺ T-cell functionality and programming. CD8⁺ T cells were activated by T-cell receptor engagement in the presence or absence of Tat. Cytokine production, killing capacity, surface phenotype and expression of transcription factors important for T-cell programming were evaluated. Tat favors the secretion of interleukin-2, interferon-γ and granzyme B in CD8⁺ T cells. Behind this functional modulation we observed that Tat increases the expression of T-bet, Eomesdermin, Blimp-1, Bcl-6 and Bcl-2 in activated but not in unstimulated CD8⁺ T lymphocytes. This effect is associated with the down-regulation of CD127 and the up-regulation of CD27. Tat deeply alters the programming and functionality of CD8⁺ T lymphocytes.

  17. HIV-1 proteins dysregulate motivational processes and dopamine circuitry.

    Science.gov (United States)

    Bertrand, Sarah J; Mactutus, Charles F; Harrod, Steven B; Moran, Landhing M; Booze, Rosemarie M

    2018-05-18

    Motivational alterations, such as apathy, in HIV-1+ individuals are associated with decreased performance on tasks involving frontal-subcortical circuitry. We used the HIV-1 transgenic (Tg) rat to assess effect of long-term HIV-1 protein exposure on motivated behavior using sucrose (1-30%, w/v) and cocaine (0.01-1.0 mg/kg/infusion) maintained responding with fixed-ratio (FR) and progressive-ratio (PR) schedules of reinforcement. For sucrose-reinforced responding, HIV-1 Tg rats displayed no change in EC 50 relative to controls, suggesting no change in sucrose reinforcement but had a downward shifted concentration-response curves, suggesting a decrease in response vigor. Cocaine-maintained responding was attenuated in HIV-1 Tg rats (FR1 0.33 mg/kg/infusion and PR 1.0 mg/kg/infusion). Dose-response tests (PR) revealed that HIV-1 Tg animals responded significantly less than F344 control rats and failed to earn significantly more infusions of cocaine as the unit dose increased. When choosing between cocaine and sucrose, control rats initially chose sucrose but with time shifted to a cocaine preference. In contrast, HIV-1 disrupted choice behaviors. DAT function was altered in the striatum of HIV-1 Tg rats; however, prior cocaine self-administration produced a unique effect on dopamine homeostasis in the HIV-1 Tg striatum. These findings of altered goal directed behaviors may determine neurobiological mechanisms of apathy in HIV-1+ patients.

  18. Effects of altered TatC proteins on protein secretion efficiency via the twin-arginine translocation pathway of Bacillus subtilis

    NARCIS (Netherlands)

    Eijlander, Robyn T.; Kolbusz, Magdalena A.; Berendsen, Erwin M.; Kuipers, Oscar P.

    Protein translocation via the Tat machinery in thylakoids and bacteria occurs through a cooperation between the TatA, TatB and TatC subunits, of which the TatC protein forms the initial Tat substrate-binding site. The Bacillus subtilis Tat machinery lacks TatB and comprises two separate TatAC

  19. HIV-1 tat promotes integrin-mediated HIV transmission to dendritic cells by binding Env spikes and competes neutralization by anti-HIV antibodies.

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    Paolo Monini

    Full Text Available Use of Env in HIV vaccine development has been disappointing. Here we show that, in the presence of a biologically active Tat subunit vaccine, a trimeric Env protein prevents in monkeys virus spread from the portal of entry to regional lymph nodes. This appears to be due to specific interactions between Tat and Env spikes that form a novel virus entry complex favoring R5 or X4 virus entry and productive infection of dendritic cells (DCs via an integrin-mediated pathway. These Tat effects do not require Tat-transactivation activity and are blocked by anti-integrin antibodies (Abs. Productive DC infection promoted by Tat is associated with a highly efficient virus transmission to T cells. In the Tat/Env complex the cysteine-rich region of Tat engages the Env V3 loop, whereas the Tat RGD sequence remains free and directs the virus to integrins present on DCs. V2 loop deletion, which unshields the CCR5 binding region of Env, increases Tat/Env complex stability. Of note, binding of Tat to Env abolishes neutralization of Env entry or infection of DCs by anti-HIV sera lacking anti-Tat Abs, which are seldom present in natural infection. This is reversed, and neutralization further enhanced, by HIV sera containing anti-Tat Abs such as those from asymptomatic or Tat-vaccinated patients, or by sera from the Tat/Env vaccinated monkeys. Thus, both anti-Tat and anti-Env Abs are required for efficient HIV neutralization. These data suggest that the Tat/Env interaction increases HIV acquisition and spreading, as a mechanism evolved by the virus to escape anti-Env neutralizing Abs. This may explain the low effectiveness of Env-based vaccines, which are also unlikely to elicit Abs against new Env epitopes exposed by the Tat/Env interaction. As Tat also binds Envs from different clades, new vaccine strategies should exploit the Tat/Env interaction for both preventative and therapeutic interventions.

  20. Effects of the TAT peptide orientation and relative location on the protein transduction efficiency.

    Science.gov (United States)

    Guo, Qingguo; Zhao, Guojie; Hao, Fengjin; Guan, Yifu

    2012-05-01

    To understand the protein transduction domain (PTD)-mediated protein transduction behavior and to explore its potential in delivering biopharmaceutic drugs, we prepared four TAT-EGFP conjugates: TAT(+)-EGFP, TAT(-)-EGFP, EGFP-TAT(+) and EGFP-TAT(-), where TAT(+) and TAT(-) represent the original and the reversed TAT sequence, respectively. These four TAT-EGFP conjugates were incubated with HeLa and PC12 cells for in vitro study as well as injected intraperitoneally to mice for in vivo study. Flow cytometric results showed that four TAT-EGFP conjugates were able to traverse HeLa and PC12 cells with almost equal transduction efficiency. The in vivo study showed that the TAT-EGFP conjugates could be delivered into different organs of mice with different transduction capabilities. Bioinformatic analyses and CD spectroscopic data revealed that the TAT peptide has no defined secondary structure, and conjugating the TAT peptide to the EGFP cargo protein would not alter the native structure and the function of the EGFP protein. These results conclude that the sequence orientation, the spatial structure, and the relative location of the TAT peptide have much less effect on the TAT-mediated protein transduction. Thus, the TAT-fused conjugates could be constructed in more convenient and flexible formats for a wide range of biopharmaceutical applications. © 2011 John Wiley & Sons A/S.

  1. Trans-activation of the JC virus late promoter by the tat protein of type 1 human immunodeficiency virus in glial cells

    International Nuclear Information System (INIS)

    Tada, Hiroomi; Lashgari, M.; Amini, S.; Khalili, K.; Rappaport, J.; Wong-Staal, F.

    1990-01-01

    Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system caused by the JC virus (JCV), a human papovavirus. PML is a relatively rare disease seen predominantly in immunocompromised individuals and is a frequent complication observed in AIDS patients. The significantly higher incidence of PML in AIDS patients than in other immunosuppressive disorders has suggested that the presence of human immunodeficiency virus type 1 (HIV-1) in the brain may directly or indirectly contribute to the pathogenesis of this disease. In the present study the authors have examined the expression of the JCV genome in both glial and non-glial cells in the presence of HIV-1 regulatory proteins. They find that the HIV-1-encoded trans-regulatory protein tat increases the basal activity of the JCV late promoter, JCV L , in glial cells. They conclude that the presence of the HIV-1-encoded tat protein may positively affect the JCV lytic cycle in glial cells by stimulating JCV gene expression. The results suggest a mechanism for the relatively high incidence of PML in AIDS patients than in other immunosuppressive disorders. Furthermore, the findings indicate that the HIV-1 regulatory protein tat may stimulate other viral and perhaps cellular promoters, in addition to its own

  2. NF90 una proteína celular que se une a RNAds inhibe la transactivación del LTR del HIV-1 mediada por TAT

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    Silvio Urcuqui Inchima

    2001-04-01

    Full Text Available

    La transactivación del LTR de HIV-1 requiere la interacción de la proteína Tat ( trans-activation transcription con la estructura TAR ( trans-activation response, que se encuentra localizada en el extremo 5’ de todos los transcriptos virales. La interacción de la proteína Tat con TAR forma un precomplejo transcripcional indispensable para la eficiente transcripción del genoma viral.
    La interacción de diferentes factores celulares (cdk9 y ciclina T, entre otros con el precomplejo Tat-TAR, constituye un complejo estable que facilita la fosforilación de la subunidad mayor de la RNA polimerasa II, asegurando una eficiente elongación de los transcriptos virales. No se han descripto proteínas capaces de regular negativamente la función de Tat con resultados nefastos para la replicación viral. También se ha descrito otro tipo de proteínas celulares con capacidad de interactuar con TAR. El ejemplo mejor caracterizado es la proteína-kinasa dependiente de RNA (PKR, se autofosforila y mediante fosforilación del factor eIF2, inhibe la síntesis de proteínas. Con el presente trabajo se
    pretendió aislar y caracterizar otras proteínas celulares capaces de interactuar con la estructura TAR y estudiar su efecto en la función de Tat.

     

  3. Hyperthermia stimulates HIV-1 replication.

    Directory of Open Access Journals (Sweden)

    Ferdinand Roesch

    Full Text Available HIV-infected individuals may experience fever episodes. Fever is an elevation of the body temperature accompanied by inflammation. It is usually beneficial for the host through enhancement of immunological defenses. In cultures, transient non-physiological heat shock (42-45°C and Heat Shock Proteins (HSPs modulate HIV-1 replication, through poorly defined mechanisms. The effect of physiological hyperthermia (38-40°C on HIV-1 infection has not been extensively investigated. Here, we show that culturing primary CD4+ T lymphocytes and cell lines at a fever-like temperature (39.5°C increased the efficiency of HIV-1 replication by 2 to 7 fold. Hyperthermia did not facilitate viral entry nor reverse transcription, but increased Tat transactivation of the LTR viral promoter. Hyperthermia also boosted HIV-1 reactivation in a model of latently-infected cells. By imaging HIV-1 transcription, we further show that Hsp90 co-localized with actively transcribing provirus, and this phenomenon was enhanced at 39.5°C. The Hsp90 inhibitor 17-AAG abrogated the increase of HIV-1 replication in hyperthermic cells. Altogether, our results indicate that fever may directly stimulate HIV-1 replication, in a process involving Hsp90 and facilitation of Tat-mediated LTR activity.

  4. "cART intensification by the HIV-1 Tat B clade vaccine: progress to phase III efficacy studies".

    Science.gov (United States)

    Cafaro, Aurelio; Sgadari, Cecilia; Picconi, Orietta; Tripiciano, Antonella; Moretti, Sonia; Francavilla, Vittorio; Pavone Cossut, Maria Rosaria; Buttò, Stefano; Cozzone, Giovanni; Ensoli, Fabrizio; Monini, Paolo; Ensoli, Barbara

    2018-02-01

    In spite of its success at suppressing HIV replication, combination antiretroviral therapy (cART) only partially reduces immune dysregulation and loss of immune functions. These cART-unmet needs appear to be due to persistent virus replication and cell-to-cell transmission in reservoirs, and are causes of increased patients' morbidity and mortality. Up to now, therapeutic interventions aimed at cART-intensification by attacking the virus reservoir have failed. Areas covered: We briefly review the rationale and clinical development of Tat therapeutic vaccine in cART-treated subjects in Italy and South Africa (SA). Vaccination with clade-B Tat induced cross-clade neutralizing antibodies, immune restoration, including CD4 + T cell increase particularly in low immunological responders, and reduction of proviral DNA. Phase III efficacy trials in SA are planned both in adult and pediatric populations. Expert commentary: We propose the Tat therapeutic vaccine as a pathogenesis-driven intervention that effectively intensifies cART and may lead to a functional cure and provide new perspectives for prevention and virus eradication strategies.

  5. Recognition of HIV-1 peptides by host CTL is related to HIV-1 similarity to human proteins.

    Directory of Open Access Journals (Sweden)

    Morgane Rolland

    Full Text Available BACKGROUND: While human immunodeficiency virus type 1 (HIV-1-specific cytotoxic T lymphocytes preferentially target specific regions of the viral proteome, HIV-1 features that contribute to immune recognition are not well understood. One hypothesis is that similarities between HIV and human proteins influence the host immune response, i.e., resemblance between viral and host peptides could preclude reactivity against certain HIV epitopes. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the extent of similarity between HIV-1 and the human proteome. Proteins from the HIV-1 B consensus sequence from 2001 were dissected into overlapping k-mers, which were then probed against a non-redundant database of the human proteome in order to identify segments of high similarity. We tested the relationship between HIV-1 similarity to host encoded peptides and immune recognition in HIV-infected individuals, and found that HIV immunogenicity could be partially modulated by the sequence similarity to the host proteome. ELISpot responses to peptides spanning the entire viral proteome evaluated in 314 individuals showed a trend indicating an inverse relationship between the similarity to the host proteome and the frequency of recognition. In addition, analysis of responses by a group of 30 HIV-infected individuals against 944 overlapping peptides representing a broad range of individual HIV-1B Nef variants, affirmed that the degree of similarity to the host was significantly lower for peptides with reactive epitopes than for those that were not recognized. CONCLUSIONS/SIGNIFICANCE: Our results suggest that antigenic motifs that are scarcely represented in human proteins might represent more immunogenic CTL targets not selected against in the host. This observation could provide guidance in the design of more effective HIV immunogens, as sequences devoid of host-like features might afford superior immune reactivity.

  6. Tat protein vaccination of cynomolgus macaques influences SHIV-89.6P cy243 epitope variability.

    Science.gov (United States)

    Ridolfi, Barbara; Genovese, Domenico; Argentini, Claudio; Maggiorella, Maria Teresa; Sernicola, Leonardo; Buttò, Stefano; Titti, Fausto; Borsetti, Alessandra; Ensoli, Barbara

    2008-02-01

    In a previous study we showed that vaccination with the native Tat protein controlled virus replication in five out of seven monkeys against challenge with the simian human immunodeficiency virus (SHIV)-89.6P cy243 and that this protection correlated with T helper (Th)-1 response and cytotoxic T lymphocyte (CTL) activity. To address the evolution of the SHIV-89.6P cy243 both in control and vaccinated infected monkeys, the sequence of the human immunodeficiency virus (HIV)-1 Tat protein and the C2-V3 Env region of the proviral-DNA-derived clones were analyzed in both control and vaccinated but unprotected animals. We also performed analysis of the T cell epitope using a predictive epitope model taking into consideration the phylogeny of the variants. Our results suggest that even though the viral evolution observed in both groups of monkeys was directed toward variations in the major histocompatibility complex (MHC)-I epitopes, in the control animals it was associated with mutational escape of such epitopes. On the contrary, it is possible that viral evolution in the vaccinated monkeys was linked to mutations that arose to keep high the viral fitness. In the vaccinated animals the reduction of epitope variability, obtained prompting the immune system by vaccination and inducing a specific immunological response against virus, was able to reduce the emergence of escape mutants. Thus the intervention of host's selective forces in driving CTL escape mutants and in modulating viral fitness appeared to be different in the two groups of monkeys. We concluded that in the vaccinated unprotected animals, vaccination with the Tat protein induced a broad antiviral response, as demonstrated by the reduced ability to develop escape mutants, which is known to help in the control of viral replication.

  7. Thermodynamic studies of a series of homologous HIV-1 TAR RNA ligands reveal that loose binders are stronger Tat competitors than tight ones.

    Science.gov (United States)

    Pascale, Lise; Azoulay, Stéphane; Di Giorgio, Audrey; Zenacker, Laura; Gaysinski, Marc; Clayette, Pascal; Patino, Nadia

    2013-06-01

    RNA is a major drug target, but the design of small molecules that modulate RNA function remains a great challenge. In this context, a series of structurally homologous 'polyamide amino acids' (PAA) was studied as HIV-1 trans-activating response (TAR) RNA ligands. An extensive thermodynamic study revealed the occurence of an enthalpy-entropy compensation phenomenon resulting in very close TAR affinities for all PAA. However, their binding modes and their ability to compete with the Tat fragment strongly differ according to their structure. Surprisingly, PAA that form loose complexes with TAR were shown to be stronger Tat competitors than those forming tight ones, and thermal denaturation studies demonstrated that loose complexes are more stable than tight ones. This could be correlated to the fact that loose and tight ligands induce distinct RNA conformational changes as revealed by circular dichroism experiments, although nuclear magnetic resonance (NMR) experiments showed that the TAR binding site is the same in all cases. Finally, some loose PAA also display promising inhibitory activities on HIV-infected cells. Altogether, these results lead to a better understanding of RNA interaction modes that could be very useful for devising new ligands of relevant RNA targets.

  8. Proteomics in the investigation of HIV-1 interactions with host proteins.

    Science.gov (United States)

    Li, Ming

    2015-02-01

    Productive HIV-1 infection depends on host machinery, including a broad array of cellular proteins. Proteomics has played a significant role in the discovery of HIV-1 host proteins. In this review, after a brief survey of the HIV-1 host proteins that were discovered by proteomic analyses, I focus on analyzing the interactions between the virion and host proteins, as well as the technologies and strategies used in those proteomic studies. With the help of proteomics, the identification and characterization of HIV-1 host proteins can be translated into novel antiretroviral therapeutics. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Epigenetic regulation of HIV-1 latency: focus on polycomb group (PcG) proteins.

    Science.gov (United States)

    Khan, Sheraz; Iqbal, Mazhar; Tariq, Muhammad; Baig, Shahid M; Abbas, Wasim

    2018-01-01

    HIV-1 latency allows the virus to persist until reactivation, in a transcriptionally silent form in its cellular reservoirs despite the presence of effective cART. Such viral persistence represents a major barrier to HIV eradication since treatment interruption leads to rebound plasma viremia. Polycomb group (PcG) proteins have recently got a considerable attention in regulating HIV-1 post-integration latency as they are involved in the repression of proviral gene expression through the methylation of histones. This epigenetic regulation plays an important role in the establishment and maintenance of HIV-1 latency. In fact, PcG proteins act in complexes and modulate the epigenetic signatures of integrated HIV-1 promoter. Key role played by PcG proteins in the molecular control of HIV-1 latency has led to hypothesize that PcG proteins may represent a valuable target for future HIV-1 therapy in purging HIV-1 reservoirs. In this regard, various small molecules have been synthesized or explored to specifically block the epigenetic activity of PcG. In this review, we will highlight the possible therapeutic approaches to achieve either a functional or sterilizing cure of HIV-1 infection with special focus on histone methylation by PcG proteins together with current and novel pharmacological approaches to reactivate HIV-1 from latency that could ultimately lead towards a better clearance of viral latent reservoirs.

  10. Therapeutic immunization with HIV-1 Tat reduces immune activation and loss of regulatory T-cells and improves immune function in subjects on HAART.

    Directory of Open Access Journals (Sweden)

    Barbara Ensoli

    2010-11-01

    Full Text Available Although HAART suppresses HIV replication, it is often unable to restore immune homeostasis. Consequently, non-AIDS-defining diseases are increasingly seen in treated individuals. This is attributed to persistent virus expression in reservoirs and to cell activation. Of note, in CD4(+ T cells and monocyte-macrophages of virologically-suppressed individuals, there is continued expression of multi-spliced transcripts encoding HIV regulatory proteins. Among them, Tat is essential for virus gene expression and replication, either in primary infection or for virus reactivation during HAART, when Tat is expressed, released extracellularly and exerts, on both the virus and the immune system, effects that contribute to disease maintenance. Here we report results of an ad hoc exploratory interim analysis (up to 48 weeks on 87 virologically-suppressed HAART-treated individuals enrolled in a phase II randomized open-label multicentric clinical trial of therapeutic immunization with Tat (ISS T-002. Eighty-eight virologically-suppressed HAART-treated individuals, enrolled in a parallel prospective observational study at the same sites (ISS OBS T-002, served for intergroup comparison. Immunization with Tat was safe, induced durable immune responses, and modified the pattern of CD4(+ and CD8(+ cellular activation (CD38 and HLA-DR together with reduction of biochemical activation markers and persistent increases of regulatory T cells. This was accompanied by a progressive increment of CD4(+ T cells and B cells with reduction of CD8(+ T cells and NK cells, which were independent from the type of antiretroviral regimen. Increase in central and effector memory and reduction in terminally-differentiated effector memory CD4(+ and CD8(+ T cells were accompanied by increases of CD4(+ and CD8(+ T cell responses against Env and recall antigens. Of note, more immune-compromised individuals experienced greater therapeutic effects. In contrast, these changes were opposite

  11. IFITM Proteins Restrict HIV-1 Infection by Antagonizing the Envelope Glycoprotein

    Directory of Open Access Journals (Sweden)

    Jingyou Yu

    2015-10-01

    Full Text Available The interferon-induced transmembrane (IFITM proteins have been recently shown to restrict HIV-1 and other viruses. Here, we provide evidence that IFITM proteins, particularly IFITM2 and IFITM3, specifically antagonize the HIV-1 envelope glycoprotein (Env, thereby inhibiting viral infection. IFITM proteins interact with HIV-1 Env in viral producer cells, leading to impaired Env processing and virion incorporation. Notably, the level of IFITM incorporation into HIV-1 virions does not strictly correlate with the extent of inhibition. Prolonged passage of HIV-1 in IFITM-expressing T lymphocytes leads to emergence of Env mutants that overcome IFITM restriction. The ability of IFITMs to inhibit cell-to-cell infection can be extended to HIV-1 primary isolates, HIV-2 and SIVs; however, the extent of inhibition appears to be virus-strain dependent. Overall, our study uncovers a mechanism by which IFITM proteins specifically antagonize HIV-1 Env to restrict HIV-1 infection and provides insight into the specialized role of IFITMs in HIV infection.

  12. Clustering patterns of cytotoxic T-lymphocyte epitopes in human immunodeficiency virus type 1 (HIV-1) proteins reveal imprints of immune evasion on HIV-1 global variation

    DEFF Research Database (Denmark)

    Yusim, K.; Kesmir, Can; Gaschen, B.

    2002-01-01

    The human cytotoxic T-lymphocyte (CTL) response to human immunodeficiency virus type 1 (HIV-1) has been intensely studied, and hundreds of CTL epitopes have been experimentally defined, published, and compiled in the HIV Molecular Immunology Database. Maps of CTL epitopes on HIV-1 protein sequenc...

  13. Backbone dynamics of the EIAV-Tat protein from 15N relaxation studies

    International Nuclear Information System (INIS)

    Ejchart, A.; Herrmann, F.; Roesch, P.; Sticht, H.; Willbold, D.

    1994-01-01

    The work investigates the mobility of EIAV-Tat protein backbone by measuring the relaxation parameters of the 15 N nitrogens. High degree of the flexibility, non-typical of rigid, well structured proteins was shown

  14. Rational design of highly potent HIV-1 fusion inhibitory proteins: Implication for developing antiviral therapeutics

    International Nuclear Information System (INIS)

    Ni Ling; Gao, George F.; Tien Po

    2005-01-01

    Recombinant protein containing one heptad-repeat 1 (HR1) segment and one HR2 segment of the HIV-1 gp41 (HR1-HR2) has been shown to fold into thermally stable six-helix bundle, representing the fusogenic core of gp41. In this study, we have used the fusogenic core as a scaffold to design HIV-1 fusion inhibitory proteins by linking another HR1 to the C terminus of HR1-HR2 (HR121) or additional HR2 to the N terminus of HR1-HR2 (HR212). Both recombinant proteins could be abundantly and solubly expressed and easily purified, exhibiting high stability and potent inhibitory activity on HIV-1 fusion with IC 50 values of 16.2 ± 2.8 and 2.8 ± 0.63 nM, respectively. These suggest that these rationally designed proteins can be further developed as novel anti-HIV-1 therapeutics

  15. Interactive HIV-1 Tat and morphine-induced synaptodendritic injury is triggered through focal disruptions in Na⁺ influx, mitochondrial instability, and Ca²⁺ overload.

    Science.gov (United States)

    Fitting, Sylvia; Knapp, Pamela E; Zou, Shiping; Marks, William D; Bowers, M Scott; Akbarali, Hamid I; Hauser, Kurt F

    2014-09-17

    Synaptodendritic injury is thought to underlie HIV-associated neurocognitive disorders and contributes to exaggerated inflammation and cognitive impairment seen in opioid abusers with HIV-1. To examine events triggering combined transactivator of transcription (Tat)- and morphine-induced synaptodendritic injury systematically, striatal neuron imaging studies were conducted in vitro. These studies demonstrated nearly identical pathologic increases in dendritic varicosities as seen in Tat transgenic mice in vivo. Tat caused significant focal increases in intracellular sodium ([Na(+)]i) and calcium ([Ca(2+)]i) in dendrites that were accompanied by the emergence of dendritic varicosities. These effects were largely, but not entirely, attenuated by the NMDA and AMPA receptor antagonists MK-801 and CNQX, respectively. Concurrent morphine treatment accelerated Tat-induced focal varicosities, which were accompanied by localized increases in [Ca(2+)]i and exaggerated instability in mitochondrial inner membrane potential. Importantly, morphine's effects were prevented by the μ-opioid receptor antagonist CTAP and were not observed in neurons cultured from μ-opioid receptor knock-out mice. Combined Tat- and morphine-induced initial losses in ion homeostasis and increases in [Ca(2+)]i were attenuated by the ryanodine receptor inhibitor ryanodine, as well as pyruvate. In summary, Tat induced increases in [Na(+)]i, mitochondrial instability, excessive Ca(2+) influx through glutamatergic receptors, and swelling along dendrites. Morphine, acting via μ-opioid receptors, exacerbates these excitotoxic Tat effects at the same subcellular locations by mobilizing additional [Ca(2+)]i and by further disrupting [Ca(2+)]i homeostasis. We hypothesize that the spatiotemporal relationship of μ-opioid and aberrant AMPA/NMDA glutamate receptor signaling is critical in defining the location and degree to which opiates exacerbate the synaptodendritic injury commonly observed in neuro

  16. Boosting of HIV-1 neutralizing antibody responses by a distally related retroviral envelope protein.

    Science.gov (United States)

    Uchtenhagen, Hannes; Schiffner, Torben; Bowles, Emma; Heyndrickx, Leo; LaBranche, Celia; Applequist, Steven E; Jansson, Marianne; De Silva, Thushan; Back, Jaap Willem; Achour, Adnane; Scarlatti, Gabriella; Fomsgaard, Anders; Montefiori, David; Stewart-Jones, Guillaume; Spetz, Anna-Lena

    2014-06-15

    Our knowledge of the binding sites for neutralizing Abs (NAb) that recognize a broad range of HIV-1 strains (bNAb) has substantially increased in recent years. However, gaps remain in our understanding of how to focus B cell responses to vulnerable conserved sites within the HIV-1 envelope glycoprotein (Env). In this article, we report an immunization strategy composed of a trivalent HIV-1 (clade B envs) DNA prime, followed by a SIVmac239 gp140 Env protein boost that aimed to focus the immune response to structurally conserved parts of the HIV-1 and simian immunodeficiency virus (SIV) Envs. Heterologous NAb titers, primarily to tier 1 HIV-1 isolates, elicited during the trivalent HIV-1 env prime, were significantly increased by the SIVmac239 gp140 protein boost in rabbits. Epitope mapping of Ab-binding reactivity revealed preferential recognition of the C1, C2, V2, V3, and V5 regions. These results provide a proof of concept that a distally related retroviral SIV Env protein boost can increase pre-existing NAb responses against HIV-1. Copyright © 2014 by The American Association of Immunologists, Inc.

  17. A triad of lys12, lys41, arg78 spatial domain, a novel identified heparin binding site on tat protein, facilitates tat-driven cell adhesion.

    Directory of Open Access Journals (Sweden)

    Jing Ai

    Full Text Available Tat protein, released by HIV-infected cells, has a battery of important biological effects leading to distinct AIDS-associated pathologies. Cell surface heparan sulfate protoglycans (HSPGs have been accepted as endogenous Tat receptors, and the Tat basic domain has been identified as the heparin binding site. However, findings that deletion or substitution of the basic domain inhibits but does not completely eliminate Tat-heparin interactions suggest that the basic domain is not the sole Tat heparin binding site. In the current study, an approach integrating computational modeling, mutagenesis, biophysical and cell-based assays was used to elucidate a novel, high affinity heparin-binding site: a Lys12, Lys41, Arg78 (KKR spatial domain. This domain was also found to facilitate Tat-driven β1 integrin activation, producing subsequent SLK cell adhesion in an HSPG-dependent manner, but was not involved in Tat internalization. The identification of this new heparin binding site may foster further insight into the nature of Tat-heparin interactions and subsequent biological functions, facilitating the rational design of new therapeutics against Tat-mediated pathological events.

  18. HIV-1 nucleocapsid protein localizes efficiently to the nucleus and nucleolus

    International Nuclear Information System (INIS)

    Yu, Kyung Lee; Lee, Sun Hee; Lee, Eun Soo; You, Ji Chang

    2016-01-01

    The HIV-1 nucleocapsid (NC) is an essential viral protein containing two highly conserved retroviral-type zinc finger (ZF) motifs, which functions in multiple stages of the HIV-1 life cycle. Although a number of functions for NC either in its mature form or as a domain of Gag have been revealed, little is known about the intracellular localization of NC and, moreover, its role in Gag protein trafficking. Here, we have investigated various forms of HIV-1 NC protein for its cellular localization and found that the NC has a strong nuclear and nucleolar localization activity. The linker region, composed of a stretch of basic amino acids between the two ZF motifs, was necessary and sufficient for the activity. - Highlights: • HIV-1 NC possess a NLS and leads to nuclear and nucleolus localization. • Mutations in basic residues between two ZFs in NC decrease the nucleus localization. • ZFs of NC affect cytoplasmic organelles localization rather than nucleus localization.

  19. HIV-1 nucleocapsid protein localizes efficiently to the nucleus and nucleolus

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Kyung Lee; Lee, Sun Hee; Lee, Eun Soo; You, Ji Chang, E-mail: jiyou@catholic.ac.kr

    2016-05-15

    The HIV-1 nucleocapsid (NC) is an essential viral protein containing two highly conserved retroviral-type zinc finger (ZF) motifs, which functions in multiple stages of the HIV-1 life cycle. Although a number of functions for NC either in its mature form or as a domain of Gag have been revealed, little is known about the intracellular localization of NC and, moreover, its role in Gag protein trafficking. Here, we have investigated various forms of HIV-1 NC protein for its cellular localization and found that the NC has a strong nuclear and nucleolar localization activity. The linker region, composed of a stretch of basic amino acids between the two ZF motifs, was necessary and sufficient for the activity. - Highlights: • HIV-1 NC possess a NLS and leads to nuclear and nucleolus localization. • Mutations in basic residues between two ZFs in NC decrease the nucleus localization. • ZFs of NC affect cytoplasmic organelles localization rather than nucleus localization.

  20. RRE-dependent HIV-1 Env RNA effects on Gag protein expression, assembly and release

    International Nuclear Information System (INIS)

    López, Claudia S.; Sloan, Rachel; Cylinder, Isabel; Kozak, Susan L.; Kabat, David; Barklis, Eric

    2014-01-01

    The HIV-1 Gag proteins are translated from the full-length HIV-1 viral RNA (vRNA), whereas the envelope (Env) protein is translated from incompletely spliced Env mRNAs. Nuclear export of vRNAs and Env mRNAs is mediated by the Rev accessory protein which binds to the rev-responsive element (RRE) present on these RNAs. Evidence has shown there is a direct or indirect interaction between the Gag protein, and the cytoplasmic tail (CT) of the Env protein. Our current work shows that env gene expression impacts HIV-1 Gag expression and function in two ways. At the protein level, full-length Env expression altered Gag protein expression, while Env CT-deletion proteins did not. At the RNA level, RRE-containing Env mRNA expression reduced Gag expression, processing, and virus particle release from cells. Our results support models in which Gag is influenced by the Env CT, and Env mRNAs compete with vRNAs for nuclear export. - Highlights: • At the protein level, full-length HIV-1 Env alters Gag protein expression. • HIV-1 Env RNA expression reduces Gag levels and virus release. • Env RNA effects on Gag are dependent on the RRE. • RRE-containing Env RNAs compete with vRNAs for nuclear export

  1. Optimizing HIV-1 protease production in Escherichia coli as fusion protein

    Directory of Open Access Journals (Sweden)

    Piubelli Luciano

    2011-06-01

    Full Text Available Abstract Background Human immunodeficiency virus (HIV is the etiological agent in AIDS and related diseases. The aspartyl protease encoded by the 5' portion of the pol gene is responsible for proteolytic processing of the gag-pol polyprotein precursor to yield the mature capsid protein and the reverse transcriptase and integrase enzymes. The HIV protease (HIV-1Pr is considered an attractive target for designing inhibitors which could be used to tackle AIDS and therefore it is still the object of a number of investigations. Results A recombinant human immunodeficiency virus type 1 protease (HIV-1Pr was overexpressed in Escherichia coli cells as a fusion protein with bacterial periplasmic protein dithiol oxidase (DsbA or glutathione S-transferase (GST, also containing a six-histidine tag sequence. Protein expression was optimized by designing a suitable HIV-1Pr cDNA (for E. coli expression and to avoid autoproteolysis and by screening six different E. coli strains and five growth media. The best expression yields were achieved in E. coli BL21-Codon Plus(DE3-RIL host and in TB or M9 medium to which 1% (w/v glucose was added to minimize basal expression. Among the different parameters assayed, the presence of a buffer system (based on phosphate salts and a growth temperature of 37°C after adding IPTG played the main role in enhancing protease expression (up to 10 mg of chimeric DsbA:HIV-1Pr/L fermentation broth. GST:HIVPr was in part (50% produced as soluble protein while the overexpressed DsbA:HIV-1Pr chimeric protein largely accumulated in inclusion bodies as unprocessed fusion protein. A simple refolding procedure was developed on HiTrap Chelating column that yielded a refolded DsbA:HIV-1Pr with a > 80% recovery. Finally, enterokinase digestion of resolubilized DsbA:HIV-1Pr gave more than 2 mg of HIV-1Pr per liter of fermentation broth with a purity ≤ 80%, while PreScission protease cleavage of soluble GST:HIVPr yielded ~ 0.15 mg of pure HIV-1

  2. Proteomic Profiling of a Primary CD4+ T Cell Model of HIV-1 Latency Identifies Proteins Whose Differential Expression Correlates with Reactivation of Latent HIV-1.

    Science.gov (United States)

    Saha, Jamaluddin Md; Liu, Hongbing; Hu, Pei-Wen; Nikolai, Bryan C; Wu, Hulin; Miao, Hongyu; Rice, Andrew P

    2018-01-01

    The latent HIV-1 reservoir of memory CD4 + T cells that persists during combination antiviral therapy prevents a cure of infection. Insight into mechanisms of latency and viral reactivation are essential for the rational design of strategies to reduce the latent reservoir. In this study, we quantified the levels of >2,600 proteins in the CCL19 primary CD4 + T cell model of HIV-1 latency. We profiled proteins under conditions that promote latent infection and after cells were treated with phorbol 12-myristate 13-acetate (PMA) + ionomycin, which is known to efficiently induce reactivation of latent HIV-1. In an analysis of cells from two healthy blood donors, we identified 61 proteins that were upregulated ≥2-fold, and 36 proteins that were downregulated ≥2-fold under conditions in which latent viruses were reactivated. These differentially expressed proteins are, therefore, candidates for cellular factors that regulate latency or viral reactivation. Two unexpected findings were obtained from the proteomic data: (1) the interactions among the majority of upregulated proteins are largely undetermined in published protein-protein interaction networks and (2) downregulated proteins are strongly associated with Gene Ontology terms related to mitochondrial protein synthesis. This proteomic data set provides a useful resource for future mechanistic studies of HIV-1 latency.

  3. RECOVERY ACT - Thylakoid Assembly and Folded Protein Transport by the Tat Pathway

    Energy Technology Data Exchange (ETDEWEB)

    Dabney-Smith, Carole [Miami Univ., Oxford, OH (United States)

    2016-07-18

    Assembly of functional photosystems complete with necessary intrinsic (membrane-bound) and extrinsic proteins requires the function of at least 3 protein transport pathways in thylakoid membranes. Our research focuses on one of those pathways, a unique and essential protein transport pathway found in the chloroplasts of plants, bacteria, and some archaebacteria, the Twin arginine translocation (Tat) system. The chloroplast Tat (cpTat) system is thought to be responsible for the proper location of ~50% of thylakoid lumen proteins, several of which are necessary for proper photosystem assembly, maintenance, and function. Specifically, cpTat systems are unique because they transport fully folded and assembled proteins across ion tight membranes using only three membrane components, Tha4, Hcf106, and cpTatC, and the protonmotive force generated by photosynthesis. Despite the importance of the cpTat system in plants, the mechanism of transport of a folded precursor is not well known. Our long-term goal is to investigate the role protein transport systems have on organelle biogenesis, particularly the assembly of membrane protein complexes in thylakoids of chloroplasts. The objective of this proposal is to correlate structural changes in the membrane-bound cpTat component, Tha4, to the mechanism of translocation of folded-precursor substrates across the membrane bilayer by using a cysteine accessibility and crosslinking approach. Our central hypothesis is that the precursor passes through a proteinaceous pore of assembled Tha4 protomers that have undergone a conformational or topological change in response to transport. This research is predicated upon the observations that Tha4 exists in molar excess in the membrane relative to the other cpTat components; its regulated assembly to the precursor-bound receptor; and our data showing oligomerization of Tha4 into very large complexes in response to transport. Our rationale for these studies is that understanding cpTat

  4. Competition between Sec- and TAT-dependent protein translocation in Escherichia coli

    DEFF Research Database (Denmark)

    Cristóbal, S.; de Gier, J.-W.; Nielsen, Henrik

    1999-01-01

    -routed into the TAT pathway, suggesting that Sec-targeting signals in Lep can override TAT-targeting information in the TorA signal peptide. We also show that the TorA signal peptide can be converted into a Sec-targeting signal peptide by increasing the hydrophobicity of its h-region. Thus, beyond the twin...... the TorA TAT-targeting signal peptide to the Sec-dependent inner membrane protein leader peptidase (Lep). We find that the soluble, periplasmic P2 domain from Lep is re-routed by the TorA signal peptide into the TAT pathway. In contrast, the full-length TorA–Lep fusion protein is not re...

  5. Solution Properties of Murine Leukemia Virus Gag Protein: Differences from HIV-1 Gag▿

    Science.gov (United States)

    Datta, Siddhartha A. K.; Zuo, Xiaobing; Clark, Patrick K.; Campbell, Stephen J.; Wang, Yun-Xing; Rein, Alan

    2011-01-01

    Immature retrovirus particles are assembled from the multidomain Gag protein. In these particles, the Gag proteins are arranged radially as elongated rods. We have previously characterized the properties of HIV-1 Gag in solution. In the absence of nucleic acid, HIV-1 Gag displays moderately weak interprotein interactions, existing in monomer-dimer equilibrium. Neutron scattering and hydrodynamic studies suggest that the protein is compact, and biochemical studies indicate that the two ends can approach close in three-dimensional space, implying the need for a significant conformational change during assembly. We now describe the properties of the Gag protein of Moloney murine leukemia virus (MLV), a gammaretrovirus. We found that this protein is very different from HIV-1 Gag: it has much weaker protein-protein interaction and is predominantly monomeric in solution. This has allowed us to study the protein by small-angle X-ray scattering and to build a low-resolution molecular envelope for the protein. We found that MLV Gag is extended in solution, with an axial ratio of ∼7, comparable to its dimensions in immature particles. Mutational analysis suggests that runs of prolines in its matrix and p12 domains and the highly charged stretch at the C terminus of its capsid domain all contribute to this extended conformation. These differences between MLV Gag and HIV-1 Gag and their implications for retroviral assembly are discussed. PMID:21917964

  6. Iron chelators ICL670 and 311 inhibit HIV-1 transcription

    International Nuclear Information System (INIS)

    Debebe, Zufan; Ammosova, Tatyana; Jerebtsova, Marina; Kurantsin-Mills, Joseph; Niu, Xiaomei; Charles, Sharroya; Richardson, Des R.; Ray, Patricio E.; Gordeuk, Victor R.; Nekhai, Sergei

    2007-01-01

    HIV-1 replication is induced by an excess of iron and iron chelation by desferrioxamine (DFO) inhibits viral replication by reducing proliferation of infected cells. Treatment of cells with DFO and 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311) inhibit expression of proteins that regulate cell-cycle progression, including cycle-dependent kinase 2 (CDK2). Our recent studies showed that CDK2 participates in HIV-1 transcription and viral replication suggesting that inhibition of CDK2 by iron chelators might also affect HIV-1 transcription. Here we evaluated the effect of a clinically approved orally effective iron chelator, 4-[3,5-bis-(hydroxyphenyl)-1,2,4-triazol-1-yl]-benzoic acid (ICL670) and 311 on HIV-1 transcription. Both ICL670 and 311 inhibited Tat-induced HIV-1 transcription in CEM-T cells, 293T and HeLa cells. Neither ICL670 nor 311 induced cytotoxicity at concentrations that inhibited HIV-1 transcription. The chelators decreased cellular activity of CDK2 and reduced HIV-1 Tat phosphorylation by CDK2. Neither ICL670A or 311 decreased CDK9 protein level but significantly reduced association of CDK9 with cyclin T1 and reduced phosphorylation of Ser-2 residues of RNA polymerase II C-terminal domain. In conclusion, our findings add to the evidence that iron chelators can inhibit HIV-1 transcription by deregulating CDK2 and CDK9. Further consideration should be given to the development of iron chelators for future anti-retroviral therapeutics

  7. HIV-1 nucleocapsid protein localizes efficiently to the nucleus and nucleolus.

    Science.gov (United States)

    Yu, Kyung Lee; Lee, Sun Hee; Lee, Eun Soo; You, Ji Chang

    2016-05-01

    The HIV-1 nucleocapsid (NC) is an essential viral protein containing two highly conserved retroviral-type zinc finger (ZF) motifs, which functions in multiple stages of the HIV-1 life cycle. Although a number of functions for NC either in its mature form or as a domain of Gag have been revealed, little is known about the intracellular localization of NC and, moreover, its role in Gag protein trafficking. Here, we have investigated various forms of HIV-1 NC protein for its cellular localization and found that the NC has a strong nuclear and nucleolar localization activity. The linker region, composed of a stretch of basic amino acids between the two ZF motifs, was necessary and sufficient for the activity. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Structural similarity-based predictions of protein interactions between HIV-1 and Homo sapiens

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    Gomez Shawn M

    2010-04-01

    Full Text Available Abstract Background In the course of infection, viruses such as HIV-1 must enter a cell, travel to sites where they can hijack host machinery to transcribe their genes and translate their proteins, assemble, and then leave the cell again, all while evading the host immune system. Thus, successful infection depends on the pathogen's ability to manipulate the biological pathways and processes of the organism it infects. Interactions between HIV-encoded and human proteins provide one means by which HIV-1 can connect into cellular pathways to carry out these survival processes. Results We developed and applied a computational approach to predict interactions between HIV and human proteins based on structural similarity of 9 HIV-1 proteins to human proteins having known interactions. Using functional data from RNAi studies as a filter, we generated over 2000 interaction predictions between HIV proteins and 406 unique human proteins. Additional filtering based on Gene Ontology cellular component annotation reduced the number of predictions to 502 interactions involving 137 human proteins. We find numerous known interactions as well as novel interactions showing significant functional relevance based on supporting Gene Ontology and literature evidence. Conclusions Understanding the interplay between HIV-1 and its human host will help in understanding the viral lifecycle and the ways in which this virus is able to manipulate its host. The results shown here provide a potential set of interactions that are amenable to further experimental manipulation as well as potential targets for therapeutic intervention.

  9. A DEAD box protein facilitates HIV-1 replication as a cellular co-factor of Rev

    International Nuclear Information System (INIS)

    Fang Jianhua; Kubota, Satoshi; Yang Bin; Zhou Naiming; Zhang Hui; Godbout, Roseline; Pomerantz, Roger J.

    2004-01-01

    HIV-1 Rev escorts unspliced viral mRNAs out of the nucleus of infected cells, which allows formation of infectious HIV-1 virions. We have identified a putative DEAD box (Asp-Glu-Ala-Asp) RNA helicase, DDX1, as a cellular co-factor of Rev, through yeast and mammalian two-hybrid systems using the N-terminal motif of Rev as 'bait'. DDX1 is not a functional homolog of HIV-1 Rev, but down-regulation of DDX1 resulted in an alternative splicing pattern of Rev-responsive element (RRE)-containing mRNA, and attenuation of Gag p24 antigen production from HLfb rev(-) cells rescued by exogenous Rev. Co-transfection of a DDX1 expression vector with HIV-1 significantly increased viral production. DDX1 binding to Rev, as well as to the RRE, strongly suggest that DDX1 affects Rev function through the Rev-RRE axis. Moreover, down-regulation of DDX1 altered the steady state subcellular distribution of Rev, from nuclear/nucleolar to cytoplasmic dominance. These findings indicate that DDX1 is a critical cellular co-factor for Rev function, which maintains the proper subcellular distribution of this lentiviral regulatory protein. Therefore, alterations in DDX1-Rev interactions could induce HIV-1 persistence and targeting DDX1 may lead to rationally designed and novel anti-HIV-1 strategies and therapeutics

  10. Nuclear Matrix protein SMAR1 represses HIV-1 LTR mediated transcription through chromatin remodeling

    International Nuclear Information System (INIS)

    Sreenath, Kadreppa; Pavithra, Lakshminarasimhan; Singh, Sandeep; Sinha, Surajit; Dash, Prasanta K.; Siddappa, Nagadenahalli B.; Ranga, Udaykumar; Mitra, Debashis; Chattopadhyay, Samit

    2010-01-01

    Nuclear Matrix and MARs have been implicated in the transcriptional regulation of host as well as viral genes but their precise role in HIV-1 transcription remains unclear. Here, we show that > 98% of HIV sequences contain consensus MAR element in their promoter. We show that SMAR1 binds to the LTR MAR and reinforces transcriptional silencing by tethering the LTR MAR to nuclear matrix. SMAR1 associated HDAC1-mSin3 corepressor complex is dislodged from the LTR upon cellular activation by PMA/TNFα leading to an increase in the acetylation and a reduction in the trimethylation of histones, associated with the recruitment of RNA Polymerase II on the LTR. Overexpression of SMAR1 lead to reduction in LTR mediated transcription, both in a Tat dependent and independent manner, resulting in a decreased virion production. These results demonstrate the role of SMAR1 in regulating viral transcription by alternative compartmentalization of LTR between the nuclear matrix and chromatin.

  11. Human immunodeficiency virus-1 protein Tat induces excitotoxic loss of presynaptic terminals in hippocampal cultures.

    Science.gov (United States)

    Shin, Angela H; Thayer, Stanley A

    2013-05-01

    Human immunodeficiency virus (HIV) infection of the CNS produces dendritic damage that correlates with cognitive decline in patients with HIV-associated neurocognitive disorders (HAND). HIV-induced neurotoxicity results in part from viral proteins shed from infected cells, including the HIV transactivator of transcription (Tat). We previously showed that Tat binds to the low density lipoprotein receptor-related protein (LRP), resulting in overactivation of NMDA receptors, activation of the ubiquitin-proteasome pathway, and subsequent loss of postsynaptic densities. Here, we show that Tat also induces a loss of presynaptic terminals. The number of presynaptic terminals was quantified using confocal imaging of synaptophysin fused to green fluorescent protein (Syn-GFP). Tat-induced loss of presynaptic terminals was secondary to excitatory postsynaptic mechanisms because treatment with an LRP antagonist or an NMDA receptor antagonist inhibited this loss. Treatment with nutlin-3, an E3 ligase inhibitor, prevented Tat-induced loss of presynaptic terminals. These data suggest that Tat-induced loss of presynaptic terminals is a consequence of excitotoxic postsynaptic activity. We previously found that ifenprodil, an NR2B subunit-selective NMDA receptor antagonist, induced recovery of postsynaptic densities. Here we show that Tat-induced loss of presynaptic terminals was reversed by ifenprodil treatment. Thus, Tat-induced loss of presynaptic terminals is reversible, and this recovery can be initiated by inhibiting a subset of postsynaptic NMDA receptors. Understanding the dynamics of synaptic changes in response to HIV infection of the CNS may lead to the design of improved pharmacotherapies for HAND patients. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. The Effects of the Recombinant CCR5 T4 Lysozyme Fusion Protein on HIV-1 Infection.

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    Qingwen Jin

    Full Text Available Insertion of T4 lysozyme (T4L into the GPCR successfully enhanced GPCR protein stability and solubilization. However, the biological functions of the recombinant GPCR protein have not been analyzed.We engineered the CCR5-T4L mutant and expressed and purified the soluble recombinant protein using an E.coli expression system. The antiviral effects of this recombinant protein in THP-1 cell lines, primary human macrophages, and PBMCs from different donors were investigated. We also explored the possible mechanisms underlying the observed antiviral effects.Our data showed the biphasic inhibitory and promotion effects of different concentrations of soluble recombinant CCR5-T4L protein on R5 tropic human immunodeficiency virus-1 (HIV-1 infection in THP-1 cell lines, human macrophages, and PBMCs from clinical isolates. We demonstrated that soluble recombinant CCR5-T4L acts as a HIV-1 co-receptor, interacts with wild type CCR5, down-regulates the surface CCR5 expression in human macrophages, and interacts with CCL5 to inhibit macrophage migration. Using binding assays, we further determined that recombinant CCR5-T4L and [125I]-CCL5 compete for the same binding site on wild type CCR5.Our results suggest that recombinant CCR5-T4L protein marginally promotes HIV-1 infection at low concentrations and markedly inhibits infection at higher concentrations. This recombinant protein may be helpful in the future development of anti-HIV-1 therapeutic agents.

  13. Super-resolution imaging of ESCRT-proteins at HIV-1 assembly sites.

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    Jens Prescher

    2015-02-01

    Full Text Available The cellular endosomal sorting complex required for transport (ESCRT machinery is involved in membrane budding processes, such as multivesicular biogenesis and cytokinesis. In HIV-infected cells, HIV-1 hijacks the ESCRT machinery to drive HIV release. Early in the HIV-1 assembly process, the ESCRT-I protein Tsg101 and the ESCRT-related protein ALIX are recruited to the assembly site. Further downstream, components such as the ESCRT-III proteins CHMP4 and CHMP2 form transient membrane associated lattices, which are involved in virus-host membrane fission. Although various geometries of ESCRT-III assemblies could be observed, the actual membrane constriction and fission mechanism is not fully understood. Fission might be driven from inside the HIV-1 budding neck by narrowing the membranes from the outside by larger lattices surrounding the neck, or from within the bud. Here, we use super-resolution fluorescence microscopy to elucidate the size and structure of the ESCRT components Tsg101, ALIX, CHMP4B and CHMP2A during HIV-1 budding below the diffraction limit. To avoid the deleterious effects of using fusion proteins attached to ESCRT components, we performed measurements on the endogenous protein or, in the case of CHMP4B, constructs modified with the small HA tag. Due to the transient nature of the ESCRT interactions, the fraction of HIV-1 assembly sites with colocalizing ESCRT complexes was low (1.5%-3.4%. All colocalizing ESCRT clusters exhibited closed, circular structures with an average size (full-width at half-maximum between 45 and 60 nm or a diameter (determined using a Ripley's L-function analysis of roughly 60 to 100 nm. The size distributions for colocalizing clusters were narrower than for non-colocalizing clusters, and significantly smaller than the HIV-1 bud. Hence, our results support a membrane scission process driven by ESCRT protein assemblies inside a confined structure, such as the bud neck, rather than by large lattices

  14. HIV-1 accessory proteins VPR and Vif modulate antiviral response by targeting IRF-3 for degradation

    International Nuclear Information System (INIS)

    Okumura, Atsushi; Alce, Tim; Lubyova, Barbora; Ezelle, Heather; Strebel, Klaus; Pitha, Paula M.

    2008-01-01

    The activation of IRF-3 during the early stages of viral infection is critical for the initiation of the antiviral response; however the activation of IRF-3 in HIV-1 infected cells has not yet been characterized. We demonstrate that the early steps of HIV-1 infection do not lead to the activation and nuclear translocation of IRF-3; instead, the relative levels of IRF-3 protein are decreased due to the ubiquitin-associated proteosome degradation. Addressing the molecular mechanism of this effect we show that the degradation is independent of HIV-1 replication and that virion-associated accessory proteins Vif and Vpr can independently degrade IRF-3. The null mutation of these two genes reduced the capacity of the HIV-1 virus to down modulate IRF-3 levels. The degradation was associated with Vif- and Vpr-mediated ubiquitination of IRF-3 and was independent of the activation of IRF-3. N-terminal lysine residues were shown to play a critical role in the Vif- and Vpr-mediated degradation of IRF-3. These data implicate Vif and Vpr in the disruption of the initial antiviral response and point to the need of HIV-1 to circumvent the antiviral response during the very early phase of replication

  15. TAT-Mediated Delivery of Tousled Protein to Salivary Glands Protects Against Radiation-Induced Hypofunction

    Energy Technology Data Exchange (ETDEWEB)

    Sunavala-Dossabhoy, Gulshan, E-mail: gsunav@lsuhsc.edu [Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, LA (United States); Palaniyandi, Senthilnathan; Richardson, Charles; De Benedetti, Arrigo [Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, LA (United States); Schrott, Lisa [Department of Pharmacology, Toxicology, and Neuroscience, Louisiana State University Health Sciences Center, Shreveport, LA (United States); Caldito, Gloria [Department of Bioinformatics and Computational Biology, Louisiana State University Health Sciences Center, Shreveport, LA (United States)

    2012-09-01

    Purpose: Patients treated with radiotherapy for head-and-neck cancer invariably suffer its deleterious side effect, xerostomia. Salivary hypofunction ensuing from the irreversible destruction of glands is the most common and debilitating oral complication affecting patients undergoing regional radiotherapy. Given that the current management of xerostomia is palliative and ineffective, efforts are now directed toward preventive measures to preserve gland function. The human homolog of Tousled protein, TLK1B, facilitates chromatin remodeling at DNA repair sites and improves cell survival against ionizing radiation (IR). Therefore, we wanted to determine whether a direct transfer of TLK1B protein to rat salivary glands could protect against IR-induced salivary hypofunction. Methods: The cell-permeable TAT-TLK1B fusion protein was generated. Rat acinar cell line and rat salivary glands were pretreated with TAT peptide or TAT-TLK1B before IR. The acinar cell survival in vitro and salivary function in vivo were assessed after radiation. Results: We demonstrated that rat acinar cells transduced with TAT-TLK1B were more resistant to radiation (D{sub 0} = 4.13 {+-} 1.0 Gy; {alpha}/{beta} = 0 Gy) compared with cells transduced with the TAT peptide (D{sub 0} = 4.91 {+-} 1.0 Gy; {alpha}/{beta} = 20.2 Gy). Correspondingly, retroductal instillation of TAT-TLK1B in rat submandibular glands better preserved salivary flow after IR (89%) compared with animals pretreated with Opti-MEM or TAT peptide (31% and 39%, respectively; p < 0.01). Conclusions: The results demonstrate that a direct transfer of TLK1B protein to the salivary glands effectively attenuates radiation-mediated gland dysfunction. Prophylactic TLK1B-protein therapy could benefit patients undergoing radiotherapy for head-and-neck cancer.

  16. Interferon-inducible protein 10 (IP-10) is associated with viremia of early HIV-1 infection in Korean patients.

    Science.gov (United States)

    Lee, SoYong; Chung, Yoon-Seok; Yoon, Cheol-Hee; Shin, YoungHyun; Kim, SeungHyun; Choi, Byeong-Sun; Kim, Sung Soon

    2015-05-01

    Cytokines/chemokines play key roles in modulating disease progression in human immunodeficiency virus (HIV) infection. Although it is known that early HIV-1 infection is associated with increased production of proinflammatory cytokines, the relationship between cytokine levels and HIV-1 pathogenesis is not clear. The concentrations of 18 cytokines/chemokines in 30 HIV-1 negative and 208 HIV-1 positive plasma samples from Korean patients were measured by the Luminex system. Early HIV-1 infection was classified according to the Fiebig stage (FS) based on the characteristics of the patients infected with HIV-1. Concentrations of interleukin-12 (IL-12), interferon-inducible protein-10 (IP-10), macrophage inflammatory protein-1α (MIP-1α) and regulated upon activation, normal T cells expressed and secreted (RANTES) were increased significantly during the early stage of HIV-1 infection (FS II-IV) compared with the HIV-1-negative group. Of these cytokines, an elevated level of IP-10 was the only factor to be correlated positively with a higher viral load during the early stages of HIV-1 infection (FS II-IV) in Koreans (R = 0.52, P IP-10 may be an indicator for HIV-1 viremia and associated closely with viral replication in patients with early HIV-1 infection. © 2015 Wiley Periodicals, Inc.

  17. Role of protein disulfide isomerase and other thiol-reactive proteins in HIV-1 envelope protein-mediated fusion

    International Nuclear Information System (INIS)

    Ou Wu; Silver, Jonathan

    2006-01-01

    Cell-surface protein disulfide isomerase (PDI) has been proposed to promote disulfide bond rearrangements in HIV-1 envelope protein (Env) that accompany Env-mediated fusion. We evaluated the role of PDI in ways that have not been previously tested by downregulating PDI with siRNA and by overexpressing wild-type or variant forms of PDI in transiently and stably transfected cells. These manipulations, as well as treatment with anti-PDI antibodies, had only small effects on infection or cell fusion mediated by NL4-3 or AD8 strains of HIV-1. However, the cell-surface thiol-reactive reagent 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB) had a much stronger inhibitory effect in our system, suggesting that cell-surface thiol-containing molecules other than PDI, acting alone or in concert, have a greater effect than PDI on HIV-1 Env-mediated fusion. We evaluated one such candidate, thioredoxin, a PDI family member reported to reduce a labile disulfide bond in CD4. We found that the ability of thioredoxin to reduce the disulfide bond in CD4 is enhanced in the presence of HIV-1 Env gp120 and that thioredoxin also reduces disulfide bonds in gp120 directly in the absence of CD4. We discuss the implications of these observations for identification of molecules involved in disulfide rearrangements in Env during fusion

  18. Mimicking protein-protein interactions through peptide-peptide interactions: HIV-1 gp120 and CXCR4

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    Andrea eGross

    2013-09-01

    Full Text Available We have recently designed a soluble synthetic peptide that functionally mimics the HIV-1 coreceptor CXCR4, which is a chemokine receptor that belongs to the family of seven-transmembrane GPCRs. This CXCR4 mimetic peptide, termed CX4-M1, presents the three extracellular loops (ECLs of the receptor. In binding assays involving recombinant proteins, as well as in cellular infection assays, CX4-M1 was found to selectively recognize gp120 from HIV-1 strains that use CXCR4 for cell entry (X4 tropic HIV-1. Furthermore, anti-HIV-1 antibodies modulate this interaction in a molecular mechanism related to that of their impact on the gp120-CXCR4 interaction. We could now show that the selectivity of CX4-M1 pertains not only to gp120 from X4 tropic HIV-1, but also to synthetic peptides presenting the V3 loops of these gp120 proteins. The V3 loop is thought to be an essential part of the coreceptor binding site of gp120 that contacts the second ECL of the coreceptor. We were able to experimentally confirm this notion in binding assays using substitution analogs of CX4-M1 and the V3 loop peptides, respectively, as well as in cellular infection assays. These results indicate that interactions of the HIV-1 Env with coreceptors can be mimicked by synthetic peptides, which may be useful to explore these interactions at the molecular level in more detail.

  19. Selection of unadapted, pathogenic SHIVs encoding newly transmitted HIV-1 envelope proteins.

    Science.gov (United States)

    Del Prete, Gregory Q; Ailers, Braiden; Moldt, Brian; Keele, Brandon F; Estes, Jacob D; Rodriguez, Anthony; Sampias, Marissa; Oswald, Kelli; Fast, Randy; Trubey, Charles M; Chertova, Elena; Smedley, Jeremy; LaBranche, Celia C; Montefiori, David C; Burton, Dennis R; Shaw, George M; Markowitz, Marty; Piatak, Michael; KewalRamani, Vineet N; Bieniasz, Paul D; Lifson, Jeffrey D; Hatziioannou, Theodora

    2014-09-10

    Infection of macaques with chimeric viruses based on SIVMAC but expressing the HIV-1 envelope (Env) glycoproteins (SHIVs) remains the most powerful model for evaluating prevention and therapeutic strategies against AIDS. Unfortunately, only a few SHIVs are currently available. Furthermore, their generation has required extensive adaptation of the HIV-1 Env sequences in macaques so they may not accurately represent HIV-1 Env proteins circulating in humans, potentially limiting their translational utility. We developed a strategy for generating large numbers of SHIV constructs expressing Env proteins from newly transmitted HIV-1 strains. By inoculating macaques with cocktails of multiple SHIV variants, we selected SHIVs that can replicate and cause AIDS-like disease in immunologically intact rhesus macaques without requiring animal-to-animal passage. One of these SHIVs could be transmitted mucosally. We demonstrate the utility of the SHIVs generated by this method for evaluating neutralizing antibody administration as a protection against mucosal SHIV challenge. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. The prion protein has RNA binding and chaperoning properties characteristic of nucleocapsid protein NCP7 of HIV-1.

    Science.gov (United States)

    Gabus, C; Derrington, E; Leblanc, P; Chnaiderman, J; Dormont, D; Swietnicki, W; Morillas, M; Surewicz, W K; Marc, D; Nandi, P; Darlix, J L

    2001-06-01

    Transmissible spongiform encephalopathies are fatal neurodegenerative diseases associated with the accumulation of a protease-resistant form of the prion protein (PrP). Although PrP is conserved in vertebrates, its function remains to be identified. In vitro PrP binds large nucleic acids causing the formation of nucleoprotein complexes resembling human immunodeficiency virus type 1 (HIV-1) nucleocapsid-RNA complexes and in vivo MuLV replication accelerates the scrapie infectious process, suggesting possible interactions between retroviruses and PrP. Retroviruses, including HIV-1 encode a major nucleic acid binding protein (NC protein) found within the virus where 2000 NC protein molecules coat the dimeric genome. NC is required in virus assembly and infection to chaperone RNA dimerization and packaging and in proviral DNA synthesis by reverse transcriptase (RT). In HIV-1, 5'-leader RNA/NC interactions appear to control these viral processes. This prompted us to compare and contrast the interactions of human and ovine PrP and HIV-1 NCp7 with HIV-1 5'-leader RNA. Results show that PrP has properties characteristic of NCp7 with respect to viral RNA dimerization and proviral DNA synthesis by RT. The NC-like properties of huPrP map to the N-terminal region of huPrP. Interestingly, PrP localizes in the membrane and cytoplasm of PrP-expressing cells. These findings suggest that PrP is a multifunctional protein possibly participating in nucleic acid metabolism.

  1. PrPC has nucleic acid chaperoning properties similar to the nucleocapsid protein of HIV-1.

    Science.gov (United States)

    Derrington, Edmund; Gabus, Caroline; Leblanc, Pascal; Chnaidermann, Jonas; Grave, Linda; Dormont, Dominique; Swietnicki, Wieslaw; Morillas, Manuel; Marck, Daniel; Nandi, Pradip; Darlix, Jean-Luc

    2002-01-01

    The function of the cellular prion protein (PrPC) remains obscure. Studies suggest that PrPC functions in several processes including signal transduction and Cu2+ metabolism. PrPC has also been established to bind nucleic acids. Therefore we investigated the properties of PrPC as a putative nucleic acid chaperone. Surprisingly, PrPC possesses all the nucleic acid chaperoning properties previously specific to retroviral nucleocapsid proteins. PrPC appears to be a molecular mimic of NCP7, the nucleocapsid protein of HIV-1. Thus PrPC, like NCP7, chaperones the annealing of tRNA(Lys) to the HIV-1 primer binding site, the initial step of retrovirus replication. PrPC also chaperones the two DNA strand transfers required for production of a complete proviral DNA with LTRs. Concerning the functions of NCP7 during budding, PrPC also mimices NCP7 by dimerizing the HIV-1 genomic RNA. These data are unprecedented because, although many cellular proteins have been identified as nucleic acid chaperones, none have the properties of retroviral nucleocapsid proteins.

  2. Ex vivo expansion of hematopoietic stem cell by fusion protein TAT-Zfx

    International Nuclear Information System (INIS)

    Xu Chong; Zhang Yanbing; Jiang Hua

    2009-01-01

    The relative inability of hemopoietic stem cells (HSCs) to reproduce themselves (self-renew) ex vivo imposes substantial limitations on the current use of HSC transplantation. Recently, the transcription factor Zfx has been demonstrated that played an important in controlling the self-renewal of hematopoietic stem cells. Here, we reported that Zfx could enable high-level expansion of HSCs in vitro, by combination of protein transduction domain, TAT. Furthermore, we also demonstrated that expanded HSCs population retains their normal in vivo potential of pluripotency. It is thus that TAT-Zfx has the potential to expand HSCs significantly in vitro, and will have enormous clinical potentials.

  3. Analysis of Select Herpes Simplex Virus 1 (HSV-1) Proteins for Restriction of Human Immunodeficiency Virus Type 1 (HIV-1): HSV-1 gM Protein Potently Restricts HIV-1 by Preventing Intracellular Transport and Processing of Env gp160.

    Science.gov (United States)

    Polpitiya Arachchige, Sachith; Henke, Wyatt; Pramanik, Ankita; Kalamvoki, Maria; Stephens, Edward B

    2018-01-15

    Virus-encoded proteins that impair or shut down specific host cell functions during replication can be used as probes to identify potential proteins/pathways used in the replication of viruses from other families. We screened nine proteins from herpes simplex virus 1 (HSV-1) for the ability to enhance or restrict human immunodeficiency virus type 1 (HIV-1) replication. We show that several HSV-1 proteins (glycoprotein M [gM], US3, and UL24) potently restricted the replication of HIV-1. Unlike UL24 and US3, which reduced viral protein synthesis, we observed that gM restriction of HIV-1 occurred through interference with the processing and transport of gp160, resulting in a significantly reduced level of mature gp120/gp41 released from cells. Finally, we show that an HSV-1 gM mutant lacking the majority of the C-terminal domain (HA-gM[Δ345-473]) restricted neither gp160 processing nor the release of infectious virus. These studies identify proteins from heterologous viruses that can restrict viruses through novel pathways. IMPORTANCE HIV-1 infection of humans results in AIDS, characterized by the loss of CD4 + T cells and increased susceptibility to opportunistic infections. Both HIV-1 and HSV-1 can infect astrocytes and microglia of the central nervous system (CNS). Thus, the identification of HSV-1 proteins that directly restrict HIV-1 or interfere with pathways required for HIV-1 replication could lead to novel antiretroviral strategies. The results of this study show that select viral proteins from HSV-1 can potently restrict HIV-1. Further, our results indicate that the gM protein of HSV-1 restricts HIV-1 through a novel pathway by interfering with the processing of gp160 and its incorporation into virus maturing from the cell. Copyright © 2018 American Society for Microbiology.

  4. Human Polycomb group EED protein negatively affects HIV-1 assembly and release

    Directory of Open Access Journals (Sweden)

    Darlix Jean-Luc

    2007-06-01

    Full Text Available Abstract Background The human EED protein, a member of the superfamily of Polycomb group (PcG proteins with WD-40 repeats, has been found to interact with three HIV-1 components, namely the structural Gag matrix protein (MA, the integrase enzyme (IN and the Nef protein. The aim of the present study was to analyze the possible biological role of EED in HIV-1 replication, using the HIV-1-based vector HIV-Luc and EED protein expressed by DNA transfection of 293T cells. Results During the early phase of HIV-1 infection, a slight negative effect on virus infectivity occurred in EED-expressing cells, which appeared to be dependent on EED-MA interaction. At late times post infection, EED caused an important reduction of virus production, from 20- to 25-fold as determined by CAp24 immunoassay, to 10- to 80-fold based on genomic RNA levels, and this decrease was not due to a reduction of Gag protein synthesis. Coexpression of WTNef, or the non-N-myristoylated mutant NefG2A, restored virus yields to levels obtained in the absence of exogenous EED protein. This effect was not observed with mutant NefΔ57 mimicking the Nef core, or with the lipid raft-retargeted fusion protein LAT-Nef. LATAA-Nef, a mutant defective in the lipid raft addressing function, had the same anti-EED effect as WTNef. Cell fractionation and confocal imaging showed that, in the absence of Nef, EED mainly localized in membrane domains different from the lipid rafts. Upon co-expression with WTNef, NefG2A or LATAA-Nef, but not with NefΔ57 or LAT-Nef, EED was found to relocate into an insoluble fraction along with Nef protein. Electron microscopy of HIV-Luc producer cells overexpressing EED showed significant less virus budding at the cell surface compared to control cells, and ectopic assembly and clustering of nuclear pore complexes within the cytoplasm. Conclusion Our data suggested that EED exerted an antiviral activity at the late stage of HIV-1 replication, which included genomic

  5. Identifying potential survival strategies of HIV-1 through virus-host protein interaction networks

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    Boucher Charles AB

    2010-07-01

    Full Text Available Abstract Background The National Institute of Allergy and Infectious Diseases has launched the HIV-1 Human Protein Interaction Database in an effort to catalogue all published interactions between HIV-1 and human proteins. In order to systematically investigate these interactions functionally and dynamically, we have constructed an HIV-1 human protein interaction network. This network was analyzed for important proteins and processes that are specific for the HIV life-cycle. In order to expose viral strategies, network motif analysis was carried out showing reoccurring patterns in virus-host dynamics. Results Our analyses show that human proteins interacting with HIV form a densely connected and central sub-network within the total human protein interaction network. The evaluation of this sub-network for connectivity and centrality resulted in a set of proteins essential for the HIV life-cycle. Remarkably, we were able to associate proteins involved in RNA polymerase II transcription with hubs and proteasome formation with bottlenecks. Inferred network motifs show significant over-representation of positive and negative feedback patterns between virus and host. Strikingly, such patterns have never been reported in combined virus-host systems. Conclusions HIV infection results in a reprioritization of cellular processes reflected by an increase in the relative importance of transcriptional machinery and proteasome formation. We conclude that during the evolution of HIV, some patterns of interaction have been selected for resulting in a system where virus proteins preferably interact with central human proteins for direct control and with proteasomal proteins for indirect control over the cellular processes. Finally, the patterns described by network motifs illustrate how virus and host interact with one another.

  6. A Tat-grafted anti-nucleic acid antibody acquires nuclear-localization property and a preference for TAR RNA

    International Nuclear Information System (INIS)

    Jeong, Jong-Geun; Kim, Dong-Sik; Kim, Yong-Sung; Kwon, Myung-Hee

    2011-01-01

    Highlights: → We generate ' H3 Tat-3D8' by grafting Tat 48-60 peptide to VH CDR of 3D8 scFv antibody. → H3 Tat-3D8 antibody retains nucleic acid binding and hydrolyzing activities. → H3 Tat-3D8 acquires a preference for TAR RNA structure. → Properties of Tat 48-60 is transferred to an antibody via Tat-grafting into a CDR. -- Abstract: The 3D8 single chain variable fragment (3D8 scFv) is an anti-nucleic acid antibody that can hydrolyze nucleic acids and enter the cytosol of cells without reaching the nucleus. The Tat peptide, derived from the basic region of the HIV-1 Tat protein, translocates to cell nuclei and has TAR RNA binding activity. In this study, we generated a Tat-grafted antibody ( H3 Tat-3D8) by replacing complementarity-determining region 3 (CDR3) within the VH domain of the 3D8 scFv with a Tat 48-60 peptide (GRKKRRQRRRPPQ). H3 Tat-3D8 retained the DNA-binding and DNA-hydrolyzing activity of the scFv, and translocated to the nuclei of HeLa cells and preferentially recognized TAR RNA. Thus, the properties associated with the Tat peptide were transferred to the antibody via Tat-grafting without loss of the intrinsic DNA-binding and hydrolyzing activities of the 3D8 scFv antibody.

  7. Human-Phosphate-Binding-Protein inhibits HIV-1 gene transcription and replication

    Directory of Open Access Journals (Sweden)

    Candolfi Ermanno

    2011-07-01

    Full Text Available Abstract The Human Phosphate-Binding protein (HPBP is a serendipitously discovered lipoprotein that binds phosphate with high affinity. HPBP belongs to the DING protein family, involved in various biological processes like cell cycle regulation. We report that HPBP inhibits HIV-1 gene transcription and replication in T cell line, primary peripherical blood lymphocytes and primary macrophages. We show that HPBP is efficient in naïve and HIV-1 AZT-resistant strains. Our results revealed HPBP as a new and potent anti HIV molecule that inhibits transcription of the virus, which has not yet been targeted by HAART and therefore opens new strategies in the treatment of HIV infection.

  8. Thermodynamic characterization of the peptide assembly inhibitor binding to HIV-1 capsid protein

    Czech Academy of Sciences Publication Activity Database

    Kožíšek, Milan; Durčák, Jindřich; Konvalinka, Jan

    2013-01-01

    Roč. 10, Suppl. 1 (2013), S37-S37 ISSN 1742-4690. [Frontiers of Retrovirology: Complex retorviruses, retroelements and their hosts. 16.09.2013-18.09.2013, Cambridge] R&D Projects: GA ČR GA13-19561S Institutional support: RVO:61388963 Keywords : HIV -1 capsid protein * CAI Subject RIV: EE - Microbiology, Virology http://www.retrovirology.com/content/10/S1/P108

  9. Mapping the binding interface between an HIV-1 inhibiting intrabody and the viral protein Rev.

    Directory of Open Access Journals (Sweden)

    Thomas Vercruysse

    Full Text Available HIV-1 Rev is the key protein in the nucleocytoplasmic export and expression of the late viral mRNAs. An important aspect for its function is its ability to multimerize on these mRNAs. We have recently identified a llama single-domain antibody (Nb190 as the first inhibitor targeting the Rev multimerization function in cells. This nanobody is a potent intracellular antibody that efficiently inhibits HIV-1 viral production. In order to gain insight into the Nb190-Rev interaction interface, we performed mutational and docking studies to map the interface between the nanobody paratope and the Rev epitope. Alanine mutants of the hyper-variable domains of Nb190 and the Rev multimerization domains were evaluated in different assays measuring Nb190-Rev interaction or viral production. Seven residues within Nb190 and five Rev residues are demonstrated to be crucial for epitope recognition. These experimental data were used to perform docking experiments and map the Nb190-Rev structural interface. This Nb190-Rev interaction model can guide further studies of the Nb190 effect on HIV-1 Rev function and could serve as starting point for the rational development of smaller entities binding to the Nb190 epitope, aimed at interfering with protein-protein interactions of the Rev N-terminal domain.

  10. Identification of novel 2-benzoxazolinone derivatives with specific inhibitory activity against the HIV-1 nucleocapsid protein.

    Science.gov (United States)

    Gamba, Elia; Mori, Mattia; Kovalenko, Lesia; Giannini, Alessia; Sosic, Alice; Saladini, Francesco; Fabris, Dan; Mély, Yves; Gatto, Barbara; Botta, Maurizio

    2018-02-10

    In this report, we present a new benzoxazole derivative endowed with inhibitory activity against the HIV-1 nucleocapsid protein (NC). NC is a 55-residue basic protein with nucleic acid chaperone properties, which has emerged as a novel and potential pharmacological target against HIV-1. In the pursuit of novel NC-inhibitor chemotypes, we performed virtual screening and in vitro biological evaluation of a large library of chemical entities. We found that compounds sharing a benzoxazolinone moiety displayed putative inhibitory properties, which we further investigated by considering a series of chemical analogues. This approach provided valuable information on the structure-activity relationships of these compounds and, in the process, demonstrated that their anti-NC activity could be finely tuned by the addition of specific substituents to the initial benzoxazolinone scaffold. This study represents the starting point for the possible development of a new class of antiretroviral agents targeting the HIV-1 NC protein. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  11. The Natural Killer Cell Cytotoxic Function Is Modulated by HIV-1 Accessory Proteins

    Directory of Open Access Journals (Sweden)

    Edward Barker

    2011-07-01

    Full Text Available Natural killer (NK cells’ major role in the control of viruses is to eliminate established infected cells. The capacity of NK cells to kill virus-infected cells is dependent on the interactions between ligands on the infected cell and receptors on the NK cell surface. Because of the importance of ligand-receptor interactions in modulating the NK cell cytotoxic response, HIV has developed strategies to regulate various NK cell ligands making the infected cell surprisingly refractory to NK cell lysis. This is perplexing because the HIV-1 accessory protein Vpr induces expression of ligands for the NK cell activating receptor, NKG2D. In addition, the accessory protein Nef removes the inhibitory ligands HLA-A and -B. The reason for the ineffective killing by NK cells despite the strong potential to eliminate infected cells is due to HIV-1 Vpu’s ability to down modulate the co-activation ligand, NTB-A, from the cell surface. Down modulation of NTB-A prevents efficient NK cell degranulation. This review will focus on the mechanisms through which the HIV-1 accessory proteins modulate their respective ligands, and its implication for NK cell killing of HIV-infected cells.

  12. Promiscuous RNA binding ensures effective encapsidation of APOBEC3 proteins by HIV-1.

    Directory of Open Access Journals (Sweden)

    Luis Apolonia

    2015-01-01

    Full Text Available The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3 proteins are cell-encoded cytidine deaminases, some of which, such as APOBEC3G (A3G and APOBEC3F (A3F, act as potent human immunodeficiency virus type-1 (HIV-1 restriction factors. These proteins require packaging into HIV-1 particles to exert their antiviral activities, but the molecular mechanism by which this occurs is incompletely understood. The nucleocapsid (NC region of HIV-1 Gag is required for efficient incorporation of A3G and A3F, and the interaction between A3G and NC has previously been shown to be RNA-dependent. Here, we address this issue in detail by first determining which RNAs are able to bind to A3G and A3F in HV-1 infected cells, as well as in cell-free virions, using the unbiased individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP method. We show that A3G and A3F bind many different types of RNA, including HIV-1 RNA, cellular mRNAs and small non-coding RNAs such as the Y or 7SL RNAs. Interestingly, A3G/F incorporation is unaffected when the levels of packaged HIV-1 genomic RNA (gRNA and 7SL RNA are reduced, implying that these RNAs are not essential for efficient A3G/F packaging. Confirming earlier work, HIV-1 particles formed with Gag lacking the NC domain (Gag ΔNC fail to encapsidate A3G/F. Here, we exploit this system by demonstrating that the addition of an assortment of heterologous RNA-binding proteins and domains to Gag ΔNC efficiently restored A3G/F packaging, indicating that A3G and A3F have the ability to engage multiple RNAs to ensure viral encapsidation. We propose that the rather indiscriminate RNA binding characteristics of A3G and A3F promote functionality by enabling recruitment into a wide range of retroviral particles whose packaged RNA genomes comprise divergent sequences.

  13. Pyridones as NNRTIs against HIV-1 mutants: 3D-QSAR and protein informatics

    Science.gov (United States)

    Debnath, Utsab; Verma, Saroj; Jain, Surabhi; Katti, Setu B.; Prabhakar, Yenamandra S.

    2013-07-01

    CoMFA and CoMSIA based 3D-QSAR of HIV-1 RT wild and mutant (K103, Y181C, and Y188L) inhibitory activities of 4-benzyl/benzoyl pyridin-2-ones followed by protein informatics of corresponding non-nucleoside inhibitors' binding pockets from pdbs 2BAN, 3MED, 1JKH, and 2YNF were analysed to discover consensus features of the compounds for broad-spectrum activity. The CoMFA/CoMSIA models indicated that compounds with groups which lend steric-cum-electropositive fields in the vicinity of C5, hydrophobic field in the vicinity of C3 of pyridone region and steric field in aryl region produce broad-spectrum anti-HIV-1 RT activity. Also, a linker rendering electronegative field between pyridone and aryl moieties is common requirement for the activities. The protein informatics showed considerable alteration in residues 181 and 188 characteristics on mutation. Also, mutants' isoelectric points shifted in acidic direction. The study offered fresh avenues for broad-spectrum anti-HIV-1 agents through designing new molecules seeded with groups satisfying common molecular fields and concerns of mutating residues.

  14. Biochemistry and biophysics of HIV-1 gp41 - membrane interactions and implications for HIV-1 envelope protein mediated viral-cell fusion and fusion inhibitor design.

    Science.gov (United States)

    Cai, Lifeng; Gochin, Miriam; Liu, Keliang

    2011-12-01

    Human immunodeficiency virus type 1 (HIV-1), the pathogen of acquired immunodeficiency syndrome (AIDS), causes ~2 millions death every year and still defies an effective vaccine. HIV-1 infects host cells through envelope protein - mediated virus-cell fusion. The transmembrane subunit of envelope protein, gp41, is the molecular machinery which facilitates fusion. Its ectodomain contains several distinguishing functional domains, fusion peptide (FP), Nterminal heptad repeat (NHR), C-terminal heptad repeat (CHR) and membrane proximal extracellular region (MPER). During the fusion process, FP inserts into the host cell membrane, and an extended gp41 prehairpin conformation bridges the viral and cell membranes through MPER and FP respectively. Subsequent conformational change of the unstable prehairpin results in a coiled-coil 6-helix bundle (6HB) structure formed between NHR and CHR. The energetics of 6HB formation drives membrane apposition and fusion. Drugs targeting gp41 functional domains to prevent 6HB formation inhibit HIV-1 infection. T20 (enfuvirtide, Fuzeon) was approved by the US FDA in 2003 as the first fusion inhibitor. It is a 36-residue peptide from the gp41 CHR, and it inhibits 6HB formation by targeting NHR and lipids. Development of new fusion inhibitors, especially small molecule drugs, is encouraged to overcome the shortcomings of T20 as a peptide drug. Hydrophobic characteristics and membrane association are critical for gp41 function and mechanism of action. Research in gp41-membrane interactions, using peptides corresponding to specific functional domains, or constructs including several interactive domains, are reviewed here to get a better understanding of gp41 mediated virus-cell fusion that can inform or guide the design of new HIV-1 fusion inhibitors.

  15. Prediction of mutational tolerance in HIV-1 protease and reverse transcriptase using flexible backbone protein design.

    Directory of Open Access Journals (Sweden)

    Elisabeth Humphris-Narayanan

    Full Text Available Predicting which mutations proteins tolerate while maintaining their structure and function has important applications for modeling fundamental properties of proteins and their evolution; it also drives progress in protein design. Here we develop a computational model to predict the tolerated sequence space of HIV-1 protease reachable by single mutations. We assess the model by comparison to the observed variability in more than 50,000 HIV-1 protease sequences, one of the most comprehensive datasets on tolerated sequence space. We then extend the model to a second protein, reverse transcriptase. The model integrates multiple structural and functional constraints acting on a protein and uses ensembles of protein conformations. We find the model correctly captures a considerable fraction of protease and reverse-transcriptase mutational tolerance and shows comparable accuracy using either experimentally determined or computationally generated structural ensembles. Predictions of tolerated sequence space afforded by the model provide insights into stability-function tradeoffs in the emergence of resistance mutations and into strengths and limitations of the computational model.

  16. Labeling of multiple HIV-1 proteins with the biarsenical-tetracysteine system.

    Directory of Open Access Journals (Sweden)

    Cândida F Pereira

    Full Text Available Due to its small size and versatility, the biarsenical-tetracysteine system is an attractive way to label viral proteins for live cell imaging. This study describes the genetic labeling of the human immunodeficiency virus type 1 (HIV-1 structural proteins (matrix, capsid and nucleocapsid, enzymes (protease, reverse transcriptase, RNAse H and integrase and envelope glycoprotein 120 with a tetracysteine tag in the context of a full-length virus. We measure the impact of these modifications on the natural virus infection and, most importantly, present the first infectious HIV-1 construct containing a fluorescently-labeled nucleocapsid protein. Furthermore, due to the high background levels normally associated with the labeling of tetracysteine-tagged proteins we have also optimized a metabolic labeling system that produces infectious virus containing the natural envelope glycoproteins and specifically labeled tetracysteine-tagged proteins that can easily be detected after virus infection of T-lymphocytes. This approach can be adapted to other viral systems for the visualization of the interplay between virus and host cell during infection.

  17. HIV-1 Nef binds with human GCC185 protein and regulates mannose 6 phosphate receptor recycling

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Manjeet; Kaur, Supinder; Nazir, Aamir; Tripathi, Raj Kamal, E-mail: rajkamalcdri@gmail.com

    2016-05-20

    HIV-1 Nef modulates cellular function that enhances viral replication in vivo which culminate into AIDS pathogenesis. With no enzymatic activity, Nef regulates cellular function through host protein interaction. Interestingly, trans-cellular introduction of recombinant Nef protein in Caenorhabditis elegans results in AIDS like pathogenesis which might share common pathophysiology because the gene sequence of C. elegans and humans share considerable homology. Therefore employing C. elegans based initial screen complemented with sequence based homology search we identified GCC185 as novel host protein interacting with HIV-1 Nef. The detailed molecular characterization revealed N-terminal EEEE{sub 65} acidic domain of Nef as key region for interaction. GCC185 is a tethering protein that binds with Rab9 transport vesicles. Our results show that Nef-GCC185 interaction disrupts Rab9 interaction resulting in delocalization of CI-MPR (cation independent Mannose 6 phosphate receptor) resulting in elevated secretion of hexosaminidase. In agreement with this, our studies identified novel host GCC185 protein that interacts with Nef EEEE65 acidic domain interfering GCC185-Rab9 vesicle membrane fusion responsible for retrograde vesicular transport of CI-MPR from late endosomes to TGN. In light of existing report suggesting critical role of Nef-GCC185 interaction reveals valuable mechanistic insights affecting specific protein transport pathway in docking of late endosome derived Rab9 bearing transport vesicle at TGN elucidating role of Nef during viral pathogenesis. -- Highlights: •Nef, an accessory protein of HIV-1 interacts with host factor and culminates into AIDS pathogenesis. •Using Caenorhabditis elegans based screen system, novel Nef interacting cellular protein GCC185 was identified. •Molecular characterization of Nef and human protein GCC185 revealed Nef EEEE{sub 65} key region interacted with full length GCC185. •Nef impeded the GCC185-Rab 9 interaction and

  18. Comparative analysis of twin-arginine (Tat)-dependent protein secretion of a heterologous model protein (GFP) in three different Gram-positive bacteria

    NARCIS (Netherlands)

    Meissner, Daniel; Vollstedt, Angela; van Dijl, Jan Maarten; Freudl, Roland

    In contrast to the general protein secretion (Sec) system, the twin-arginine translocation (Tat) export pathway allows the translocation of proteins across the bacterial plasma membrane in a fully folded conformation. Due to this feature, the Tat pathway provides an attractive alternative to the

  19. Virus-producing cells determine the host protein profiles of HIV-1 virion cores

    Science.gov (United States)

    2012-01-01

    Background Upon HIV entry into target cells, viral cores are released and rearranged into reverse transcription complexes (RTCs), which support reverse transcription and also protect and transport viral cDNA to the site of integration. RTCs are composed of viral and cellular proteins that originate from both target and producer cells, the latter entering the target cell within the viral core. However, the proteome of HIV-1 viral cores in the context of the type of producer cells has not yet been characterized. Results We examined the proteomic profiles of the cores purified from HIV-1 NL4-3 virions assembled in Sup-T1 cells (T lymphocytes), PMA and vitamin D3 activated THP1 (model of macrophages, mMΦ), and non-activated THP1 cells (model of monocytes, mMN) and assessed potential involvement of identified proteins in the early stages of infection using gene ontology information and data from genome-wide screens on proteins important for HIV-1 replication. We identified 202 cellular proteins incorporated in the viral cores (T cells: 125, mMΦ: 110, mMN: 90) with the overlap between these sets limited to 42 proteins. The groups of RNA binding (29), DNA binding (17), cytoskeleton (15), cytoskeleton regulation (21), chaperone (18), vesicular trafficking-associated (12) and ubiquitin-proteasome pathway-associated proteins (9) were most numerous. Cores of the virions from SupT1 cells contained twice as many RNA binding proteins as cores of THP1-derived virus, whereas cores of virions from mMΦ and mMN were enriched in components of cytoskeleton and vesicular transport machinery, most probably due to differences in virion assembly pathways between these cells. Spectra of chaperones, cytoskeletal proteins and ubiquitin-proteasome pathway components were similar between viral cores from different cell types, whereas DNA-binding and especially RNA-binding proteins were highly diverse. Western blot analysis showed that within the group of overlapping proteins, the level of

  20. Quantitative analysis of differentially expressed saliva proteins in human immunodeficiency virus type 1 (HIV-1) infected individuals

    International Nuclear Information System (INIS)

    Zhang, Nawei; Zhang, Zhenyu; Feng, Shan; Wang, Qingtao; Malamud, Daniel; Deng, Haiteng

    2013-01-01

    Highlights: ► A high-throughput method for profiling and quantification of the differentially expressed proteins in saliva samples was developed. ► Identified that DMBT1, S100A7, S100A8, S100A9 and alpha defensin were up-regulated in saliva from HIV-1 seropositive patients. ► Established analytical strategies are translatable to the clinical setting. -- Abstract: In the present study, we have established a new methodology to analyze saliva proteins from HIV-1-seropositive patients before highly active antiretroviral therapy (HAART) and seronegative controls. A total of 593 and 601 proteins were identified in the pooled saliva samples from 5 HIV-1 subjects and 5 controls, respectively. Forty-one proteins were found to be differentially expressed. Bioinformatic analysis of differentially expressed salivary proteins showed an increase of antimicrobial proteins and decrease of protease inhibitors upon HIV-1 infection. To validate some of these differentially expressed proteins, a high-throughput quantitation method was established to determine concentrations of 10 salivary proteins in 40 individual saliva samples from 20 seropositive patients before HAART and 20 seronegative subjects. This method was based on limited protein separation within the zone of the stacking gel of the 1D SDS PAGE and using isotope-coded synthetic peptides as internal standards. The results demonstrated that a combination of protein profiling and targeted quantitation is an efficient method to identify and validate differentially expressed salivary proteins. Expression levels of members of the calcium-binding S100 protein family and deleted in malignant brain tumors 1 protein (DMBT1) were up-regulated while that of Mucin 5B was down-regulated in HIV-1 seropositive saliva samples, which may provide new perspectives for monitoring HIV-infection and understanding the mechanism of HIV-1 infectivity

  1. Quantitative analysis of differentially expressed saliva proteins in human immunodeficiency virus type 1 (HIV-1) infected individuals

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Nawei; Zhang, Zhenyu [Beijing Chaoyang Hospital Affiliated Capital Medical University, Beijing (China); Feng, Shan [MOE Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University, Beijing (China); Wang, Qingtao [Beijing Chaoyang Hospital Affiliated Capital Medical University, Beijing (China); Malamud, Daniel [NYU College of Dentistry, 345 East 24th Street, New York, NY 10010 (United States); Deng, Haiteng, E-mail: dht@mail.tsinghua.edu.cn [MOE Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University, Beijing (China)

    2013-04-24

    Highlights: ► A high-throughput method for profiling and quantification of the differentially expressed proteins in saliva samples was developed. ► Identified that DMBT1, S100A7, S100A8, S100A9 and alpha defensin were up-regulated in saliva from HIV-1 seropositive patients. ► Established analytical strategies are translatable to the clinical setting. -- Abstract: In the present study, we have established a new methodology to analyze saliva proteins from HIV-1-seropositive patients before highly active antiretroviral therapy (HAART) and seronegative controls. A total of 593 and 601 proteins were identified in the pooled saliva samples from 5 HIV-1 subjects and 5 controls, respectively. Forty-one proteins were found to be differentially expressed. Bioinformatic analysis of differentially expressed salivary proteins showed an increase of antimicrobial proteins and decrease of protease inhibitors upon HIV-1 infection. To validate some of these differentially expressed proteins, a high-throughput quantitation method was established to determine concentrations of 10 salivary proteins in 40 individual saliva samples from 20 seropositive patients before HAART and 20 seronegative subjects. This method was based on limited protein separation within the zone of the stacking gel of the 1D SDS PAGE and using isotope-coded synthetic peptides as internal standards. The results demonstrated that a combination of protein profiling and targeted quantitation is an efficient method to identify and validate differentially expressed salivary proteins. Expression levels of members of the calcium-binding S100 protein family and deleted in malignant brain tumors 1 protein (DMBT1) were up-regulated while that of Mucin 5B was down-regulated in HIV-1 seropositive saliva samples, which may provide new perspectives for monitoring HIV-infection and understanding the mechanism of HIV-1 infectivity.

  2. Identification of a human protein-derived HIV-1 fusion inhibitor targeting the gp41 fusion core structure.

    Directory of Open Access Journals (Sweden)

    Lijun Chao

    Full Text Available The HIV-1 envelope glycoprotein (Env gp41 plays a crucial role in the viral fusion process. The peptides derived from the C-terminal heptad repeat (CHR of gp41 are potent HIV fusion inhibitors. However, the activity of these anti-HIV-1 peptides in vivo may be attenuated by their induction of anti-gp41 antibodies. Thus, it is essential to identify antiviral peptides or proteins with low, or no, immunogenicity to humans. Here, we found that the C-terminal fragment (aa 462-521 of the human POB1 (the partner of RalBP1, designated C60, is an HIV-1 fusion inhibitor. It bound to N36, the peptide derived from the N-terminal heptad repeat (NHR of gp41, and to the six-helix bundle (6-HB formed by N36 and C34, a CHR-peptide, but it did not bind to C34. Unlike the CHR-peptides, C60 did not block gp41 6-HB formation. Rather, results suggest that C60 inhibits HIV-1 fusion by binding to the 6-HB, in particular, the residues in the gp41 NHR domain that are exposed on the surface of 6-HB. Since 6-HB plays a crucial role in the late stage of fusion between the viral envelope and endosomal membrane during the endocytic process of HIV-1, C60 may serve as a host restriction factor to suppress HIV-1 entry into CD4+ T lymphocytes. Taken together, it can be concluded from these results that C60 can be used as a lead for the development of anti-HIV-1 therapeutics or microbicides for the treatment and prevention of HIV-1 infection, as well as a molecular probe to study the fusogenic mechanism of HIV-1.

  3. Systematic Analysis of the Functions of Lysine Acetylation in the Regulation of Tat Activity.

    Directory of Open Access Journals (Sweden)

    Minghao He

    Full Text Available The Tat protein of HIV-1 has several well-known properties, such as nucleocytoplasmic trafficking, transactivation of transcription, interaction with tubulin, regulation of mitotic progression, and induction of apoptosis. Previous studies have identified a couple of lysine residues in Tat that are essential for its functions. In order to analyze the functions of all the lysine residues in Tat, we mutated them individually to alanine, glutamine, and arginine. Through systematic analysis of the lysine mutants, we discovered several previously unidentified characteristics of Tat. We found that lysine acetylation could modulate the subcellular localization of Tat, in addition to the regulation of its transactivation activity. Our data also revealed that lysine mutations had distinct effects on microtubule assembly and Tat binding to bromodomain proteins. By correlation analysis, we further found that the effects of Tat on apoptosis and mitotic progression were not entirely attributed to its effect on microtubule assembly. Our findings suggest that Tat may regulate diverse cellular activities through binding to different proteins and that the acetylation of distinct lysine residues in Tat may modulate its interaction with various partners.

  4. HIV-1 uncoating: connection to nuclear entry and regulation by host proteins

    Energy Technology Data Exchange (ETDEWEB)

    Ambrose, Zandrea, E-mail: zaa4@pitt.edu [Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15261 (United States); Aiken, Christopher [Department of Pathology, Microbiology and Immunology, Vanderbilt University, School of Medicine, Nashville, TN 37232 (United States)

    2014-04-15

    The RNA genome of human immunodeficiency virus type 1 (HIV-1) is enclosed by a capsid shell that dissociates within the cell in a multistep process known as uncoating, which influences completion of reverse transcription of the viral genome. Double-stranded viral DNA is imported into the nucleus for integration into the host genome, a hallmark of retroviral infection. Reverse transcription, nuclear entry, and integration are coordinated by a capsid uncoating process that is regulated by cellular proteins. Although uncoating is not well understood, recent studies have revealed insights into the process, particularly with respect to nuclear import pathways and protection of the viral genome from DNA sensors. Understanding uncoating will be valuable toward developing novel antiretroviral therapies for HIV-infected individuals.

  5. Human Immunodeficiency Virus Tat-Activated Expression of Poliovirus Protein 2A Inhibits mRNA Translation

    Science.gov (United States)

    Sun, Xiao-Hong; Baltimore, David

    1989-04-01

    To study the effect of poliovirus protein 2A on cellular RNA translation, the tat control system of human immunodeficiency virus (HIV) was used. Protein 2A was expressed from a plasmid construct (pHIV/2A) incorporating the HIV long terminal repeat. Protein synthesis was measured by using chloramphenicol acetyltransferase as a reporter gene driven by the Rous sarcoma virus long terminal repeat. When HIV/2A was contransfected with the reporter, addition of a tat-producing plasmid caused at least a 50-fold drop in chloramphenicol acetyltransferase synthesis. A HeLa cell line carrying HIV/2A was established. In it, tat expression caused more than a 10-fold drop in chloramphenicol acetyltransferase synthesis from the reporter plasmid. Furthermore, 2A induction by tat caused cleavage of the cellular translation factor P220, a part of eukaryotic translation initiation factor 4F. Thus protein 2A can, by itself, carry out the inhibition of cellular protein synthesis characteristic of a poliovirus infection. Also, the HIV tat activation provides a very effective method to control gene expression in mammalian cells.

  6. The mitochondrial translocator protein, TSPO, inhibits HIV-1 envelope glycoprotein biosynthesis via the endoplasmic reticulum-associated protein degradation pathway.

    Science.gov (United States)

    Zhou, Tao; Dang, Ying; Zheng, Yong-Hui

    2014-03-01

    The HIV-1 Env glycoprotein is folded in the endoplasmic reticulum (ER), which is necessary for viral entry and replication. Currently, it is still unclear how this process is regulated. The glycoprotein folding in the ER is controlled by the ER-associated protein degradation (ERAD) pathway, which specifically targets misfolded proteins for degradation. Previously, we reported that HIV-1 replication is restricted in the human CD4(+) T cell line CEM.NKR (NKR). To understand this mechanism, we first analyzed cellular protein expression in NKR cells and discovered that levels of the mitochondrial translocator protein TSPO were upregulated by ∼64-fold. Notably, when NKR cells were treated with TSPO antagonist PK-11195, Ro5-4864, or diazepam, HIV restriction was completely disrupted, and TSPO knockdown by short hairpin RNAs (shRNAs) achieved a similar effect. We next analyzed viral protein expression, and, interestingly, we discovered that Env expression was specifically inhibited. Both TSPO knockdown and treatment with TSPO antagonist could restore Env expression in NKR cells. We further discovered that Env proteins were rapidly degraded and that kifunensine, an ERAD pathway inhibitor, could restore Env expression and viral replication, indicating that Env proteins were misfolded and degraded through the ERAD pathway in NKR cells. We also knocked out the TSPO gene in 293T cells using CRISPR/Cas9 (clustered, regularly interspaced, short palindromic repeat [CRISPR]/CRISPR-associated-9) technology and found that TSPO could similarly inhibit Env expression in these cells. Taken together, these results demonstrate that TSPO inhibits Env protein expression through the ERAD pathway and suggest that mitochondria play an important role in regulating the Env folding process. The HIV-1 Env glycoprotein is absolutely required for viral infection, and an understanding of its expression pathway in infected cells will identify new targets for antiretroviral therapies. Env proteins

  7. The human immunodeficiency virus-1 protein Tat and its discrete fragments evoke selective release of acetylcholine from human and rat cerebrocortical terminals through species-specific mechanisms.

    Science.gov (United States)

    Feligioni, Marco; Raiteri, Luca; Pattarini, Roberto; Grilli, Massimo; Bruzzone, Santina; Cavazzani, Paolo; Raiteri, Maurizio; Pittaluga, Anna

    2003-07-30

    The effect of the human immunodeficiency virus-1 protein Tat was investigated on neurotransmitter release from human and rat cortical nerve endings. Tat failed to affect the release of several neurotransmitters, such as glutamate, GABA, norepinephrine, and others, but it evoked the release of [3H]ACh via increase of cytosolic [Ca2+]. In human nerve terminals, the Tat effect partly depends on Ca2+ entry through voltage-sensitive Ca2+ channels, because Cd2+ halved the Tat-evoked release. Activation of group I metabotropic glutamate receptors (mGluR) and mobilization of Ca2+ from IP3-sensitive intraterminal stores are also involved, because the Tat effect was prevented by mGluR antagonists 2-methyl-6-(phenylethynyl)pyridine hydrochloride and 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester and by the IP3 receptor antagonists heparin and xestospongin C. Furthermore, the group I selective mGlu agonist (RS)-3,5-dihydroxyphenylglycine enhanced [3H]ACh release. In rat nerve terminals, the Tat-evoked release neither depends on external Ca2+ ions entry nor on IP3-mediated mechanisms. Tat seems to cause mobilization of Ca2+ from ryanodine-sensitive internal stores because its effect was prevented by both 8-bromo-cyclic adenosine diphosphate-ribose and dantrolene. The Tat-evoked release from human synaptosomes was mimicked by the peptide sequences Tat 32-62, Tat 49-86, and Tat 41-60. In contrast, the Tat 49-86 and Tat 61-80 fragments, but not the Tat 32-62 fragment, were active in rat synaptosomes. In conclusion, Tat elicits Ca2+-dependent [3H]ACh release by species-specific intraterminal mechanisms by binding via discrete amino acid sequences to different receptive sites on human and rat cholinergic terminals.

  8. Brain transcriptome-wide screen for HIV-1 Nef protein interaction partners reveals various membrane-associated proteins.

    Directory of Open Access Journals (Sweden)

    Ellen C Kammula

    Full Text Available HIV-1 Nef protein contributes essentially to the pathology of AIDS by a variety of protein-protein-interactions within the host cell. The versatile functionality of Nef is partially attributed to different conformational states and posttranslational modifications, such as myristoylation. Up to now, many interaction partners of Nef have been identified using classical yeast two-hybrid screens. Such screens rely on transcriptional activation of reporter genes in the nucleus to detect interactions. Thus, the identification of Nef interaction partners that are integral membrane proteins, membrane-associated proteins or other proteins that do not translocate into the nucleus is hampered. In the present study, a split-ubiquitin based yeast two-hybrid screen was used to identify novel membrane-localized interaction partners of Nef. More than 80% of the hereby identified interaction partners of Nef are transmembrane proteins. The identified hits are GPM6B, GPM6A, BAP31, TSPAN7, CYB5B, CD320/TCblR, VSIG4, PMEPA1, OCIAD1, ITGB1, CHN1, PH4, CLDN10, HSPA9, APR-3, PEBP1 and B3GNT, which are involved in diverse cellular processes like signaling, apoptosis, neurogenesis, cell adhesion and protein trafficking or quality control. For a subfraction of the hereby identified proteins we present data supporting their direct interaction with HIV-1 Nef. We discuss the results with respect to many phenotypes observed in HIV infected cells and patients. The identified Nef interaction partners may help to further elucidate the molecular basis of HIV-related diseases.

  9. Similarities and differences in the nucleic acid chaperone activity of HIV-2 and HIV-1 nucleocapsid proteins in vitro.

    Science.gov (United States)

    Pachulska-Wieczorek, Katarzyna; Stefaniak, Agnieszka K; Purzycka, Katarzyna J

    2014-07-03

    The nucleocapsid domain of Gag and mature nucleocapsid protein (NC) act as nucleic acid chaperones and facilitate folding of nucleic acids at critical steps of retroviral replication cycle. The basic N-terminus of HIV-1 NC protein was shown most important for the chaperone activity. The HIV-2 NC (NCp8) and HIV-1 NC (NCp7) proteins possess two highly conserved zinc fingers, flanked by basic residues. However, the NCp8 N-terminal domain is significantly shorter and contains less positively charged residues. This study characterizes previously unknown, nucleic acid chaperone activity of the HIV-2 NC protein. We have comparatively investigated the in vitro nucleic acid chaperone properties of the HIV-2 and HIV-1 NC proteins. Using substrates derived from the HIV-1 and HIV-2 genomes, we determined the ability of both proteins to chaperone nucleic acid aggregation, annealing and strand exchange in duplex structures. Both NC proteins displayed comparable, high annealing activity of HIV-1 TAR DNA and its complementary nucleic acid. Interesting differences between the two NC proteins were discovered when longer HIV substrates, particularly those derived from the HIV-2 genome, were used in chaperone assays. In contrast to NCp7, NCp8 weakly facilitates annealing of HIV-2 TAR RNA to its complementary TAR (-) DNA. NCp8 is also unable to efficiently stimulate tRNALys3 annealing to its respective HIV-2 PBS motif. Using truncated NCp8 peptide, we demonstrated that despite the fact that the N-terminus of NCp8 differs from that of NCp7, this domain is essential for NCp8 activity. Our data demonstrate that the HIV-2 NC protein displays reduced nucleic acid chaperone activity compared to that of HIV-1 NC. We found that NCp8 activity is limited by substrate length and stability to a greater degree than that of NCp7. This is especially interesting in light of the fact that the HIV-2 5'UTR is more structured than that of HIV-1. The reduced chaperone activity observed with NCp8 may

  10. Boosting of HIV-1 neutralizing antibody responses by a distally related retroviral envelope protein

    DEFF Research Database (Denmark)

    Uchtenhagen, Hannes; Schiffner, Torben; Bowles, Emma

    2014-01-01

    Our knowledge of the binding sites for neutralizing Abs (NAb) that recognize a broad range of HIV-1 strains (bNAb) has substantially increased in recent years. However, gaps remain in our understanding of how to focus B cell responses to vulnerable conserved sites within the HIV-1 envelope glycop...

  11. The HIV-1 Vpu protein induces apoptosis in Drosophila via activation of JNK signaling.

    Directory of Open Access Journals (Sweden)

    Christelle Marchal

    Full Text Available The genome of the human immunodeficiency virus type 1 (HIV-1 encodes the canonical retroviral proteins, as well as additional accessory proteins that enhance the expression of viral genes, the infectivity of the virus and the production of virions. The accessory Viral Protein U (Vpu, in particular, enhances viral particle production, while also promoting apoptosis of HIV-infected human T lymphocytes. Some Vpu effects rely on its interaction with the ubiquitin-proteasome protein degradation system, but the mechanisms responsible for its pro-apoptotic effects in vivo are complex and remain largely to be elucidated.We took advantage of the Drosophila model to study the effects of Vpu activity in vivo. Expression of Vpu in the developing Drosophila wing provoked tissue loss due to caspase-dependent apoptosis. Moreover, Vpu induced expression of the pro-apoptotic gene reaper, known to down-regulate Inhibitor of Apoptosis Proteins (IAPs which are caspase-antagonizing E3 ubiquitin ligases. Indeed, Vpu also reduced accumulation of Drosophila IAP1 (DIAP1. Though our results demonstrate a physical interaction between Vpu and the proteasome-addressing SLIMB/β-TrCP protein, as in mammals, both SLIMB/βTrCP-dependent and -independent Vpu effects were observed in the Drosophila wing. Lastly, the pro-apoptotic effect of Vpu in this tissue was abrogated upon inactivation of the c-Jun N-terminal Kinase (JNK pathway. Our results in the fly thus provide the first functional evidence linking Vpu pro-apoptotic effects to activation of the conserved JNK pathway.

  12. Methamphetamine and HIV-Tat alter murine cardiac DNA methylation and gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Koczor, Christopher A., E-mail: ckoczor@emory.edu; Fields, Earl; Jedrzejczak, Mark J.; Jiao, Zhe; Ludaway, Tomika; Russ, Rodney; Shang, Joan; Torres, Rebecca A.; Lewis, William

    2015-11-01

    This study addresses the individual and combined effects of HIV-1 and methamphetamine (N-methyl-1-phenylpropan-2-amine, METH) on cardiac dysfunction in a transgenic mouse model of HIV/AIDS. METH is abused epidemically and is frequently associated with acquisition of HIV-1 infection or AIDS. We employed microarrays to identify mRNA differences in cardiac left ventricle (LV) gene expression following METH administration (10 d, 3 mg/kg/d, subcutaneously) in C57Bl/6 wild-type littermates (WT) and Tat-expressing transgenic (TG) mice. Arrays identified 880 differentially expressed genes (expression fold change > 1.5, p < 0.05) following METH exposure, Tat expression, or both. Using pathway enrichment analysis, mRNAs encoding polypeptides for calcium signaling and contractility were altered in the LV samples. Correlative DNA methylation analysis revealed significant LV DNA methylation changes following METH exposure and Tat expression. By combining these data sets, 38 gene promoters (27 related to METH, 11 related to Tat) exhibited differences by both methods of analysis. Among those, only the promoter for CACNA1C that encodes L-type calcium channel Cav1.2 displayed DNA methylation changes concordant with its gene expression change. Quantitative PCR verified that Cav1.2 LV mRNA abundance doubled following METH. Correlative immunoblots specific for Cav1.2 revealed a 3.5-fold increase in protein abundance in METH LVs. Data implicate Cav1.2 in calcium dysregulation and hypercontractility in the murine LV exposed to METH. They suggest a pathogenetic role for METH exposure to promote LV dysfunction that outweighs Tat-induced effects. - Highlights: • HIV-1 Tat and methamphetamine (METH) alter cardiac gene expression and epigenetics. • METH impacts gene expression or epigenetics more significantly than Tat expression. • METH alters cardiac mitochondrial function and calcium signaling independent of Tat. • METH alters DNA methylation, expression, and protein abundance of

  13. Methamphetamine and HIV-Tat alter murine cardiac DNA methylation and gene expression

    International Nuclear Information System (INIS)

    Koczor, Christopher A.; Fields, Earl; Jedrzejczak, Mark J.; Jiao, Zhe; Ludaway, Tomika; Russ, Rodney; Shang, Joan; Torres, Rebecca A.; Lewis, William

    2015-01-01

    This study addresses the individual and combined effects of HIV-1 and methamphetamine (N-methyl-1-phenylpropan-2-amine, METH) on cardiac dysfunction in a transgenic mouse model of HIV/AIDS. METH is abused epidemically and is frequently associated with acquisition of HIV-1 infection or AIDS. We employed microarrays to identify mRNA differences in cardiac left ventricle (LV) gene expression following METH administration (10 d, 3 mg/kg/d, subcutaneously) in C57Bl/6 wild-type littermates (WT) and Tat-expressing transgenic (TG) mice. Arrays identified 880 differentially expressed genes (expression fold change > 1.5, p < 0.05) following METH exposure, Tat expression, or both. Using pathway enrichment analysis, mRNAs encoding polypeptides for calcium signaling and contractility were altered in the LV samples. Correlative DNA methylation analysis revealed significant LV DNA methylation changes following METH exposure and Tat expression. By combining these data sets, 38 gene promoters (27 related to METH, 11 related to Tat) exhibited differences by both methods of analysis. Among those, only the promoter for CACNA1C that encodes L-type calcium channel Cav1.2 displayed DNA methylation changes concordant with its gene expression change. Quantitative PCR verified that Cav1.2 LV mRNA abundance doubled following METH. Correlative immunoblots specific for Cav1.2 revealed a 3.5-fold increase in protein abundance in METH LVs. Data implicate Cav1.2 in calcium dysregulation and hypercontractility in the murine LV exposed to METH. They suggest a pathogenetic role for METH exposure to promote LV dysfunction that outweighs Tat-induced effects. - Highlights: • HIV-1 Tat and methamphetamine (METH) alter cardiac gene expression and epigenetics. • METH impacts gene expression or epigenetics more significantly than Tat expression. • METH alters cardiac mitochondrial function and calcium signaling independent of Tat. • METH alters DNA methylation, expression, and protein abundance of

  14. Neuroprotective effect of TAT-14-3-3ε fusion protein against cerebral ischemia/reperfusion injury in rats.

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    Yuanjun Zhu

    Full Text Available Stroke is the major cause of death and disability worldwide, and the thrombolytic therapy currently available was unsatisfactory. 14-3-3ε is a well characterized member of 14-3-3 family, and has been reported to protect neurons against apoptosis in cerebral ischemia. However, it cannot transverse blood brain barrier (BBB due to its large size. A protein transduction domain (PTD of HIV TAT protein, is capable of delivering a large variety of proteins into the brain. In this study, we generated a fusion protein TAT-14-3-3ε, and evaluated its potential neuroprotective effect in rat focal ischemia/reperfusion (I/R model. Western blot analysis validated the efficient transduction of TAT-14-3-3ε fusion protein into brain via a route of intravenous injection. TAT-14-3-3ε pre-treatment 2 h before ischemia significantly reduced cerebral infarction volume and improved neurologic score, while post-treatment 2 h after ischemia was less effective. Importantly, pre- or post-ischemic treatment with TAT-14-3-3ε significantly increased the number of surviving neurons as determined by Nissl staining, and attenuated I/R-induced neuronal apoptosis as showed by the decrease in apoptotic cell numbers and the inhibition of caspase-3 activity. Moreover, the introduction of 14-3-3ε into brain by TAT-mediated delivering reduced the formation of autophagosome, attenuated LC3B-II upregulation and reversed p62 downregulation induced by ischemic injury. Such inhibition of autophagy was reversed by treatment with an autophagy inducer rapamycin (RAP, which also attenuated the neuroprotective effect of TAT-14-3-3ε. Conversely, autophagy inhibitor 3-methyladenine (3-MA inhibited I/R-induced the increase in autophagic activity, and attenuated I/R-induced brain infarct. These results suggest that TAT-14-3-3ε can be efficiently transduced into brain and exert significantly protective effect against brain ischemic injury through inhibiting neuronal apoptosis and autophagic

  15. Temporal expression of HIV-1 envelope proteins in baculovirus-infected insect cells: Implications for glycosylation and CD4 binding

    International Nuclear Information System (INIS)

    Murphy, C.I.; Lennick, M.; Lehar, S.M.; Beltz, G.A.; Young, E.

    1990-01-01

    Three different human immunodeficiency virus type I (HIV-1) envelope derived recombinant proteins and the full length human CD4 polypeptide were expressed in Spodoptera frugiperda (Sf9) cells. DNA constructs encoding CD4, gp120, gp160, and gp160 delta were cloned into the baculovirus expression vector pVL941 or a derivative and used to generate recombinant viruses in a cotransfection with DNA from Autographa californica nuclear polyhedrosis virus (AcMNPV). Western blotting of cell extracts of the recombinant HIV-1 proteins showed that for each construct two major bands specifically reacted with anti-HIV-1 envelope antiserum. These bands corresponded to glycosylated and nonglycosylated versions of the HIV proteins as determined by 3H-mannose labeling and tunicamycin treatment of infected cells. A time course of HIV envelope expression revealed that at early times post-infection (24 hours) the proteins were fully glycosylated and soluble in nonionic detergents. However, at later times postinfection (48 hours), expression levels of recombinant protein reached a maximum but most of the increase was due to a rise in the level of the nonglycosylated species, which was largely insoluble in nonionic detergents. Thus, it appears that Sf9 cells cannot process large amounts of glycosylated recombinant proteins efficiently. As a measure of biological activity, the CD4 binding ability of both glycosylated and nonglycosylated recombinant HIV envelope proteins was tested in a coimmunoprecipitation assay. The results showed that CD4 and the glycosylated versions of recombinant gp120 or gp160 delta specifically associated with one another in this analysis. Nonglycosylated gp120 or gp160 delta proteins from tunicamycin-treated cultures did immunoprecipitate with anti-HIV-1 antiserum but did not interact with CD4

  16. Improved innate and adaptive immunostimulation by genetically modified HIV-1 protein expressing NYVAC vectors.

    Directory of Open Access Journals (Sweden)

    Esther D Quakkelaar

    Full Text Available Attenuated poxviruses are safe and capable of expressing foreign antigens. Poxviruses are applied in veterinary vaccination and explored as candidate vaccines for humans. However, poxviruses express multiple genes encoding proteins that interfere with components of the innate and adaptive immune response. This manuscript describes two strategies aimed to improve the immunogenicity of the highly attenuated, host-range restricted poxvirus NYVAC: deletion of the viral gene encoding type-I interferon-binding protein and development of attenuated replication-competent NYVAC. We evaluated these newly generated NYVAC mutants, encoding HIV-1 env, gag, pol and nef, for their ability to stimulate HIV-specific CD8 T-cell responses in vitro from blood mononuclear cells of HIV-infected subjects. The new vectors were evaluated and compared to the parental NYVAC vector in dendritic cells (DCs, RNA expression arrays, HIV gag expression and cross-presentation assays in vitro. Deletion of type-I interferon-binding protein enhanced expression of interferon and interferon-induced genes in DCs, and increased maturation of infected DCs. Restoration of replication competence induced activation of pathways involving antigen processing and presentation. Also, replication-competent NYVAC showed increased Gag expression in infected cells, permitting enhanced cross-presentation to HIV-specific CD8 T cells and proliferation of HIV-specific memory CD8 T-cells in vitro. The recombinant NYVAC combining both modifications induced interferon-induced genes and genes involved in antigen processing and presentation, as well as increased Gag expression. This combined replication-competent NYVAC is a promising candidate for the next generation of HIV vaccines.

  17. Anti-inflammatory effects of Tat-Annexin protein on ovalbumin-induced airway inflammation in a mouse model of asthma

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Sun Hwa; Kim, Dae Won; Kim, Hye Ri; Woo, Su Jung; Kim, So Mi; Jo, Hyo Sang [Department of Biomedical Science and Research Institute of Bioscience and Biotechnology, Hallym University, Chunchon 200-702 (Korea, Republic of); Jeon, Seong Gyu [Department of Life Science, Pohang University of Science and Technology, Pohang 790-784 (Korea, Republic of); Cho, Sung-Woo [Department of Biochemistry and Molecular Biology, University of Ulsan, College of Medicine, Seoul 138-736 (Korea, Republic of); Park, Jong Hoon [Department of Biological Science, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Won, Moo Ho [Department of Neurobiology, School of Medicine, Kangwon National University, Chuncheon 200-701 (Korea, Republic of); Park, Jinseu [Department of Biomedical Science and Research Institute of Bioscience and Biotechnology, Hallym University, Chunchon 200-702 (Korea, Republic of); Eum, Won Sik, E-mail: wseum@hallym.ac.kr [Department of Biomedical Science and Research Institute of Bioscience and Biotechnology, Hallym University, Chunchon 200-702 (Korea, Republic of); Choi, Soo Young, E-mail: sychoi@hallym.ac.kr [Department of Biomedical Science and Research Institute of Bioscience and Biotechnology, Hallym University, Chunchon 200-702 (Korea, Republic of)

    2012-01-20

    Highlights: Black-Right-Pointing-Pointer We construct a cell permeable Tat-ANX1 fusion protein. Black-Right-Pointing-Pointer We examined the protective effects of Tat-ANX1 protein on OVA-induced asthma in animal models. Black-Right-Pointing-Pointer Transduced Tat-ANX1 protein protects from the OVA-induced production of cytokines and eosinophils in BAL fluid. Black-Right-Pointing-Pointer Tat-ANX1 protein markedly reduced OVA-induced MAPK in lung tissues. Black-Right-Pointing-Pointer Tat-ANX1 protein could be useful as a therapeutic agent for lung disorders including asthma. -- Abstract: Chronic airway inflammation is a key feature of bronchial asthma. Annexin-1 (ANX1) is an anti-inflammatory protein that is an important modulator and plays a key role in inflammation. Although the precise action of ANX1 remains unclear, it has emerged as a potential drug target for inflammatory diseases such as asthma. To examine the protective effects of ANX1 protein on ovalbumin (OVA)-induced asthma in animal models, we used a cell-permeable Tat-ANX1 protein. Mice sensitized and challenged with OVA antigen had an increased amount of cytokines and eosinophils in their bronchoalveolar lavage (BAL) fluid. However, administration of Tat-ANX1 protein before OVA challenge significantly decreased the levels of cytokines (interleukin (IL)-4, IL-5, and IL-13) and BAL fluid in lung tissues. Furthermore, OVA significantly increased the activation of mitogen-activated protein kinase (MAPK) in lung tissues, whereas Tat-ANX1 protein markedly reduced phosphorylation of MAPKs such as extracellular signal-regulated protein kinase, p38, and stress-activated protein kinase/c-Jun N-terminal kinase. These results suggest that transduced Tat-ANX1 protein may be a potential protein therapeutic agent for the treatment of lung disorders including asthma.

  18. Overproduction of a Model Sec- and Tat-Dependent Secretory Protein Elicits Different Cellular Responses in Streptomyces lividans.

    Directory of Open Access Journals (Sweden)

    Sonia Gullón

    Full Text Available Streptomyces lividans is considered an efficient host for the secretory production of homologous and heterologous proteins. To identify possible bottlenecks in the protein production process, a comparative transcriptomic approach was adopted to study cellular responses during the overproduction of a Sec-dependent model protein (alpha-amylase and a Tat-dependent model protein (agarase in Streptomyces lividans. The overproduction of the model secretory proteins via the Sec or the Tat route in S. lividans does elicit a different major cell response in the bacterium. The stringent response is a bacterial response to nutrients' depletion, which naturally occurs at late times of the bacterial cell growth. While the induction of the stringent response at the exponential phase of growth may limit overall productivity in the case of the Tat route, the induction of that response does not take place in the case of the Sec route, which comparatively is an advantage in secretory protein production processes. Hence, this study identifies a potential major drawback in the secretory protein production process depending on the secretory route, and provides clues to improving S. lividans as a protein production host.

  19. Intracellular trafficking of superparamagnetic iron oxide nanoparticles conjugated with TAT peptide: 3-dimensional electron tomography analysis

    Energy Technology Data Exchange (ETDEWEB)

    Nair, Baiju G.; Fukuda, Takahiro; Mizuki, Toru; Hanajiri, Tatsuro [Bio-Nano Electronics Research Centre, Toyo University, Saitama 350-8585 (Japan); Maekawa, Toru, E-mail: maekawa@toyo.jp [Bio-Nano Electronics Research Centre, Toyo University, Saitama 350-8585 (Japan)

    2012-05-18

    Highlights: Black-Right-Pointing-Pointer We study the intracellular localisation of TAT-SPIONs using 3-D electron tomography. Black-Right-Pointing-Pointer 3-D images of TAT-SPIONs in a cell are clearly shown. Black-Right-Pointing-Pointer Release of TAT-SPIONs from endocytic vesicles into the cytoplasm is clearly shown. -- Abstract: Internalisation of nanoparticles conjugated with cell penetrating peptides is a promising approach to various drug delivery applications. Cell penetrating peptides such as transactivating transcriptional activator (TAT) peptides derived from HIV-1 proteins are effective intracellular delivery vectors for a wide range of nanoparticles and pharmaceutical agents thanks to their amicable ability to enter cells and minimum cytotoxicity. Although different mechanisms of intracellular uptake and localisation have been proposed for TAT conjugated nanoparticles, it is necessary to visualise the particles on a 3-D plane in order to investigate the actual intracellular uptake and localisation. Here, we study the intracellular localisation and trafficking of TAT peptide conjugated superparamagnetic ion oxide nanoparticles (TAT-SPIONs) using 3-D electron tomography. 3-D tomograms clearly show the location of TAT-SPIONs in a cell and their slow release from the endocytic vesicles into the cytoplasm. The present methodology may well be utilised for further investigations of the behaviours of nanoparticles in cells and eventually for the development of nano drug delivery systems.

  20. Intracellular trafficking of superparamagnetic iron oxide nanoparticles conjugated with TAT peptide: 3-dimensional electron tomography analysis

    International Nuclear Information System (INIS)

    Nair, Baiju G.; Fukuda, Takahiro; Mizuki, Toru; Hanajiri, Tatsuro; Maekawa, Toru

    2012-01-01

    Highlights: ► We study the intracellular localisation of TAT-SPIONs using 3-D electron tomography. ► 3-D images of TAT-SPIONs in a cell are clearly shown. ► Release of TAT-SPIONs from endocytic vesicles into the cytoplasm is clearly shown. -- Abstract: Internalisation of nanoparticles conjugated with cell penetrating peptides is a promising approach to various drug delivery applications. Cell penetrating peptides such as transactivating transcriptional activator (TAT) peptides derived from HIV-1 proteins are effective intracellular delivery vectors for a wide range of nanoparticles and pharmaceutical agents thanks to their amicable ability to enter cells and minimum cytotoxicity. Although different mechanisms of intracellular uptake and localisation have been proposed for TAT conjugated nanoparticles, it is necessary to visualise the particles on a 3-D plane in order to investigate the actual intracellular uptake and localisation. Here, we study the intracellular localisation and trafficking of TAT peptide conjugated superparamagnetic ion oxide nanoparticles (TAT-SPIONs) using 3-D electron tomography. 3-D tomograms clearly show the location of TAT-SPIONs in a cell and their slow release from the endocytic vesicles into the cytoplasm. The present methodology may well be utilised for further investigations of the behaviours of nanoparticles in cells and eventually for the development of nano drug delivery systems.

  1. The HIV1 protein Vpr acts to enhance constitutive DCAF1-dependent UNG2 turnover.

    Directory of Open Access Journals (Sweden)

    Xiaoyun Wen

    Full Text Available The HIV1 protein Vpr assembles with and acts through an ubiquitin ligase complex that includes DDB1 and cullin 4 (CRL4 to cause G2 cell cycle arrest and to promote degradation of both uracil DNA glycosylase 2 (UNG2 and single-strand selective mono-functional uracil DNA glycosylase 1 (SMUG1. DCAF1, an adaptor protein, is required for Vpr-mediated G2 arrest through the ubiquitin ligase complex. In work described here, we used UNG2 as a model substrate to study how Vpr acts through the ubiquitin ligase complex. We examined whether DCAF1 is essential for Vpr-mediated degradation of UNG2 and SMUG1. We further investigated whether Vpr is required for recruiting substrates to the ubiquitin ligase or acts to enhance its function and whether this parallels Vpr-mediated G2 arrest.We found that DCAF1 plays an important role in Vpr-independent UNG2 and SMUG1 depletion. UNG2 assembled with the ubiquitin ligase complex in the absence of Vpr, but Vpr enhanced this interaction. Further, Vpr-mediated enhancement of UNG2 degradation correlated with low Vpr expression levels. Vpr concentrations exceeding a threshold blocked UNG2 depletion and enhanced its accumulation in the cell nucleus. A similar dose-dependent trend was seen for Vpr-mediated cell cycle arrest.This work identifies UNG2 and SMUG1 as novel targets for CRL4(DCAF1-mediated degradation. It further shows that Vpr enhances rather than enables the interaction between UNG2 and the ubiquitin ligase. Vpr augments CRL4(DCAF1-mediated UNG2 degradation at low concentrations but antagonizes it at high concentrations, allowing nuclear accumulation of UNG2. Further, the protein that is targeted to cause G2 arrest behaves much like UNG2. Our findings provide the basis for determining whether the CRL4(DCAF1 complex is alone responsible for cell cycle-dependent UNG2 turnover and will also aid in establishing conditions necessary for the identification of additional targets of Vpr-enhanced degradation.

  2. Fluorescent protein-tagged Vpr dissociates from HIV-1 core after viral fusion and rapidly enters the cell nucleus.

    Science.gov (United States)

    Desai, Tanay M; Marin, Mariana; Sood, Chetan; Shi, Jiong; Nawaz, Fatima; Aiken, Christopher; Melikyan, Gregory B

    2015-10-29

    HIV-1 Vpr is recruited into virions during assembly and appears to remain associated with the viral core after the reverse transcription and uncoating steps of entry. This feature has prompted the use of fluorescently labeled Vpr to visualize viral particles and to follow trafficking of post-fusion HIV-1 cores in the cytoplasm. Here, we tracked single pseudovirus entry and fusion and observed that fluorescently tagged Vpr gradually dissociates from post-fusion viral cores over the course of several minutes and accumulates in the nucleus. Kinetics measurements showed that fluorescent Vpr released from the cores very rapidly entered the cell nucleus. More than 10,000 Vpr molecules can be delivered into the cell nucleus within 45 min of infection by HIV-1 particles pseudotyped with the avian sarcoma and leukosis virus envelope glycoprotein. The fraction of Vpr from cell-bound viruses that accumulated in the nucleus was proportional to the extent of virus-cell fusion and was fully blocked by viral fusion inhibitors. Entry of virus-derived Vpr into the nucleus occurred independently of envelope glycoproteins or target cells. Fluorescence correlation spectroscopy revealed two forms of nuclear Vpr-monomers and very large complexes, likely involving host factors. The kinetics of viral Vpr entering the nucleus after fusion was not affected by point mutations in the capsid protein that alter the stability of the viral core. The independence of Vpr shedding of capsid stability and its relatively rapid dissociation from post-fusion cores suggest that this process may precede capsid uncoating, which appears to occur on a slower time scale. Our results thus demonstrate that a bulk of fluorescently labeled Vpr incorporated into HIV-1 particles is released shortly after fusion. Future studies will address the question whether the quick and efficient nuclear delivery of Vpr derived from incoming viruses can regulate subsequent steps of HIV-1 infection.

  3. Regulatory CD4 T cells inhibit HIV-1 expression of other CD4 T cell subsets via interactions with cell surface regulatory proteins.

    Science.gov (United States)

    Zhang, Mingce; Robinson, Tanya O; Duverger, Alexandra; Kutsch, Olaf; Heath, Sonya L; Cron, Randy Q

    2018-03-01

    During chronic HIV-1 infection, regulatory CD4 T cells (Tregs) frequently represent the largest subpopulation of CD4 T cell subsets, implying relative resistant to HIV-1. When HIV-1 infection of CD4 T cells was explored in vitro and ex vivo from patient samples, Tregs possessed lower levels of HIV-1 DNA and RNA in comparison with conventional effector and memory CD4 T cells. Moreover, Tregs suppressed HIV-1 expression in other CD4 T cells in an in vitro co-culture system. This suppression was mediated in part via multiple inhibitory surface proteins expressed on Tregs. Antibody blockade of CTLA-4, PD-1, and GARP on Tregs resulted in increased HIV-1 DNA integration and mRNA expression in neighboring CD4 T cells. Moreover, antibody blockade of Tregs inhibitory proteins resulted in increased HIV-1 LTR transcription in co-cultured CD4 T cells. Thus, Tregs inhibit HIV-1 infection of other CD4 T cell subsets via interactions with inhibitory cell surface proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

  4. Mining the Human Complexome Database Identifies RBM14 as an XPO1-Associated Protein Involved in HIV-1 Rev Function

    OpenAIRE

    Budhiraja, Sona; Liu, Hongbing; Couturier, Jacob; Malovannaya, Anna; Qin, Jun; Lewis, Dorothy E.; Rice, Andrew P.

    2015-01-01

    By recruiting the host protein XPO1 (CRM1), the HIV-1 Rev protein mediates the nuclear export of incompletely spliced viral transcripts. We mined data from the recently described human nuclear complexome to identify a host protein, RBM14, which associates with XPO1 and Rev and is involved in Rev function. Using a Rev-dependent p24 reporter plasmid, we found that RBM14 depletion decreased Rev activity and Rev-mediated enhancement of the cytoplasmic levels of unspliced viral transcripts. RBM14 ...

  5. Necroptosis takes place in human immunodeficiency virus type-1 (HIV-1-infected CD4+ T lymphocytes.

    Directory of Open Access Journals (Sweden)

    Ting Pan

    Full Text Available Human immunodeficiency virus type 1 (HIV-1 infection is characterized by progressive depletion of CD4+ T lymphocytes and dysfunction of the immune system. The numbers of CD4+ T lymphocytes in the human body are maintained constantly by homeostatic mechanisms that failed during HIV-1 infection, resulting in progressive loss of CD4+ T cells mainly via apoptosis. Recently, a non-apoptotic form of necrotic programmed cell death, named necroptosis, has been investigated in many biological and pathological processes. We then determine whether HIV-1-infected cells also undergo necroptosis. In this report, we demonstrate that HIV-1 not only induces apoptosis, but also mediates necroptosis in the infected primary CD4+ T lymphocytes and CD4+ T-cell lines. Necroptosis-dependent cytopathic effects are significantly increased in HIV-1-infected Jurkat cells that is lack of Fas-associated protein-containing death domain (FADD, indicating that necroptosis occurs as an alternative cell death mechanism in the absence of apoptosis. Unlike apoptosis, necroptosis mainly occurs in HIV-infected cells and spares bystander damage. Treatment with necrostatin-1(Nec-1, a RIP1 inhibitor that specifically blocks the necroptosis pathway, potently restrains HIV-1-induced cytopathic effect and interestingly, inhibits the formation of HIV-induced syncytia in CD4+ T-cell lines. This suggests that syncytia formation is mediated, at least partially, by necroptosis-related processes. Furthermore, we also found that the HIV-1 infection-augmented tumor necrosis factor-alpha (TNF-α plays a key role in inducing necroptosis and HIV-1 Envelope and Tat proteins function as its co-factors. Taken together,necroptosis can function as an alternative cell death pathway in lieu of apoptosis during HIV-1 infection, thereby also contributing to HIV-1-induced cytopathic effects. Our results reveal that in addition to apoptosis, necroptosis also plays an important role in HIV-1-induced pathogenesis.

  6. Effects of Inner Nuclear Membrane Proteins SUN1/UNC-84A and SUN2/UNC-84B on the Early Steps of HIV-1 Infection.

    Science.gov (United States)

    Schaller, Torsten; Bulli, Lorenzo; Pollpeter, Darja; Betancor, Gilberto; Kutzner, Juliane; Apolonia, Luis; Herold, Nikolas; Burk, Robin; Malim, Michael H

    2017-10-01

    Human immunodeficiency virus type 1 (HIV-1) infection of dividing and nondividing cells involves regulatory interactions with the nuclear pore complex (NPC), followed by translocation to the nucleus and preferential integration into genomic areas in proximity to the inner nuclear membrane (INM). To identify host proteins that may contribute to these processes, we performed an overexpression screen of known membrane-associated NE proteins. We found that the integral transmembrane proteins SUN1/UNC84A and SUN2/UNC84B are potent or modest inhibitors of HIV-1 infection, respectively, and that suppression corresponds to defects in the accumulation of viral cDNA in the nucleus. While laboratory strains (HIV-1 NL4.3 and HIV-1 IIIB ) are sensitive to SUN1-mediated inhibition, the transmitted founder viruses RHPA and ZM247 are largely resistant. Using chimeric viruses, we identified the HIV-1 capsid (CA) protein as a major determinant of sensitivity to SUN1, and in vitro -assembled capsid-nucleocapsid (CANC) nanotubes captured SUN1 and SUN2 from cell lysates. Finally, we generated SUN1 -/- and SUN2 -/- cells by using CRISPR/Cas9 and found that the loss of SUN1 had no effect on HIV-1 infectivity, whereas the loss of SUN2 had a modest suppressive effect. Taken together, these observations suggest that SUN1 and SUN2 may function redundantly to modulate postentry, nuclear-associated steps of HIV-1 infection. IMPORTANCE HIV-1 causes more than 1 million deaths per year. The life cycle of HIV-1 has been studied extensively, yet important steps that occur between viral capsid release into the cytoplasm and the expression of viral genes remain elusive. We propose here that the INM components SUN1 and SUN2, two members of the linker of nucleoskeleton and cytoskeleton (LINC) complex, may interact with incoming HIV-1 replication complexes and affect key steps of infection. While overexpression of these proteins reduces HIV-1 infection, disruption of the individual SUN2 and SUN1 genes

  7. Solution Structure and Membrane Interaction of the Cytoplasmic Tail of HIV-1 gp41 Protein.

    Science.gov (United States)

    Murphy, R Elliot; Samal, Alexandra B; Vlach, Jiri; Saad, Jamil S

    2017-11-07

    The cytoplasmic tail of gp41 (gp41CT) remains the last HIV-1 domain with an unknown structure. It plays important roles in HIV-1 replication such as mediating envelope (Env) intracellular trafficking and incorporation into assembling virions, mechanisms of which are poorly understood. Here, we present the solution structure of gp41CT in a micellar environment and characterize its interaction with the membrane. We show that the N-terminal 45 residues are unstructured and not associated with the membrane. However, the C-terminal 105 residues form three membrane-bound amphipathic α helices with distinctive structural features such as variable degree of membrane penetration, hydrophobic and basic surfaces, clusters of aromatic residues, and a network of cation-π interactions. This work fills a major gap by providing the structure of the last segment of HIV-1 Env, which will provide insights into the mechanisms of Gag-mediated Env incorporation as well as the overall Env mobility and conformation on the virion surface. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. DmsD, a Tat system specific chaperone, interacts with other general chaperones and proteins involved in the molybdenum cofactor biosynthesis

    OpenAIRE

    Li, Haiming; Chang, Limei; Howell, Jenika M.; Turner, Raymond J.

    2010-01-01

    Many bacterial oxidoreductases depend on the Tat translocase for correct cell localization. Substrates for the Tat translocase possess twin-arginine leaders. System specific chaperones or redox enzyme maturation proteins (REMPs) are a group of proteins implicated in oxidoreductase maturation. DmsD is a REMP discovered in Escherichia coli, which interacts with the twin-arginine leader sequence of DmsA, the catalytic subunit of DMSO reductase. In this study, we identified several potential inte...

  9. Nuclear retention of multiply spliced HIV-1 RNA in resting CD4+ T cells.

    Directory of Open Access Journals (Sweden)

    Kara G Lassen

    2006-07-01

    Full Text Available HIV-1 latency in resting CD4+ T cells represents a major barrier to virus eradication in patients on highly active antiretroviral therapy (HAART. We describe here a novel post-transcriptional block in HIV-1 gene expression in resting CD4+ T cells from patients on HAART. This block involves the aberrant localization of multiply spliced (MS HIV-1 RNAs encoding the critical positive regulators Tat and Rev. Although these RNAs had no previously described export defect, we show that they exhibit strict nuclear localization in resting CD4+ T cells from patients on HAART. Overexpression of the transcriptional activator Tat from non-HIV vectors allowed virus production in these cells. Thus, the nuclear retention of MS HIV-1 RNA interrupts a positive feedback loop and contributes to the non-productive nature of infection of resting CD4+ T cells. To define the mechanism of nuclear retention, proteomic analysis was used to identify proteins that bind MS HIV-1 RNA. Polypyrimidine tract binding protein (PTB was identified as an HIV-1 RNA-binding protein differentially expressed in resting and activated CD4+ T cells. Overexpression of PTB in resting CD4+ T cells from patients on HAART allowed cytoplasmic accumulation of HIV-1 RNAs. PTB overexpression also induced virus production by resting CD4+ T cells. Virus culture experiments showed that overexpression of PTB in resting CD4+ T cells from patients on HAART allowed release of replication-competent virus, while preserving a resting cellular phenotype. Whether through effects on RNA export or another mechanism, the ability of PTB to reverse latency without inducing cellular activation is a result with therapeutic implications.

  10. Mucosal Immunogenicity of Genetically Modified Lactobacillus acidophilus Expressing an HIV-1 Epitope within the Surface Layer Protein.

    Directory of Open Access Journals (Sweden)

    Akinobu Kajikawa

    Full Text Available Surface layer proteins of probiotic lactobacilli are theoretically efficient epitope-displaying scaffolds for oral vaccine delivery due to their high expression levels and surface localization. In this study, we constructed genetically modified Lactobacillus acidophilus strains expressing the membrane proximal external region (MPER from human immunodeficiency virus type 1 (HIV-1 within the context of the major S-layer protein, SlpA. Intragastric immunization of mice with the recombinants induced MPER-specific and S-layer protein-specific antibodies in serum and mucosal secretions. Moreover, analysis of systemic SlpA-specific cytokines revealed that the responses appeared to be Th1 and Th17 dominant. These findings demonstrated the potential use of the Lactobacillus S-layer protein for development of oral vaccines targeting specific peptides.

  11. Developing HIV-1 Protease Inhibitors through Stereospecific Reactions in Protein Crystals.

    Science.gov (United States)

    Olajuyigbe, Folasade M; Demitri, Nicola; De Zorzi, Rita; Geremia, Silvano

    2016-10-31

    Protease inhibitors are key components in the chemotherapy of HIV infection. However, the appearance of viral mutants routinely compromises their clinical efficacy, creating a constant need for new and more potent inhibitors. Recently, a new class of epoxide-based inhibitors of HIV-1 protease was investigated and the configuration of the epoxide carbons was demonstrated to play a crucial role in determining the binding affinity. Here we report the comparison between three crystal structures at near-atomic resolution of HIV-1 protease in complex with the epoxide-based inhibitor, revealing an in-situ epoxide ring opening triggered by a pH change in the mother solution of the crystal. Increased pH in the crystal allows a stereospecific nucleophile attack of an ammonia molecule onto an epoxide carbon, with formation of a new inhibitor containing amino-alcohol functions. The described experiments open a pathway for the development of new stereospecific protease inhibitors from a reactive lead compound.

  12. Developing HIV-1 Protease Inhibitors through Stereospecific Reactions in Protein Crystals

    Directory of Open Access Journals (Sweden)

    Folasade M. Olajuyigbe

    2016-10-01

    Full Text Available Protease inhibitors are key components in the chemotherapy of HIV infection. However, the appearance of viral mutants routinely compromises their clinical efficacy, creating a constant need for new and more potent inhibitors. Recently, a new class of epoxide-based inhibitors of HIV-1 protease was investigated and the configuration of the epoxide carbons was demonstrated to play a crucial role in determining the binding affinity. Here we report the comparison between three crystal structures at near-atomic resolution of HIV-1 protease in complex with the epoxide-based inhibitor, revealing an in-situ epoxide ring opening triggered by a pH change in the mother solution of the crystal. Increased pH in the crystal allows a stereospecific nucleophile attack of an ammonia molecule onto an epoxide carbon, with formation of a new inhibitor containing amino-alcohol functions. The described experiments open a pathway for the development of new stereospecific protease inhibitors from a reactive lead compound.

  13. First large scale chemical synthesis of the 72 amino acid HIV-1 nucleocapsid protein NCp7 in an active form.

    Science.gov (United States)

    de Rocquigny, H; Ficheux, D; Gabus, C; Fournié-Zaluski, M C; Darlix, J L; Roques, B P

    1991-10-31

    The nucleocapsid protein (NC) of the human immunodeficiency virus type 1 plays a crucial role in the formation of infectious viral particles and therefore should be a major target for the development of antiviral agents. This requires an investigation of NC protein structure and of its interactions with both primer tRNA(Lys,3) and genomic RNA. Nucleocapsid protein NCp7, which results from the maturation of NCp15, contains two zinc fingers flanked by sequences rich in basic and proline residues. Here we report the first synthesis of large quantities of NCp7 able to activate HIV-1 RNA dimerization and replication primer tRNA(Lys,3) annealing to the initiation site of reverse transcription. In addition UV spectroscopic analyses performed to characterize the Co2+ binding properties of each zinc finger suggest that the two fingers probably interact in NCp7.

  14. The utility of protein structure as a predictor of site-wise dN/dS varies widely among HIV-1 proteins.

    Science.gov (United States)

    Meyer, Austin G; Wilke, Claus O

    2015-10-06

    Protein structure acts as a general constraint on the evolution of viral proteins. One widely recognized structural constraint explaining evolutionary variation among sites is the relative solvent accessibility (RSA) of residues in the folded protein. In influenza virus, the distance from functional sites has been found to explain an additional portion of the evolutionary variation in the external antigenic proteins. However, to what extent RSA and distance from a reference site in the protein can be used more generally to explain protein adaptation in other viruses and in the different proteins of any given virus remains an open question. To address this question, we have carried out an analysis of the distribution and structural predictors of site-wise dN/dS in HIV-1. Our results indicate that the distribution of dN/dS in HIV follows a smooth gamma distribution, with no special enrichment or depletion of sites with dN/dS at or above one. The variation in dN/dS can be partially explained by RSA and distance from a reference site in the protein, but these structural constraints do not act uniformly among the different HIV-1 proteins. Structural constraints are highly predictive in just one of the three enzymes and one of three structural proteins in HIV-1. For these two proteins, the protease enzyme and the gp120 structural protein, structure explains between 30 and 40% of the variation in dN/dS. Finally, for the gp120 protein of the receptor-binding complex, we also find that glycosylation sites explain just 2% of the variation in dN/dS and do not explain gp120 evolution independently of either RSA or distance from the apical surface. © 2015 The Author(s).

  15. Opening of the TAR hairpin in the HIV-1 genome causes aberrant RNA dimerization and packaging

    Directory of Open Access Journals (Sweden)

    Das Atze T

    2012-07-01

    Full Text Available Abstract Background The TAR hairpin is present at both the 5′ and 3′ end of the HIV-1 RNA genome. The 5′ element binds the viral Tat protein and is essential for Tat-mediated activation of transcription. We recently observed that complete TAR deletion is allowed in the context of an HIV-1 variant that does not depend on this Tat-TAR axis for transcription. Mutations that open the 5′ stem-loop structure did however affect the leader RNA conformation and resulted in a severe replication defect. In this study, we set out to analyze which step of the HIV-1 replication cycle is affected by this conformational change of the leader RNA. Results We demonstrate that opening the 5′ TAR structure through a deletion in either side of the stem region caused aberrant dimerization and reduced packaging of the unspliced viral RNA genome. In contrast, truncation of the TAR hairpin through deletions in both sides of the stem did not affect RNA dimer formation and packaging. Conclusions These results demonstrate that, although the TAR hairpin is not essential for RNA dimerization and packaging, mutations in TAR can significantly affect these processes through misfolding of the relevant RNA signals.

  16. The RNA binding protein HuR does not interact directly with HIV-1 reverse transcriptase and does not affect reverse transcription in vitro

    Directory of Open Access Journals (Sweden)

    Gronenborn Angela M

    2010-05-01

    Full Text Available Abstract Background Lemay et al recently reported that the RNA binding protein HuR directly interacts with the ribonuclease H (RNase H domain of HIV-1 reverse transcriptase (RT and influences the efficiency of viral reverse transcription (Lemay et al., 2008, Retrovirology 5:47. HuR is a member of the embryonic lethal abnormal vision protein family and contains 3 RNA recognition motifs (RRMs that bind AU-rich elements (AREs. To define the structural determinants of the HuR-RT interaction and to elucidate the mechanism(s by which HuR influences HIV-1 reverse transcription activity in vitro, we cloned and purified full-length HuR as well as three additional protein constructs that contained the N-terminal and internal RRMs, the internal and C-terminal RRMs, or the C-terminal RRM only. Results All four HuR proteins were purified and characterized by biophysical methods. They are well structured and exist as monomers in solution. No direct protein-protein interaction between HuR and HIV-1 RT was detected using NMR titrations with 15N labeled HuR variants or the 15N labeled RNase H domain of HIV-1 RT. Furthermore, HuR did not significantly affect the kinetics of HIV-1 reverse transcription in vitro, even on RNA templates that contain AREs. Conclusions Our results suggest that HuR does not impact HIV-1 replication through a direct protein-protein interaction with the viral RT.

  17. Hit-and-run stimulation: a novel concept to reactivate latent HIV-1 infection without cytokine gene induction.

    Science.gov (United States)

    Wolschendorf, Frank; Duverger, Alexandra; Jones, Jennifer; Wagner, Frederic H; Huff, Jason; Benjamin, William H; Saag, Michael S; Niederweis, Michael; Kutsch, Olaf

    2010-09-01

    Current antiretroviral therapy (ART) efficiently controls HIV-1 replication but fails to eradicate the virus. Even after years of successful ART, HIV-1 can conceal itself in a latent state in long-lived CD4(+) memory T cells. From this latent reservoir, HIV-1 rebounds during treatment interruptions. Attempts to therapeutically eradicate this viral reservoir have yielded disappointing results. A major problem with previously utilized activating agents is that at the concentrations required for efficient HIV-1 reactivation, these stimuli trigger high-level cytokine gene expression (hypercytokinemia). Therapeutically relevant HIV-1-reactivating agents will have to trigger HIV-1 reactivation without the induction of cytokine expression. We present here a proof-of-principle study showing that this is a possibility. In a high-throughput screening effort, we identified an HIV-1-reactivating protein factor (HRF) secreted by the nonpathogenic bacterium Massilia timonae. In primary T cells and T-cell lines, HRF triggered a high but nonsustained peak of nuclear factor kappa B (NF-kappaB) activity. While this short NF-kappaB peak potently reactivated latent HIV-1 infection, it failed to induce gene expression of several proinflammatory NF-kappaB-dependent cellular genes, such as those for tumor necrosis factor alpha (TNF-alpha), interleukin-8 (IL-8), and gamma interferon (IFN-gamma). Dissociation of cellular and viral gene induction was achievable, as minimum amounts of Tat protein, synthesized following application of a short NF-kappaB pulse, triggered HIV-1 transactivation and subsequent self-perpetuated HIV-1 expression. In the absence of such a positive feedback mechanism, cellular gene expression was not sustained, suggesting that strategies modulating the NF-kappaB activity profile could be used to selectively trigger HIV-1 reactivation.

  18. A triclinic crystal structure of the carboxy-terminal domain of HIV-1 capsid protein with four molecules in the asymmetric unit reveals a novel packing interface

    International Nuclear Information System (INIS)

    Lampel, Ayala; Yaniv, Oren; Berger, Or; Bacharach, Eran; Gazit, Ehud; Frolow, Felix

    2013-01-01

    The triclinic structure of the HIV-1 capsid protein contains four molecules in the asymmetric unit that form a novel packing interface that could conceivably resemble an intermediate structure that is involved in the early steps of HIV-1 assembly. The Gag precursor is the major structural protein of the virion of human immunodeficiency virus-1 (HIV-1). Capsid protein (CA), a cleavage product of Gag, plays an essential role in virus assembly both in Gag-precursor multimerization and in capsid core formation. The carboxy-terminal domain (CTD) of CA contains 20 residues that are highly conserved across retroviruses and constitute the major homology region (MHR). Genetic evidence implies a role for the MHR in interactions between Gag precursors during the assembly of the virus, but the structural basis for this role remains elusive. This paper describes a novel triclinic structure of the HIV-1 CA CTD at 1.6 Å resolution with two canonical dimers of CA CTD in the asymmetric unit. The canonical dimers form a newly identified packing interface where interactions of four conserved MHR residues take place. This is the first structural indication that these MHR residues participate in the putative CTD–CTD interactions. These findings suggest that the molecules forming this novel interface resemble an intermediate structure that participates in the early steps of HIV-1 assembly. This interface may therefore provide a novel target for antiviral drugs

  19. Anti-cancer effect of HIV-1 viral protein R on doxorubicin resistant neuroblastoma.

    Directory of Open Access Journals (Sweden)

    Richard Y Zhao

    Full Text Available Several unique biological features of HIV-1 Vpr make it a potentially powerful agent for anti-cancer therapy. First, Vpr inhibits cell proliferation by induction of cell cycle G2 arrest. Second, it induces apoptosis through multiple mechanisms, which could be significant as it may be able to overcome apoptotic resistance exhibited by many cancerous cells, and, finally, Vpr selectively kills fast growing cells in a p53-independent manner. To demonstrate the potential utility of Vpr as an anti-cancer agent, we carried out proof-of-concept studies in vitro and in vivo. Results of our preliminary studies demonstrated that Vpr induces cell cycle G2 arrest and apoptosis in a variety of cancer types. Moreover, the same Vpr effects could also be detected in some cancer cells that are resistant to anti-cancer drugs such as doxorubicin (DOX. To further illustrate the potential value of Vpr in tumor growth inhibition, we adopted a DOX-resistant neuroblastoma model by injecting SK-N-SH cells into C57BL/6N and C57BL/6J-scid/scid mice. We hypothesized that Vpr is able to block cell proliferation and induce apoptosis regardless of the drug resistance status of the tumors. Indeed, production of Vpr via adenoviral delivery to neuroblastoma cells caused G2 arrest and apoptosis in both drug naïve and DOX-resistant cells. In addition, pre-infection or intratumoral injection of vpr-expressing adenoviral particles into neuroblastoma tumors in SCID mice markedly inhibited tumor growth. Therefore, Vpr could possibly be used as a supplemental viral therapeutic agent for selective inhibition of tumor growth in anti-cancer therapy especially when other therapies stop working.

  20. HIV-1 protein induced modulation of primary human osteoblast differentiation and function via a Wnt/β-catenin-dependent mechanism.

    LENUS (Irish Health Repository)

    Butler, Joseph S

    2013-02-01

    HIV infection is associated with metabolic bone disease resulting in bone demineralization and reduced bone mass. The molecular mechanisms driving this disease process have yet to be elucidated. Wnt\\/β-catenin signaling plays a key role in bone development and remodeling. We attempted to determine the effects of the HIV-1 protein, gp120, on Wnt\\/β-catenin signaling at an intracellular and transcriptional level in primary human osteoblasts (HOBs). This work, inclusive of experimental controls, was part of a greater project assessing the effects of a variety of different agents on Wnt\\/β-catenin signaling (BMC Musculoskelet Disord 2010;11:210).We examined the phenotypic effects of silencing and overexpressing the Wnt antagonist, Dickkopf-1 (Dkk1) in HOBs treated with gp120. HOBs exposed to gp120 displayed a significant reduction in alkaline phosphatase activity (ALP) activity and cell proliferation and increased cellular apoptosis over a 48 h time course. Immunocytochemistry demonstrated a significant reduction in intracytosolic and intranuclear β-catenin in response to HIV-1 protein exposure. These changes were associated with a reduction of TCF\\/LEF-mediated transcription, the transcriptional outcome of canonical Wnt β-catenin signaling. Silencing Dkk1 expression in HOBs exposed to gp120 resulted in increased ALP activity and cell proliferation, and decreased cellular apoptosis relative to scrambled control. Dkk1 overexpression exacerbated the inhibitory effect of gp120 on HOB function, with decreases in ALP activity and cell proliferation and increased cellular apoptosis relative to vector control. Wnt\\/β-catenin signaling plays a key regulatory role in HIV-associated bone loss, with Dkk1, aputative central mediator in this degenerative process. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 218-226, 2013.

  1. Possible roles of HIV-1 nucleocapsid protein in the specificity of proviral DNA synthesis and in its variability.

    Science.gov (United States)

    Lapadat-Tapolsky, M; Gabus, C; Rau, M; Darlix, J L

    1997-05-02

    Retroviral nucleocapsid (NC) protein is an integral part of the virion nucleocapsid where it coats the dimeric RNA genome. Due to its nucleic acid binding and annealing activities, NC protein directs the annealing of the tRNA primer to the primer binding site and greatly facilitates minus strand DNA elongation and transfer while protecting the nucleic acids against nuclease degradation. To understand the role of NCp7 in viral DNA synthesis, we examined the influence of NCp7 on self-primed versus primer-specific reverse transcription. The results show that HIV-1 NCp7 can extensively inhibit self-primed reverse transcription of viral and cellular RNAs while promoting primer-specific synthesis of proviral DNA. The role of NCp7 vis-a-vis the presence of mutations in the viral DNA during minus strand elongation was examined. NCp7 maximized the annealing between a cDNA(-) primer containing one to five consecutive errors and an RNA representing the 3' end of the genome. The ability of reverse transcriptase (RT) in the presence of NCp7 to subsequently extend the mutated primers depended upon the position of the mismatch within the primer:template complex. When the mutations were at the polymerisation site, primer extension by RT in the presence of NCp7 was very high, about 40% for one mismatch and 3% for five consecutive mismatches. Mutations within the DNA primer or at its 5' end had little effect on the extension of viral DNA by RT. Taken together these results indicate that NCp7 plays major roles in proviral DNA synthesis within the virion core due to its ability to promote prime-specific proviral DNA synthesis while concurrently inhibiting non-specific reverse transcription of viral and cellular RNAs. Moreover, the observation that NCp7 enhances the incorporation of mutations during minus strand DNA elongation favours the notion that NCp7 is a factor contributing to the high mutation rate of HIV-1.

  2. HIV-1 vaccines

    Science.gov (United States)

    Excler, Jean-Louis; Robb, Merlin L; Kim, Jerome H

    2014-01-01

    The development of a safe and effective preventive HIV-1 vaccine remains a public health priority. Despite scientific difficulties and disappointing results, HIV-1 vaccine clinical development has, for the first time, established proof-of-concept efficacy against HIV-1 acquisition and identified vaccine-associated immune correlates of risk. The correlate of risk analysis showed that IgG antibodies against the gp120 V2 loop correlated with decreased risk of HIV infection, while Env-specific IgA directly correlated with increased risk. The development of vaccine strategies such as improved envelope proteins formulated with potent adjuvants and DNA and vectors expressing mosaics, or conserved sequences, capable of eliciting greater breadth and depth of potentially relevant immune responses including neutralizing and non-neutralizing antibodies, CD4+ and CD8+ cell-mediated immune responses, mucosal immune responses, and immunological memory, is now proceeding quickly. Additional human efficacy trials combined with other prevention modalities along with sustained funding and international collaboration remain key to bring an HIV-1 vaccine to licensure. PMID:24637946

  3. Quantitative Characterization of Configurational Space Sampled by HIV-1 Nucleocapsid Using Solution NMR, X-ray Scattering and Protein Engineering.

    Science.gov (United States)

    Deshmukh, Lalit; Schwieters, Charles D; Grishaev, Alexander; Clore, G Marius

    2016-06-03

    Nucleic-acid-related events in the HIV-1 replication cycle are mediated by nucleocapsid, a small protein comprising two zinc knuckles connected by a short flexible linker and flanked by disordered termini. Combining experimental NMR residual dipolar couplings, solution X-ray scattering and protein engineering with ensemble simulated annealing, we obtain a quantitative description of the configurational space sampled by the two zinc knuckles, the linker and disordered termini in the absence of nucleic acids. We first compute the conformational ensemble (with an optimal size of three members) of an engineered nucleocapsid construct lacking the N- and C-termini that satisfies the experimental restraints, and then validate this ensemble, as well as characterize the disordered termini, using the experimental data from the full-length nucleocapsid construct. The experimental and computational strategy is generally applicable to multidomain proteins. Differential flexibility within the linker results in asymmetric motion of the zinc knuckles which may explain their functionally distinct roles despite high sequence identity. One of the configurations (populated at a level of ≈40 %) closely resembles that observed in various ligand-bound forms, providing evidence for conformational selection and a mechanistic link between protein dynamics and function. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. HIV-1 transgene expression in rats causes oxidant stress and alveolar epithelial barrier dysfunction

    Directory of Open Access Journals (Sweden)

    Jacob Barbara A

    2009-02-01

    Full Text Available Abstract Background HIV-infected individuals are at increased risk for acute and chronic airway disease even though there is no evidence that the virus can infect the lung epithelium. Although HIV-related proteins including gp120 and Tat can directly cause oxidant stress and cellular dysfunction, their effects in the lung are unknown. The goal of this study was to determine the effects of HIV-1 transgene expression in rats on alveolar epithelial barrier function. Alveolar epithelial barrier function was assessed by determining lung liquid clearance in vivo and alveolar epithelial monolayer permeability in vitro. Oxidant stress in the alveolar space was determined by measuring the glutathione redox couple by high performance liquid chromatography, and the expression and membrane localization of key tight junction proteins were assessed. Finally, the direct effects of the HIV-related proteins gp120 and Tat on alveolar epithelial barrier formation and tight junction protein expression were determined. Results HIV-1 transgene expression caused oxidant stress within the alveolar space and impaired epithelial barrier function even though there was no evidence of overt inflammation within the airways. The expression and membrane localization of the tight junction proteins zonula occludens-1 and occludin were decreased in alveolar epithelial cells from HIV-1 transgenic rats. Further, treating alveolar epithelial monolayers from wild type rats in vitro with recombinant gp120 or Tat for 24 hours reproduced many of the effects on zonula occludens-1 and occludin expression and membrane localization. Conclusion Taken together, these data indicate that HIV-related proteins cause oxidant stress and alter the expression of critical tight junction proteins in the alveolar epithelium, resulting in barrier dysfunction.

  5. Detection of HIV-1 and Human Proteins in Urinary Extracellular Vesicles from HIV+ Patients

    Directory of Open Access Journals (Sweden)

    Samuel I. Anyanwu

    2018-01-01

    Full Text Available Background. Extracellular vesicles (EVs are membrane bound, secreted by cells, and detected in bodily fluids, including urine, and contain proteins, RNA, and DNA. Our goal was to identify HIV and human proteins (HPs in urinary EVs from HIV+ patients and compare them to HIV− samples. Methods. Urine samples were collected from HIV+ (n=35 and HIV− (n=12 individuals. EVs were isolated by ultrafiltration and characterized using transmission electron microscopy, tandem mass spectrometry (LC/MS/MS, and nanoparticle tracking analysis (NTA. Western blots confirmed the presence of HIV proteins. Gene ontology (GO analysis was performed using FunRich and HIV Human Interaction database (HHID. Results. EVs from urine were 30–400 nm in size. More EVs were in HIV+ patients, P<0.05, by NTA. HIV+ samples had 14,475 HPs using LC/MS/MS, while only 111 were in HIV−. HPs in the EVs were of exosomal origin. LC/MS/MS showed all HIV+ samples contained at least one HIV protein. GO analysis showed differences in proteins between HIV+ and HIV− samples and more than 50% of the published HPs in the HHID interacted with EV HIV proteins. Conclusion. Differences in the proteomic profile of EVs from HIV+ versus HIV− samples were found. HIV and HPs in EVs could be used to detect infection and/or diagnose HIV disease syndromes.

  6. Probability weighted ensemble transfer learning for predicting interactions between HIV-1 and human proteins.

    Directory of Open Access Journals (Sweden)

    Suyu Mei

    Full Text Available Reconstruction of host-pathogen protein interaction networks is of great significance to reveal the underlying microbic pathogenesis. However, the current experimentally-derived networks are generally small and should be augmented by computational methods for less-biased biological inference. From the point of view of computational modelling, data scarcity, data unavailability and negative data sampling are the three major problems for host-pathogen protein interaction networks reconstruction. In this work, we are motivated to address the three concerns and propose a probability weighted ensemble transfer learning model for HIV-human protein interaction prediction (PWEN-TLM, where support vector machine (SVM is adopted as the individual classifier of the ensemble model. In the model, data scarcity and data unavailability are tackled by homolog knowledge transfer. The importance of homolog knowledge is measured by the ROC-AUC metric of the individual classifiers, whose outputs are probability weighted to yield the final decision. In addition, we further validate the assumption that only the homolog knowledge is sufficient to train a satisfactory model for host-pathogen protein interaction prediction. Thus the model is more robust against data unavailability with less demanding data constraint. As regards with negative data construction, experiments show that exclusiveness of subcellular co-localized proteins is unbiased and more reliable than random sampling. Last, we conduct analysis of overlapped predictions between our model and the existing models, and apply the model to novel host-pathogen PPIs recognition for further biological research.

  7. Intracellular high mobility group B1 protein (HMGB1) represses HIV-1 LTR-directed transcription in a promoter- and cell-specific manner

    International Nuclear Information System (INIS)

    Naghavi, Mojgan H.; Nowak, Piotr; Andersson, Jan; Soennerborg, Anders; Yang Huan; Tracey, Kevin J.; Vahlne, Anders

    2003-01-01

    We investigated whether the high mobility group B 1 (HMGB1), an abundant nuclear protein in all mammalian cells, affects HIV-1 transcription. Intracellular expression of human HMGB1 repressed HIV-1 gene expression in epithelial cells. This inhibitory effect of HMGB1 was caused by repression of long terminal repeat (LTR)-mediated transcription. Other viral promoters/enhancers, including simian virus 40 or cytomegalovirus, were not inhibited by HMGB1. In addition, HMGB1 inhibition of HIV-1 subtype C expression was dependent on the number of NFκB sites in the LTR region. The inhibitory effect of HMGB1 on viral gene expression observed in HeLa cells was confirmed by an upregulation of viral replication in the presence of antisense HMGB1 in monocytic cells. In contrast to what was found in HeLa cells and monocytic cells, endogenous HMGB1 expression did not affect HIV-1 replication in unstimulated Jurkat cells. Thus, intracellular HMGB1 affects HIV-1 LTR-directed transcription in a promoter- and cell-specific manner

  8. Immunization with Clinical HIV-1 Env Proteins Induces Broad Antibody Dependent Cellular Cytotoxicity-Mediating Antibodies in a Rabbit Vaccination Model.

    Science.gov (United States)

    Karlsson, Ingrid; Borggren, Marie; Jensen, Sanne Skov; Heyndrickx, Leo; Stewart-Jones, Guillaume; Scarlatti, Gabriella; Fomsgaard, Anders

    2017-11-17

    The induction of both neutralizing antibodies and non-neutralizing antibodies with effector functions, for example, antibody-dependent cellular cytotoxicity (ADCC), is desired in the search for effective vaccines against HIV-1. In the pursuit of novel immunogens capable of inducing an efficient antibody response, rabbits were immunized with selected antigens using different prime-boost strategies. We immunized 35 different groups of rabbits with Env antigens from clinical HIV-1 subtypes A and B, including immunization with DNA alone, protein alone, and DNA prime with protein boost. The rabbit sera were screened for ADCC activity using a GranToxiLux-based assay with human peripheral blood mononuclear cells as effector cells and CEM.NKR CCR5 cells coated with HIV-1 envelope as target cells. The groups with the highest ADCC activity were further characterized for cross-reactivity between HIV-1 subtypes. The immunogen inducing the most potent and broadest ADCC response was a trimeric gp140. The ADCC activity was highest against the HIV-1 subtype corresponding to the immunogen. The ADCC activity did not necessarily reflect neutralizing activity in the pseudovirus-TZMbl assay, but there was an overall correlation between the two antiviral activities. We present a rabbit vaccination model and an assay suitable for screening HIV-1 vaccine candidates for the induction of ADCC-mediating antibodies in addition to neutralizing antibodies. The antigens and/or immunization strategies capable of inducing antibodies with ADCC activity did not necessarily induce neutralizing activity and vice versa. Nevertheless, we identified vaccine candidates that were able to concurrently induce both types of responses and that had ADCC activity that was cross-reactive between different subtypes. When searching for an effective vaccine candidate, it is important to evaluate the antibody response using a model and an assay measuring the desired function.

  9. Heterologous prime-boost regimens with a recombinant chimpanzee adenoviral vector and adjuvanted F4 protein elicit polyfunctional HIV-1-specific T-Cell responses in macaques.

    Science.gov (United States)

    Lorin, Clarisse; Vanloubbeeck, Yannick; Baudart, Sébastien; Ska, Michaël; Bayat, Babak; Brauers, Geoffroy; Clarinval, Géraldine; Donner, Marie-Noëlle; Marchand, Martine; Koutsoukos, Marguerite; Mettens, Pascal; Cohen, Joe; Voss, Gerald

    2015-01-01

    HIV-1-specific CD4+ and CD8+ T lymphocytes are important for HIV-1 replication control. F4/AS01 consists of F4 recombinant fusion protein (containing clade B Gag/p24, Pol/RT, Nef and Gag/p17) formulated in AS01 Adjuvant System, and was shown to induce F4-specific polyfunctional CD4+ T-cell responses in humans. While replication-incompetent recombinant HIV-1/SIV antigen-expressing human adenoviral vectors can elicit high-frequency antigen-specific CD8+ T-cell responses, their use is hampered by widespread pre-existing immunity to human serotypes. Non-human adenovirus serotypes associated with lower prevalence may offer an alternative strategy. We evaluated the immunogenicity of AdC7-GRN ('A'), a recombinant chimpanzee adenovirus type 7 vector expressing clade B Gag, RT and Nef, and F4/AS01 ('P'), when delivered intramuscularly in homologous (PP or AA) and heterologous (AAPP or PPAA) prime-boost regimens, in macaques and mice. Vaccine-induced HIV-1-antigen-specific T cells in peripheral blood (macaques), liver, spleen, and intestinal and genital mucosa (mice) were characterized by intracellular cytokine staining. Vaccine-specific IgG antibodies (macaques) were detected using ELISA. In macaques, only the heterologous prime-boost regimens induced polyfunctional, persistent and balanced CD4+ and CD8+ T-cell responses specific to each HIV-1 vaccine antigen. AdC7-GRN priming increased the polyfunctionality of F4/AS01-induced CD4+ T cells. Approximately 50% of AdC7-GRN-induced memory CD8+ T cells exhibited an effector-memory phenotype. HIV-1-specific antibodies were detected with each regimen. In mice, antigen-specific CD4+ and CD8+ T-cell responses were detected in the mucosal and systemic anatomical compartments assessed. When administered in heterologous prime-boost regimens, AdC7-GRN and F4/AS01 candidate vaccines acted complementarily in inducing potent and persistent peripheral blood HIV-1-specific CD4+ and CD8+ T-cell responses and antibodies in macaques. Besides

  10. HDAC inhibition induces HIV-1 protein and enables immune-based clearance following latency reversal

    DEFF Research Database (Denmark)

    Wu, Guoxin; Swanson, Michael; Talla, Aarthi

    2017-01-01

    Promising therapeutic approaches for eradicating HIV include transcriptional activation of provirus from latently infected cells using latency-reversing agents (LRAs) and immune-mediated clearance to purge reservoirs. Accurate detection of cells capable of producing viral antigens and virions......, and the measurement of clearance of infected cells, is essential to assessing therapeutic efficacy. Here, we apply enhanced methodology extending the sensitivity limits for the rapid detection of subfemtomolar HIV gag p24 capsid protein in CD4+ T cells from ART-suppressed HIV+ individuals, and we show viral protein...... induction following treatment with LRAs. Importantly, we demonstrate that clinical administration of histone deacetylase inhibitors (HDACis; vorinostat and panobinostat) induced HIV gag p24, and ex vivo stimulation produced sufficient viral antigen to elicit immune-mediated cell killing using anti-gp120/CD3...

  11. Dynamics of Linker Residues Modulate the Nucleic Acid Binding Properties of the HIV-1 Nucleocapsid Protein Zinc Fingers

    Science.gov (United States)

    Zargarian, Loussiné; Tisné, Carine; Barraud, Pierre; Xu, Xiaoqian; Morellet, Nelly; René, Brigitte; Mély, Yves; Fossé, Philippe; Mauffret, Olivier

    2014-01-01

    The HIV-1 nucleocapsid protein (NC) is a small basic protein containing two zinc fingers (ZF) separated by a short linker. It is involved in several steps of the replication cycle and acts as a nucleic acid chaperone protein in facilitating nucleic acid strand transfers occurring during reverse transcription. Recent analysis of three-dimensional structures of NC-nucleic acids complexes established a new property: the unpaired guanines targeted by NC are more often inserted in the C-terminal zinc finger (ZF2) than in the N-terminal zinc finger (ZF1). Although previous NMR dynamic studies were performed with NC, the dynamic behavior of the linker residues connecting the two ZF domains remains unclear. This prompted us to investigate the dynamic behavior of the linker residues. Here, we collected 15N NMR relaxation data and used for the first time data at several fields to probe the protein dynamics. The analysis at two fields allows us to detect a slow motion occurring between the two domains around a hinge located in the linker at the G35 position. However, the amplitude of motion appears limited in our conditions. In addition, we showed that the neighboring linker residues R29, A30, P31, R32, K33 displayed restricted motion and numerous contacts with residues of ZF1. Our results are fully consistent with a model in which the ZF1-linker contacts prevent the ZF1 domain to interact with unpaired guanines, whereas the ZF2 domain is more accessible and competent to interact with unpaired guanines. In contrast, ZF1 with its large hydrophobic plateau is able to destabilize the double-stranded regions adjacent to the guanines bound by ZF2. The linker residues and the internal dynamics of NC regulate therefore the different functions of the two zinc fingers that are required for an optimal chaperone activity. PMID:25029439

  12. Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells

    Directory of Open Access Journals (Sweden)

    Q. Sun

    2012-10-01

    Full Text Available The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3 can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.

  13. Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells

    International Nuclear Information System (INIS)

    Sun, Q.; Xiong, J.; Lu, J.; Xu, S.; Li, Y.; Zhong, X.P.; Gao, G.K.; Liu, H.Q.

    2012-01-01

    The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy

  14. Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Q. [Department of Hyperbaric Medicine, No. 401 Hospital of PLA, Qingdao (China); Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China); Xiong, J. [Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China); Lu, J. [Office of Medical Education, Training Department, Second Military Medical University, Shanghai (China); Xu, S. [Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China); Li, Y. [State Food and Drug Administration of China,Huangdao Branch, Qingdao (China); Zhong, X.P.; Gao, G.K. [Department of Hyperbaric Medicine, No. 401 Hospital of PLA, Qingdao (China); Liu, H.Q. [2Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China)

    2012-06-22

    The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.

  15. Cytoplasmic HIV-1 RNA is mainly transported by diffusion in the presence or absence of Gag protein

    DEFF Research Database (Denmark)

    Chen, Jianbo; Grunwald, David; Sardo, Luca

    2014-01-01

    . In this report, we visualized HIV-1 RNA and monitored its movement in the cytoplasm by using single-molecule tracking. We observed that most of the HIV-1 RNA molecules move in a nondirectional, random-walk manner, which does not require an intact cytoskeletal structure, and that the mean-squared distance...... traveled by the RNA increases linearly with time, indicative of diffusive movement. We also observed that a single HIV-1 RNA molecule can move at various speeds when traveling through the cytoplasm, indicating that its movement is strongly affected by the immediate environment. To examine the effect of Gag...

  16. Cardiac Dysfunction in HIV-1 Transgenic Mouse: Role of Stress and BAG3.

    Science.gov (United States)

    Cheung, Joseph Y; Gordon, Jennifer; Wang, JuFang; Song, Jianliang; Zhang, Xue-Qian; Tilley, Douglas G; Gao, Erhe; Koch, Walter J; Rabinowitz, Joseph; Klotman, Paul E; Khalili, Kamel; Feldman, Arthur M

    2015-08-01

    Since highly active antiretroviral therapy improved long-term survival of acquired immunodeficiency syndrome (AIDS) patients, AIDS cardiomyopathy has become an increasingly relevant clinical problem. We used human immunodeficiency virus (HIV)-1 transgenic (Tg26) mouse to explore molecular mechanisms of AIDS cardiomyopathy. Tg26 mice had significantly lower left ventricular (LV) mass and smaller end-diastolic and end-systolic LV volumes. Under basal conditions, cardiac contractility and relaxation and single myocyte contraction dynamics were not different between wild-type (WT) and Tg26 mice. Ten days after open heart surgery, contractility and relaxation remained significantly depressed in Tg26 hearts, suggesting that Tg26 mice did not tolerate surgical stress well. To simulate heart failure in which expression of Bcl2-associated athanogene 3 (BAG3) is reduced, we down-regulated BAG3 by small hairpin ribonucleic acid in WT and Tg26 hearts. BAG3 down-regulation significantly reduced contractility in Tg26 hearts. BAG3 overexpression rescued contractile abnormalities in myocytes expressing the HIV-1 protein Tat. We conclude: (i) Tg26 mice exhibit normal contractile function at baseline; (ii) Tg26 mice do not tolerate surgical stress well; (iii) BAG3 down-regulation exacerbated cardiac dysfunction in Tg26 mice; (iv) BAG3 overexpression rescued contractile abnormalities in myocytes expressing HIV-1 protein Tat; and (v) BAG3 may occupy a role in pathogenesis of AIDS cardiomyopathy. © 2015 Wiley Periodicals, Inc.

  17. Fusion proteins of HIV-1 envelope glycoprotein gp120 with CD4-induced antibodies showed enhanced binding to CD4 and CD4 binding site antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Weizao, E-mail: chenw3@mail.nih.gov [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); Feng, Yang [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); Wang, Yanping [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); The Basic Research Program, Science Applications International Corporation-Frederick, Inc., National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); Zhu, Zhongyu; Dimitrov, Dimiter S. [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States)

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Some recombinant HIV-1 gp120s do not preserve their conformations on gp140s. Black-Right-Pointing-Pointer We hypothesize that CD4i antibodies could induce conformational changes in gp120. Black-Right-Pointing-Pointer CD4i antibodies enhance binding of CD4 and CD4bs antibodies to gp120. Black-Right-Pointing-Pointer CD4i antibody-gp120 fusion proteins could have potential as vaccine immunogens. -- Abstract: Development of successful AIDS vaccine immunogens continues to be a major challenge. One of the mechanisms by which HIV-1 evades antibody-mediated neutralizing responses is the remarkable conformational flexibility of its envelope glycoprotein (Env) gp120. Some recombinant gp120s do not preserve their conformations on gp140s and functional viral spikes, and exhibit decreased recognition by CD4 and neutralizing antibodies. CD4 binding induces conformational changes in gp120 leading to exposure of the coreceptor-binding site (CoRbs). In this study, we test our hypothesis that CD4-induced (CD4i) antibodies, which target the CoRbs, could also induce conformational changes in gp120 leading to better exposed conserved neutralizing antibody epitopes including the CD4-binding site (CD4bs). We found that a mixture of CD4i antibodies with gp120 only weakly enhanced CD4 binding. However, such interactions in single-chain fusion proteins resulted in gp120 conformations which bound to CD4 and CD4bs antibodies better than the original or mutagenically stabilized gp120s. Moreover, the two molecules in the fusion proteins synergized with each other in neutralizing HIV-1. Therefore, fusion proteins of gp120 with CD4i antibodies could have potential as components of HIV-1 vaccines and inhibitors of HIV-1 entry, and could be used as reagents to explore the conformational flexibility of gp120 and mechanisms of entry and immune evasion.

  18. ReFlexIn: a flexible receptor protein-ligand docking scheme evaluated on HIV-1 protease.

    Directory of Open Access Journals (Sweden)

    Simon Leis

    Full Text Available For many targets of pharmaceutical importance conformational changes of the receptor protein are relevant during the ligand binding process. A new docking approach, ReFlexIn (Receptor Flexibility by Interpolation, that combines receptor flexibility with the computationally efficient potential grid representation of receptor molecules has been evaluated on the retroviral HIV-1 (Human Immunodeficiency Virus 1 protease system. An approximate inclusion of receptor flexibility is achieved by using interpolation between grid representations of individual receptor conformations. For the retroviral protease the method was tested on an ensemble of protease structures crystallized in the presence of different ligands and on a set of structures obtained from morphing between the unbound and a ligand-bound protease structure. Docking was performed on ligands known to bind to the protease and several non-binders. For the binders the ReFlexIn method yielded in almost all cases ligand placements in similar or closer agreement with experiment than docking to any of the ensemble members without degrading the discrimination with respect to non-binders. The improved docking performance compared to docking to rigid receptors allows for systematic virtual screening applications at very small additional computational cost.

  19. Molecular dynamics simulations of conformation changes of HIV-1 regulatory protein on graphene

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Daohui; Li, Libo; He, Daohang; Zhou, Jian, E-mail: jianzhou@scut.edu.cn

    2016-07-30

    Graphical abstract: Preferential adsorption of Vpr13-33 on graphene accompanied by early conformational change from α-helix to β-sheet structures was observed by molecular simulations. This work presents the molecular mechanism of graphene-induced peptide conformational alteration and sheds light on developing graphene-based materials to inhibit HIV. - Highlights: • Graphene induced early structural transition of Vpr13-33 is studied by MD simulations. • Both π-π stacking and hydrophobic interactions orchestrate the peptide adsorption. • Vpr has an increased propensity of β-sheet content on graphene surface. • To develop graphene-based materials to inhibit HIV is possible. - Abstract: The fragment of viral protein R (Vpr), Vpr13-33, plays an important role in regulating nuclear importing of HIV genes through channel formation in which it adopts a leucine-zipper-like alpha-helical conformation. A recent experimental study reported that helical Vpr13-33 would transform to β-sheet or random coil structures and aggregate on the surface of graphene or graphene oxide through hydrophobic interactions. Due to experimental limitations, however, there is still a considerable lack of understanding on the adsorption dynamics at the early stage of the conformational transition at water-graphene interface and the underlying driving force at molecular level. In this study, atomistic molecular dynamics simulations were used to explore the conformation transition phenomena. Vpr13-33 kept α-helical structure in solution, but changed to β-sheet structure when strongly adsorbed onto graphene. Preferential adsorption of Vpr13-33 on graphene is dominated by hydrophobic interactions. The cluster analysis identified the most significant populated conformation and the early stage of structure conversion from α-helical to β-sheet was found, but the full β-sheet propagation was not observed. Free energy landscape analysis further complemented the transformation analysis of

  20. Molecular dynamics simulations of conformation changes of HIV-1 regulatory protein on graphene

    International Nuclear Information System (INIS)

    Zhao, Daohui; Li, Libo; He, Daohang; Zhou, Jian

    2016-01-01

    Graphical abstract: Preferential adsorption of Vpr13-33 on graphene accompanied by early conformational change from α-helix to β-sheet structures was observed by molecular simulations. This work presents the molecular mechanism of graphene-induced peptide conformational alteration and sheds light on developing graphene-based materials to inhibit HIV. - Highlights: • Graphene induced early structural transition of Vpr13-33 is studied by MD simulations. • Both π-π stacking and hydrophobic interactions orchestrate the peptide adsorption. • Vpr has an increased propensity of β-sheet content on graphene surface. • To develop graphene-based materials to inhibit HIV is possible. - Abstract: The fragment of viral protein R (Vpr), Vpr13-33, plays an important role in regulating nuclear importing of HIV genes through channel formation in which it adopts a leucine-zipper-like alpha-helical conformation. A recent experimental study reported that helical Vpr13-33 would transform to β-sheet or random coil structures and aggregate on the surface of graphene or graphene oxide through hydrophobic interactions. Due to experimental limitations, however, there is still a considerable lack of understanding on the adsorption dynamics at the early stage of the conformational transition at water-graphene interface and the underlying driving force at molecular level. In this study, atomistic molecular dynamics simulations were used to explore the conformation transition phenomena. Vpr13-33 kept α-helical structure in solution, but changed to β-sheet structure when strongly adsorbed onto graphene. Preferential adsorption of Vpr13-33 on graphene is dominated by hydrophobic interactions. The cluster analysis identified the most significant populated conformation and the early stage of structure conversion from α-helical to β-sheet was found, but the full β-sheet propagation was not observed. Free energy landscape analysis further complemented the transformation analysis of

  1. Recombinant TAT-BMI-1 fusion protein induces ex vivo expansion of human umbilical cord blood-derived hematopoietic stem cells.

    Science.gov (United States)

    Codispoti, Bruna; Rinaldo, Nicola; Chiarella, Emanuela; Lupia, Michela; Spoleti, Cristina Barbara; Marafioti, Maria Grazia; Aloisio, Annamaria; Scicchitano, Stefania; Giordano, Marco; Nappo, Giovanna; Lucchino, Valeria; Moore, Malcolm A S; Zhou, Pengbo; Mesuraca, Maria; Bond, Heather Mandy; Morrone, Giovanni

    2017-07-04

    Transplantation of hematopoietic stem cells (HSCs) is a well-established therapeutic approach for numerous disorders. HSCs are typically derived from bone marrow or peripheral blood after cytokine-induced mobilization. Umbilical cord blood (CB) represents an appealing alternative HSC source, but the small amounts of the individual CB units have limited its applications. The availability of strategies for safe ex vivo expansion of CB-derived HSCs (CB-HSCs) may allow to extend the use of these cells in adult patients and to avoid the risk of insufficient engraftment or delayed hematopoietic recovery.Here we describe a system for the ex vivo expansion of CB-HSCs based on their transient exposure to a recombinant TAT-BMI-1 chimeric protein. BMI-1 belongs to the Polycomb family of epigenetic modifiers and is recognized as a central regulator of HSC self-renewal. Recombinant TAT-BMI-1 produced in bacteria was able to enter the target cells via the HIV TAT-derived protein transduction peptide covalently attached to BMI-1, and conserved its biological activity. Treatment of CB-CD34+ cells for 3 days with repeated addition of 10 nM purified TAT-BMI-1 significantly enhanced total cell expansion as well as that of primitive hematopoietic progenitors in culture. Importantly, TAT-BMI-1-treated CB-CD34+ cells displayed a consistently higher rate of multi-lineage long-term repopulating activity in primary and secondary xenotransplants in immunocompromised mice. Thus, recombinant TAT-BMI-1 may represent a novel, effective reagent for ex vivo expansion of CB-HSC for therapeutic purposes.

  2. Conformational preferences of a chimeric peptide HIV-1 immunogen from the C4-V3 domains of gp120 envelope protein of HIV-1 CAN0A based on solution NMR: comparison to a related immunogenic peptide from HIV-1 RF.

    Science.gov (United States)

    Vu, H M; de Lorimier, R; Moody, M A; Haynes, B F; Spicer, L D

    1996-04-23

    A critical problem to overcome on HIV vaccine design is the variability among HIV strains. One strategy to solve this problem is the construction of multicomponent immunogens reflective of common HIV motifs. Currently, it is not known if these motifs should be based primarily on amino acid sequence or higher-order structure of the viral proteins of a combination of the two. In this paper, we report NMR-derived solution conformations for a sympathetic peptide taken from the C4 and V3 domains of HIV-1 CAN0A gp120 envelope protein. This peptide, designated T1-SP10CAN0(A), is compared to a recently reported C4-V3 peptide. T1-SP10RF(A) from the HIV-1 RF strain [de Lorimier et al. (1994) Biochemistry 33, 2055-2062], in terms of conformational features and immune responses in mice [Haynes et al. (1995) AIDS Res. Hum. Retroviruses 11, 211-221]. The T1 segment of 16 amino acids from the gp120 C4 domain is identical in both peptides and exhibits nascent helical character. The SP10 region, taken from the gp120 V3 loop, differs from that of T1-SP10RF(A) in both sequence and conformations. A reverse turn is observed at the conserved GPGX sequence. The rest of the Sp10 domain is extended with the exception of the last three residues which show evidence for a helical arrangement. Modeling of the turn region of the T1-SP10CAN0(A) peptide shows exposure of a continuous apolar stretch of side chains similar to that reported in the crystal structure of a V3 peptide from HIV-1 MN complexed with a monoclonal antibody [Rini et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6325-6329]. this hydrophobic patch is interrupted by a charged Lys residue in the T1-SP10RF(A) peptide. This observation suggests that the HIV-1 CAN0A and HIV-1 RF C4-V3 peptides can induce widely different anti-HIV antibodies. consistent with immunogenic results.

  3. Excision of HIV-1 proviral DNA by recombinant cell permeable tre-recombinase.

    Directory of Open Access Journals (Sweden)

    Lakshmikanth Mariyanna

    Full Text Available Over the previous years, comprehensive studies on antiretroviral drugs resulted in the successful introduction of highly active antiretroviral therapy (HAART into clinical practice for treatment of HIV/AIDS. However, there is still need for new therapeutic approaches, since HAART cannot eradicate HIV-1 from the infected organism and, unfortunately, can be associated with long-term toxicity and the development of drug resistance. In contrast, novel gene therapy strategies may have the potential to reverse the infection by eradicating HIV-1. For example, expression of long terminal repeat (LTR-specific recombinase (Tre-recombinase has been shown to result in chromosomal excision of proviral DNA and, in consequence, in the eradication of HIV-1 from infected cell cultures. However, the delivery of Tre-recombinase currently depends on the genetic manipulation of target cells, a process that is complicating such therapeutic approaches and, thus, might be undesirable in a clinical setting. In this report we demonstrate that E.coli expressed Tre-recombinases, tagged either with the protein transduction domain (PTD from the HIV-1 Tat trans-activator or the translocation motif (TLM of the Hepatitis B virus PreS2 protein, were able to translocate efficiently into cells and showed significant recombination activity on HIV-1 LTR sequences. Tre activity was observed using episomal and stable integrated reporter constructs in transfected HeLa cells. Furthermore, the TLM-tagged enzyme was able to excise the full-length proviral DNA from chromosomal integration sites of HIV-1-infected HeLa and CEM-SS cells. The presented data confirm Tre-recombinase activity on integrated HIV-1 and provide the basis for the non-genetic transient application of engineered recombinases, which may be a valuable component of future HIV eradication strategies.

  4. Elevation of intact and proteolytic fragments of acute phase proteins constitutes the earliest systemic antiviral response in HIV-1 infection.

    Directory of Open Access Journals (Sweden)

    Holger B Kramer

    2010-05-01

    Full Text Available The earliest immune responses activated in acute human immunodeficiency virus type 1 infection (AHI exert a critical influence on subsequent virus spread or containment. During this time frame, components of the innate immune system such as macrophages and DCs, NK cells, beta-defensins, complement and other anti-microbial factors, which have all been implicated in modulating HIV infection, may play particularly important roles. A proteomics-based screen was performed on a cohort from whom samples were available at time points prior to the earliest positive HIV detection. The ability of selected factors found to be elevated in the plasma during AHI to inhibit HIV-1 replication was analyzed using in vitro PBMC and DC infection models. Analysis of unique plasma donor panels spanning the eclipse and viral expansion phases revealed very early alterations in plasma proteins in AHI. Induction of acute phase protein serum amyloid A (A-SAA occurred as early as 5-7 days prior to the first detection of plasma viral RNA, considerably prior to any elevation in systemic cytokine levels. Furthermore, a proteolytic fragment of alpha-1-antitrypsin (AAT, termed virus inhibitory peptide (VIRIP, was observed in plasma coincident with viremia. Both A-SAA and VIRIP have anti-viral activity in vitro and quantitation of their plasma levels indicated that circulating concentrations are likely to be within the range of their inhibitory activity. Our results provide evidence for a first wave of host anti-viral defense occurring in the eclipse phase of AHI prior to systemic activation of other immune responses. Insights gained into the mechanism of action of acute-phase reactants and other innate molecules against HIV and how they are induced could be exploited for the future development of more efficient prophylactic vaccine strategies.

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

    Science.gov (United States)

    Li, Ge; Park, Hyeon U; Liang, Dong; Zhao, Richard Y

    2010-07-07

    Cell 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. To 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. These 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.

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

    Directory of Open Access Journals (Sweden)

    Liang Dong

    2010-07-01

    Full Text Available Abstract Background Cell 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. Results To 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. Conclusions These 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.

  7. Biocompatible anionic polymeric microspheres as priming delivery system for effetive HIV/AIDS Tat-based vaccines.

    Directory of Open Access Journals (Sweden)

    Fausto Titti

    Full Text Available Here we describe a prime-boost regimen of vaccination in Macaca fascicularis that combines priming with novel anionic microspheres designed to deliver the biologically active HIV-1 Tat protein and boosting with Tat in Alum. This regimen of immunization modulated the IgG subclass profile and elicited a balanced Th1-Th2 type of humoral and cellular responses. Remarkably, following intravenous challenge with SHIV89.6Pcy243, vaccinees significantly blunted acute viremia, as compared to control monkeys, and this control was associated with significantly lower CD4+ T cell depletion rate during the acute phase of infection and higher ability to resume the CD4+ T cell counts in the post-acute and chronic phases of infection. The long lasting control of viremia was associated with the persistence of high titers anti-Tat antibodies whose profile clearly distinguished vaccinees in controllers and viremics. Controllers, as opposed to vaccinated and viremic cynos, exhibited significantly higher pre-challenge antibody responses to peptides spanning the glutamine-rich and the RGD-integrin-binding regions of Tat. Finally, among vaccinees, titers of anti-Tat IgG1, IgG3 and IgG4 subclasses had a significant association with control of viremia in the acute and post-acute phases of infection. Altogether these findings indicate that the Tat/H1D/Alum regimen of immunization holds promise for next generation vaccines with Tat protein or other proteins for which maintenance of the native conformation and activity are critical for optimal immunogenicity. Our results also provide novel information on the role of anti-Tat responses in the prevention of HIV pathogenesis and for the design of new vaccine candidates.

  8. Novel PI3K/Akt Inhibitors Screened by the Cytoprotective Function of Human Immunodeficiency Virus Type 1 Tat

    Science.gov (United States)

    Kim, Dong-Hyun; Kim, Baek

    2011-01-01

    The PI3K/Akt pathway regulates various stress-related cellular responses such as cell survival, cell proliferation, metabolism and protein synthesis. Many cancer cell types display the activation of this pathway, and compounds inhibiting this cell survival pathway have been extensively evaluated as anti-cancer agents. In addition to cancers, several human viruses, such as HTLV, HPV, HCV and HIV-1, also modulate this pathway, presumably in order to extend the life span of the infected target cells for productive viral replication. The expression of HIV-1 Tat protein exhibited the cytoprotective effect in macrophages and a human microglial cell line by inhibiting the negative regulator of this pathway, PTEN. This cytoprotective effect of HIV-1 appears to contribute to the long-term survival and persistent HIV-1 production in human macrophage reservoirs. In this study we exploited the PI3K/Akt dependent cytoprotective effect of Tat-expressing CHME5 cells. We screened a collection of compounds known to modulate inflammation, and identified three novel compounds: Lancemaside A, Compound K and Arctigenin that abolished the cytoprotective phenotype of Tat-expressing CHME5 cells. All three compounds antagonized the kinase activity of Akt. Further detailed signaling studies revealed that each of these three compounds targeted different steps of the PI3K/Akt pathway. Arctigenin regulates the upstream PI3K enzyme from converting PIP2 to PIP3. Lancemaside A1 inhibited the movement of Akt to the plasma membrane, a critical step for Akt activation. Compound K inhibited Akt phosphorylation. This study supports that Tat-expressing CHME5 cells are an effective model system for screening novel PI3K/Akt inhibitors. PMID:21765914

  9. Novel PI3K/Akt inhibitors screened by the cytoprotective function of human immunodeficiency virus type 1 Tat.

    Directory of Open Access Journals (Sweden)

    Yuri Kim

    Full Text Available The PI3K/Akt pathway regulates various stress-related cellular responses such as cell survival, cell proliferation, metabolism and protein synthesis. Many cancer cell types display the activation of this pathway, and compounds inhibiting this cell survival pathway have been extensively evaluated as anti-cancer agents. In addition to cancers, several human viruses, such as HTLV, HPV, HCV and HIV-1, also modulate this pathway, presumably in order to extend the life span of the infected target cells for productive viral replication. The expression of HIV-1 Tat protein exhibited the cytoprotective effect in macrophages and a human microglial cell line by inhibiting the negative regulator of this pathway, PTEN. This cytoprotective effect of HIV-1 appears to contribute to the long-term survival and persistent HIV-1 production in human macrophage reservoirs. In this study we exploited the PI3K/Akt dependent cytoprotective effect of Tat-expressing CHME5 cells. We screened a collection of compounds known to modulate inflammation, and identified three novel compounds: Lancemaside A, Compound K and Arctigenin that abolished the cytoprotective phenotype of Tat-expressing CHME5 cells. All three compounds antagonized the kinase activity of Akt. Further detailed signaling studies revealed that each of these three compounds targeted different steps of the PI3K/Akt pathway. Arctigenin regulates the upstream PI3K enzyme from converting PIP2 to PIP3. Lancemaside A1 inhibited the movement of Akt to the plasma membrane, a critical step for Akt activation. Compound K inhibited Akt phosphorylation. This study supports that Tat-expressing CHME5 cells are an effective model system for screening novel PI3K/Akt inhibitors.

  10. The yeast Ty3 retrotransposon contains a 5'-3' bipartite primer-binding site and encodes nucleocapsid protein NCp9 functionally homologous to HIV-1 NCp7.

    Science.gov (United States)

    Gabus, C; Ficheux, D; Rau, M; Keith, G; Sandmeyer, S; Darlix, J L

    1998-08-17

    Retroviruses, including HIV-1 and the distantly related yeast retroelement Ty3, all encode a nucleoprotein required for virion structure and replication. During an in vitro comparison of HIV-1 and Ty3 nucleoprotein function in RNA dimerization and cDNA synthesis, we discovered a bipartite primer-binding site (PBS) for Ty3 composed of sequences located at opposite ends of the genome. Ty3 cDNA synthesis requires the 3' PBS for primer tRNAiMet annealing to the genomic RNA, and the 5' PBS, in cis or in trans, as the reverse transcription start site. Ty3 RNA alone is unable to dimerize, but formation of dimeric tRNAiMet bound to the PBS was found to direct dimerization of Ty3 RNA-tRNAiMet. Interestingly, HIV-1 nucleocapsid protein NCp7 and Ty3 NCp9 were interchangeable using HIV-1 and Ty3 RNA template-primer systems. Our findings impact on the understanding of non-canonical reverse transcription as well as on the use of Ty3 systems to screen for anti-NCp7 drugs.

  11. Inhibition of HIV Replication by Cyclic and Hairpin PNAs Targeting the HIV-1 TAR RNA Loop

    Science.gov (United States)

    Upert, Gregory; Di Giorgio, Audrey; Upadhyay, Alok; Manvar, Dinesh; Pandey, Nootan; Pandey, Virendra N.; Patino, Nadia

    2012-01-01

    Human immunodeficiency virus-1 (HIV-1) replication and gene expression entails specific interaction of the viral protein Tat with its transactivation responsive element (TAR), to form a highly stable stem-bulge-loop structure. Previously, we described triphenylphosphonium (TPP) cation-based vectors that efficiently deliver nucleotide analogs (PNAs) into the cytoplasm of cells. In particular, we showed that the TPP conjugate of a linear 16-mer PNA targeting the apical stem-loop region of TAR impedes Tat-mediated transactivation of the HIV-1 LTR in vitro and also in cell culture systems. In this communication, we conjugated TPP to cyclic and hairpin PNAs targeting the loop region of HIV-1 TAR and evaluated their antiviral efficacy in a cell culture system. We found that TPP-cyclic PNAs containing only 8 residues, showed higher antiviral potency compared to hairpin PNAs of 12 or 16 residues. We further noted that the TPP-conjugates of the 8-mer cyclic PNA as well as the 16-mer linear PNA displayed similar antiviral efficacy. However, cyclic PNAs were shown to be highly specific to their target sequences. This communication emphasizes on the importance of small constrained cyclic PNAs over both linear and hairpin structures for targeting biologically relevant RNA hairpins. PMID:23029603

  12. Inhibition of HIV Replication by Cyclic and Hairpin PNAs Targeting the HIV-1 TAR RNA Loop

    Directory of Open Access Journals (Sweden)

    Gregory Upert

    2012-01-01

    Full Text Available Human immunodeficiency virus-1 (HIV-1 replication and gene expression entails specific interaction of the viral protein Tat with its transactivation responsive element (TAR, to form a highly stable stem-bulge-loop structure. Previously, we described triphenylphosphonium (TPP cation-based vectors that efficiently deliver nucleotide analogs (PNAs into the cytoplasm of cells. In particular, we showed that the TPP conjugate of a linear 16-mer PNA targeting the apical stem-loop region of TAR impedes Tat-mediated transactivation of the HIV-1 LTR in vitro and also in cell culture systems. In this communication, we conjugated TPP to cyclic and hairpin PNAs targeting the loop region of HIV-1 TAR and evaluated their antiviral efficacy in a cell culture system. We found that TPP-cyclic PNAs containing only 8 residues, showed higher antiviral potency compared to hairpin PNAs of 12 or 16 residues. We further noted that the TPP-conjugates of the 8-mer cyclic PNA as well as the 16-mer linear PNA displayed similar antiviral efficacy. However, cyclic PNAs were shown to be highly specific to their target sequences. This communication emphasizes on the importance of small constrained cyclic PNAs over both linear and hairpin structures for targeting biologically relevant RNA hairpins.

  13. ERManI (Endoplasmic Reticulum Class I α-Mannosidase) Is Required for HIV-1 Envelope Glycoprotein Degradation via Endoplasmic Reticulum-associated Protein Degradation Pathway.

    Science.gov (United States)

    Zhou, Tao; Frabutt, Dylan A; Moremen, Kelley W; Zheng, Yong-Hui

    2015-09-04

    Previously, we reported that the mitochondrial translocator protein (TSPO) induces HIV-1 envelope (Env) degradation via the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway, but the mechanism was not clear. Here we investigated how the four ER-associated glycoside hydrolase family 47 (GH47) α-mannosidases, ERManI, and ER-degradation enhancing α-mannosidase-like (EDEM) proteins 1, 2, and 3, are involved in the Env degradation process. Ectopic expression of these four α-mannosidases uncovers that only ERManI inhibits HIV-1 Env expression in a dose-dependent manner. In addition, genetic knock-out of the ERManI gene MAN1B1 using CRISPR/Cas9 technology disrupts the TSPO-mediated Env degradation. Biochemical studies show that HIV-1 Env interacts with ERManI, and between the ERManI cytoplasmic, transmembrane, lumenal stem, and lumenal catalytic domains, the catalytic domain plays a critical role in the Env-ERManI interaction. In addition, functional studies show that inactivation of the catalytic sites by site-directed mutagenesis disrupts the ERManI activity. These studies identify ERManI as a critical GH47 α-mannosidase in the ER-associated protein degradation pathway that initiates the Env degradation and suggests that its catalytic domain and enzymatic activity play an important role in this process. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Effects of nucleic acid local structure and magnesium ions on minus-strand transfer mediated by the nucleic acid chaperone activity of HIV-1 nucleocapsid protein

    OpenAIRE

    Wu, Tiyun; Heilman-Miller, Susan L.; Levin, Judith G.

    2007-01-01

    HIV-1 nucleocapsid protein (NC) is a nucleic acid chaperone, which is required for highly specific and efficient reverse transcription. Here, we demonstrate that local structure of acceptor RNA at a potential nucleation site, rather than overall thermodynamic stability, is a critical determinant for the minus-strand transfer step (annealing of acceptor RNA to (−) strong-stop DNA followed by reverse transcriptase (RT)-catalyzed DNA extension). In our system, destabilization of a stem-loop stru...

  15. Intermolecular masking of the HIV-1 Rev NLS by the cellular protein HIC: novel insights into the regulation of Rev nuclear import.

    LENUS (Irish Health Repository)

    Gu, Lili

    2011-01-01

    The HIV-1 regulatory protein Rev, which is essential for viral replication, mediates the nuclear export of unspliced viral transcripts. Rev nuclear function requires active nucleocytoplasmic shuttling, and Rev nuclear import is mediated by the recognition of its Nuclear Localisation Signal (NLS) by multiple import factors, which include transportin and importin β. However, it remains unclear which nuclear import pathway(s) predominate in vivo, and the cellular environment that modulates Rev nucleocytoplasmic shuttling remains to be characterised.

  16. Editing of HIV-1 RNA by the double-stranded RNA deaminase ADAR1 stimulates viral infection

    Science.gov (United States)

    Doria, Margherita; Neri, Francesca; Gallo, Angela; Farace, Maria Giulia; Michienzi, Alessandro

    2009-01-01

    Adenosine deaminases that act on dsRNA (ADARs) are enzymes that target double-stranded regions of RNA converting adenosines into inosines (A-to-I editing) thus contributing to genome complexity and fine regulation of gene expression. It has been described that a member of the ADAR family, ADAR1, can target viruses and affect their replication process. Here we report evidence showing that ADAR1 stimulates human immuno deficiency virus type 1 (HIV-1) replication by using both editing-dependent and editing-independent mechanisms. We show that over-expression of ADAR1 in HIV-1 producer cells increases viral protein accumulation in an editing-independent manner. Moreover, HIV-1 virions generated in the presence of over-expressed ADAR1 but not an editing-inactive ADAR1 mutant are released more efficiently and display enhanced infectivity, as demonstrated by challenge assays performed with T cell lines and primary CD4+ T lymphocytes. Finally, we report that ADAR1 associates with HIV-1 RNAs and edits adenosines in the 5′ untranslated region (UTR) and the Rev and Tat coding sequence. Overall these results suggest that HIV-1 has evolved mechanisms to take advantage of specific RNA editing activity of the host cell and disclose a stimulatory function of ADAR1 in the spread of HIV-1. PMID:19651874

  17. Mapping of immunogenic and protein-interacting regions at the surface of the seven-bladed β-propeller domain of the HIV-1 cellular interactor EED

    Directory of Open Access Journals (Sweden)

    Gouet Patrice

    2008-02-01

    Full Text Available Abstract Background The human EED protein, a member of the superfamily of Polycomb group proteins, is involved in multiple cellular protein complexes. Its C-terminal domain, which is common to the four EED isoforms, contains seven repeats of a canonical WD-40 motif. EED is an interactor of three HIV-1 proteins, matrix (MA, integrase (IN and Nef. An antiviral activity has been found to be associated with isoforms EED3 and EED4 at the late stage of HIV-1 replication, due to a negative effect on virus assembly and genomic RNA packaging. The aim of the present study was to determine the regions of the EED C-terminal core domain which were accessible and available to protein interactions, using three-dimensional (3D protein homology modelling with a WD-40 protein of known structure, and epitope mapping of anti-EED antibodies. Results Our data suggested that the C-terminal domain of EED was folded as a seven-bladed β-propeller protein. During the completion of our work, crystallographic data of EED became available from co-crystals of the EED C-terminal core with the N-terminal domain of its cellular partner EZH2. Our 3D-model was in good congruence with the refined structural model determined from crystallographic data, except for a unique α-helix in the fourth β-blade. More importantly, the position of flexible loops and accessible β-strands on the β-propeller was consistent with our mapping of immunogenic epitopes and sites of interaction with HIV-1 MA and IN. Certain immunoreactive regions were found to overlap with the EZH2, MA and IN binding sites, confirming their accessibility and reactivity at the surface of EED. Crystal structure of EED showed that the two discrete regions of interaction with MA and IN did not overlap with each other, nor with the EZH2 binding pocket, but were contiguous, and formed a continuous binding groove running along the lateral face of the β-propeller. Conclusion Identification of antibody-, MA-, IN- and EZH2

  18. A Cell Internalizing Antibody Targeting Capsid Protein (p24 Inhibits the Replication of HIV-1 in T Cells Lines and PBMCs: A Proof of Concept Study.

    Directory of Open Access Journals (Sweden)

    Syed A Ali

    Full Text Available There remains a need for newer therapeutic approaches to combat HIV/AIDS. Viral capsid protein p24 plays important roles in HIV pathogenesis. Peptides and small molecule inhibitors targeting p24 have shown to inhibit virus replication in treated cell. High specificity and biological stability of monoclonal antibodies (mAbs make them an attractive contender for in vivo treatments. However, mAbs do not enter into cells, thus are restricted to target surface molecules. This also makes targeting intracellular HIV-1 p24 a challenge. A mAb specific to p24 that can internalize into the HIV-infected cells is hypothesized to inhibit the virus replication. We selected a mAb that has previously shown to inhibit p24 polymerization in an in vitro assay and chemically conjugated it with cell penetrating peptides (CPP to generate cell internalizing anti-p24 mAbs. Out of 8 CPPs tested, κFGF-MTS -conjugated mAbs internalized T cells most efficiently. At nontoxic concentration, the κFGF-MTS-anti-p24-mAbs reduced the HIV-1 replication up to 73 and 49% in T-lymphocyte and PBMCs respectively. Marked inhibition of HIV-1 replication in relevant cells by κFGF-MTS-anti-p24-mAbs represents a viable strategy to target HIV proteins present inside the cells.

  19. Systematic Protein-Protein Docking and Molecular Dynamics Studies of HIV-1 gp120 and CD4: Insights for New Drug Development

    Directory of Open Access Journals (Sweden)

    M. Rizman-Idid

    2011-12-01

    Full Text Available Background and the purpose of the study: The interactions between HIV-1 gp120 and mutated CD4 proteins were investigated in order to identify a lead structure for therapy based on competitive blocking of the HIV binding receptor for human T-cells. Crystal structures of HIV gp120-CD4 complexes reveal a close interaction of the virus receptor with CD4 Phe43, which is embedded in a pocket of the virus protein.Methods: This study applies computer simulations to determine the best binding of amino acid 43 CD4 mutants to HIV gp120. Besides natural CD4, three mutants carrying alternate aromatic residues His, Trp and Tyr at position 43 were investigated. Several docking programs were applied on isolated proteins based on selected crystal structures of gp120-CD4 complexes, as well as a 5 ns molecular dynamics study on the protein complexes. The initial structures were minimized in Gromacs to avoid crystal packing effects, and then subjected to docking experiments using AutoDock4, FireDock, ClusPro and ZDock. In molecular dynamics, the Gibbs free binding energy was calculated for the gp120-CD4 complexes. The docking outputs were analyzed on energy within the respective docking software.Results and conclusion: Visualization and hydrogen bonding analysis were performed using the Swiss-PdbViewer. Strong binding to HIV gp120 can be achieved with an extended aromatic group (Trp. However, the sterical demand of the interaction affects the binding kinetics. In conclusion, a ligand for an efficient blocking of HIV gp120 should involve an extended but conformational flexible aromatic group, i.e. a biphenyl. A docking study on biphenylalanine-43 confirms this expectation

  20. Prime-boost vaccination with heterologous live vectors encoding SIV gag and multimeric HIV-1 gp160 protein: efficacy against repeated mucosal R5 clade C SHIV challenges.

    Science.gov (United States)

    Lakhashe, Samir K; Velu, Vijayakumar; Sciaranghella, Gaia; Siddappa, Nagadenahalli B; Dipasquale, Janet M; Hemashettar, Girish; Yoon, John K; Rasmussen, Robert A; Yang, Feng; Lee, Sandra J; Montefiori, David C; Novembre, Francis J; Villinger, François; Amara, Rama Rao; Kahn, Maria; Hu, Shiu-Lok; Li, Sufen; Li, Zhongxia; Frankel, Fred R; Robert-Guroff, Marjorie; Johnson, Welkin E; Lieberman, Judy; Ruprecht, Ruth M

    2011-08-05

    We sought to induce primate immunodeficiency virus-specific cellular and neutralizing antibody (nAb) responses in rhesus macaques (RM) through a bimodal vaccine approach. RM were immunized intragastrically (i.g.) with the live-attenuated Listeria monocytogenes (Lm) vector Lmdd-BdopSIVgag encoding SIVmac239 gag. SIV Gag-specific cellular responses were boosted by intranasal and intratracheal administration of replication-competent adenovirus (Ad5hr-SIVgag) encoding the same gag. To broaden antiviral immunity, the RM were immunized with multimeric HIV clade C (HIV-C) gp160 and HIV Tat. SIV Gag-specific cellular immune responses and HIV-1 nAb developed in some RM. The animals were challenged intrarectally with five low doses of R5 SHIV-1157ipEL-p, encoding a heterologous HIV-C Env (22.1% divergent to the Env immunogen). All five controls became viremic. One out of ten vaccinees was completely protected and another had low peak viremia. Sera from the completely and partially protected RM neutralized the challenge virus > 90%; these RM also had strong SIV Gag-specific proliferation of CD8⁺ T cells. Peak and area under the curve of plasma viremia (during acute phase) among vaccinees was lower than for controls, but did not attain significance. The completely protected RM showed persistently low numbers of the α4β7-expressing CD4⁺ T cells; the latter have been implicated as preferential virus targets in vivo. Thus, vaccine-induced immune responses and relatively lower numbers of potential target cells were associated with protection. Copyright © 2011 Elsevier Ltd. All rights reserved.

  1. Genetic incorporation of the protein transduction domain of Tat into Ad5 fiber enhances gene transfer efficacy

    Directory of Open Access Journals (Sweden)

    Siegal Gene P

    2007-10-01

    Full Text Available Abstract Background Human adenovirus serotype 5 (Ad5 has been widely explored as a gene delivery vector for a variety of diseases. Many target cells, however, express low levels of Ad5 native receptor, the Coxsackie-Adenovirus Receptor (CAR, and thus are resistant to Ad5 infection. The Protein Transduction Domain of the HIV Tat protein, namely PTDtat, has been shown to mediate protein transduction in a wide range of cells. We hypothesize that re-targeting Ad5 vector via the PTDtat motif would improve the efficacy of Ad5-mediated gene delivery. Results In this study, we genetically incorporated the PTDtat motif into the knob domain of Ad5 fiber, and rescued the resultant viral vector, Ad5.PTDtat. Our data showed the modification did not interfere with Ad5 binding to its native receptor CAR, suggesting Ad5 infection via the CAR pathway is retained. In addition, we found that Ad5.PTDtat exhibited enhanced gene transfer efficacy in all of the cell lines that we have tested, which included both low-CAR and high-CAR decorated cells. Competitive inhibition assays suggested the enhanced infectivity of Ad5.PTDtat was mediated by binding of the positively charged PTDtat peptide to the negatively charged epitopes on the cells' surface. Furthermore, we investigated in vivo gene delivery efficacy of Ad5.PTDtat using subcutaneous tumor models established with U118MG glioma cells, and found that Ad5.PTDtat exhibited enhanced gene transfer efficacy compared to unmodified Ad5 vector as analyzed by a non-invasive fluorescence imaging technique. Conclusion Genetic incorporation of the PTDtat motif into Ad5 fiber allowed Ad5 vectors to infect cells via an alternative PTDtat targeting motif while retaining the native CAR-mediated infection pathway. The enhanced infectivity was demonstrated in both cultured cells and in in vivo tumor models. Taken together, our study identifies a novel tropism expanded Ad5 vector that may be useful for clinical gene therapy

  2. DNA vaccines expressing soluble CD4-envelope proteins fused to C3d elicit cross-reactive neutralizing antibodies to HIV-1

    International Nuclear Information System (INIS)

    Bower, Joseph F.; Green, Thomas D.; Ross, Ted M.

    2004-01-01

    DNA vaccines expressing the envelope (Env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, DNA vaccines were constructed to express a fusion protein of the soluble human CD4 (sCD4) and the gp120 subunit of the HIV-1 envelope. To enhance the immunogenicity of the expressed fusion protein, three copies of the murine C3d (mC3d 3 ) were added to the carboxyl terminus of the complex. Monoclonal antibodies that recognize CD4-induced epitopes on gp120 efficiently bound to sCD4-gp120 or sCD4-gp120-mC3d 3 . In addition, both sCD4-gp120 and sCD4-gp120-mC3d 3 bound to cells expressing appropriate coreceptors in the absence of cell surface hCD4. Mice (BALB/c) vaccinated with DNA vaccines expressing either gp120-mC3d 3 or sCD4-gp120-mC3d 3 elicited antibodies that neutralized homologous virus infection. However, the use of sCD4-gp120-mC3d 3 -DNA elicited the highest titers of neutralizing antibodies that persisted after depletion of anti-hCD4 antibodies. Interestingly, only mice vaccinated with DNA expressing sCD4-gp120-mC3d 3 had antibodies that elicited cross-protective neutralizing antibodies. The fusion of sCD4 to the HIV-1 envelope exposes neutralizing epitopes that elicit broad protective immunity when the fusion complex is coupled with the molecular adjuvant, C3d

  3. HIV-1 envelope glycoprotein

    Science.gov (United States)

    Caulfield, Michael; Cupo, Albert; Dean, Hansi; Hoffenberg, Simon; King, C. Richter; Klasse, P. J.; Marozsan, Andre; Moore, John P.; Sanders, Rogier W.; Ward, Andrew; Wilson, Ian; Julien, Jean-Philippe

    2017-08-22

    The present application relates to novel HIV-1 envelope glycoproteins, which may be utilized as HIV-1 vaccine immunogens, and antigens for crystallization, electron microscopy and other biophysical, biochemical and immunological studies for the identification of broad neutralizing antibodies. The present invention encompasses the preparation and purification of immunogenic compositions, which are formulated into the vaccines of the present invention.

  4. Imperatorin inhibits HIV-1 replication through an Sp1-dependent pathway.

    Science.gov (United States)

    Sancho, Rocío; Márquez, Nieves; Gómez-Gonzalo, Marta; Calzado, Marco A; Bettoni, Giorgio; Coiras, Maria Teresa; Alcamí, José; López-Cabrera, Manuel; Appendino, Giovanni; Muñoz, Eduardo

    2004-09-03

    Coumarins and structurally related compounds have been recently shown to present anti-human immunodeficiency virus, type 1 (HIV-1) activity. Among them, the dietary furanocoumarin imperatorin is present in citrus fruits, in culinary herbs, and in some medicinal plants. In this study we report that imperatorin inhibits either vesicular stomatitis virus-pseudotyped or gp160-enveloped recombinant HIV-1 infection in several T cell lines and in HeLa cells. These recombinant viruses express luciferase as a marker of viral replication. Imperatorin did not inhibit the reverse transcription nor the integration steps in the viral cell cycle. Using several 5' long terminal repeat-HIV-1 constructs where critical response elements were either deleted or mutated, we found that the transcription factor Sp1 is critical for the inhibitory activity of imperatorin induced by both phorbol 12-myristate 13-acetate and HIV-1 Tat. Moreover in transient transfections imperatorin specifically inhibited phorbol 12-myristate 13-acetate-induced transcriptional activity of the Gal4-Sp1 fusion protein. Since Sp1 is also implicated in cell cycle progression we further studied the effect of imperatorin on cyclin D1 gene transcription and protein expression and in HeLa cell cycle progression. We found that imperatorin strongly inhibited cyclin D1 expression and arrested the cells at the G(1) phase of the cell cycle. These results highlight the potential of Sp1 transcription factor as a target for natural anti-HIV-1 compounds such as furanocoumarins that might have a potential therapeutic role in the management of AIDS.

  5. Identification of a novel splice acceptor in the HIV-1 genome: independent expression of the cytoplasmic tail of the envelope protein

    NARCIS (Netherlands)

    Berkhout, B.; van Wamel, J. L.

    1996-01-01

    Multiple splicing sites exist in the RNA genome of the human immunodeficiency virus type 1 (HIV-1). In a screen for subgenomic forms of the HIV-1 genome that could be transferred to fresh cells by virus infection, we identified a novel spliced variant of HIV-1 RNA that uses a hitherto unknown splice

  6. Picomolar dichotomous activity of gnidimacrin against HIV-1.

    Directory of Open Access Journals (Sweden)

    Li Huang

    Full Text Available Highly active antiretroviral therapy (HAART has offered a promising approach for controlling HIV-1 replication in infected individuals. However, with HARRT, HIV-1 is suppressed rather than eradicated due to persistence of HIV-1 in latent viral reservoirs. Thus, purging the virus from latent reservoirs is an important strategy toward eradicating HIV-1 infection. In this study, we discovered that the daphnane diterpene gnidimacrin, which was previously reported to have potent anti-cancer cell activity, activated HIV-1 replication and killed persistently-infected cells at picomolar concentrations. In addition to its potential to purge HIV-1 from latently infected cells, gnidimacrin potently inhibited a panel of HIV-1 R5 virus infection of peripheral blood mononuclear cells (PBMCs at an average concentration lower than 10 pM. In contrast, gnidimacrin only partially inhibited HIV-1 ×4 virus infection of PBMCs. The strong anti-HIV-1 R5 virus activity of gnidimacrin was correlated with its effect on down-regulation of the HIV-1 coreceptor CCR5. The anti-R5 virus activity of gnidimacrin was completely abrogated by a selective protein kinase C beta inhibitor enzastaurin, which suggests that protein kinase C beta plays a key role in the potent anti-HIV-1 activity of gnidimacrin in PBMCs. In summary, these results suggest that gnidimacrin could activate latent HIV-1, specifically kill HIV-1 persistently infected cells, and inhibit R5 viruses at picomolar concentrations.

  7. HIV-1 Vif's Capacity To Manipulate the Cell Cycle Is Species Specific.

    Science.gov (United States)

    Evans, Edward L; Becker, Jordan T; Fricke, Stephanie L; Patel, Kishan; Sherer, Nathan M

    2018-04-01

    Cells derived from mice and other rodents exhibit profound blocks to HIV-1 virion production, reflecting species-specific incompatibilities between viral Tat and Rev proteins and essential host factors cyclin T1 (CCNT1) and exportin-1 (XPO1, also known as CRM1), respectively. To determine if mouse cell blocks other than CCNT1 and XPO1 affect HIV's postintegration stages, we studied HIV-1 NL4-3 gene expression in mouse NIH 3T3 cells modified to constitutively express HIV-1-compatible versions of CCNT1 and XPO1 (3T3.CX cells). 3T3.CX cells supported both Rev-independent and Rev-dependent viral gene expression and produced relatively robust levels of virus particles, confirming that CCNT1 and XPO1 represent the predominant blocks to these stages. Unexpectedly, however, 3T3.CX cells were remarkably resistant to virus-induced cytopathic effects observed in human cell lines, which we mapped to the viral protein Vif and its apparent species-specific capacity to induce G 2 /M cell cycle arrest. Vif was able to mediate rapid degradation of human APOBEC3G and the PPP2R5D regulatory B56 subunit of the PP2A phosphatase holoenzyme in mouse cells, thus demonstrating that Vif NL4-3 's modulation of the cell cycle can be functionally uncoupled from some of its other defined roles in CUL5-dependent protein degradation. Vif was also unable to induce G 2 /M cell cycle arrest in other nonhuman cell types, including cells derived from nonhuman primates, leading us to propose that one or more human-specific cofactors underpin Vif's ability to modulate the cell cycle. IMPORTANCE Cells derived from mice and other rodents exhibit profound blocks to HIV-1 replication, thus hindering the development of a low-cost small-animal model for studying HIV/AIDS. Here, we engineered otherwise-nonpermissive mouse cells to express HIV-1-compatible versions of two species-specific host dependency factors, cyclin T1 (CCNT1) and exportin-1 (XPO1) (3T3.CX cells). We show that 3T3.CX cells rescue HIV-1

  8. Structural basis for TatA oligomerization: an NMR study of Escherichia coli TatA dimeric structure.

    Directory of Open Access Journals (Sweden)

    Yi Zhang

    Full Text Available Many proteins are transported across lipid membranes by protein translocation systems in living cells. The twin-arginine transport (Tat system identified in bacteria and plant chloroplasts is a unique system that transports proteins across membranes in their fully-folded states. Up to date, the detailed molecular mechanism of this process remains largely unclear. The Escherichia coli Tat system consists of three essential transmembrane proteins: TatA, TatB and TatC. Among them, TatB and TatC form a tight complex and function in substrate recognition. The major component TatA contains a single transmembrane helix followed by an amphipathic helix, and is suggested to form the translocation pore via self-oligomerization. Since the TatA oligomer has to accommodate substrate proteins of various sizes and shapes, the process of its assembly stands essential for understanding the translocation mechanism. A structure model of TatA oligomer was recently proposed based on NMR and EPR observations, revealing contacts between the transmembrane helices from adjacent subunits. Herein we report the construction and stabilization of a dimeric TatA, as well as the structure determination by solution NMR spectroscopy. In addition to more extensive inter-subunit contacts between the transmembrane helices, we were also able to observe interactions between neighbouring amphipathic helices. The side-by-side packing of the amphipathic helices extends the solvent-exposed hydrophilic surface of the protein, which might be favourable for interactions with substrate proteins. The dimeric TatA structure offers more detailed information of TatA oligomeric interface and provides new insights on Tat translocation mechanism.

  9. Trans-activation of the 5' to 3' viral DNA strand transfer by nucleocapsid protein during reverse transcription of HIV1 RNA.

    Science.gov (United States)

    Darlix, J L; Vincent, A; Gabus, C; de Rocquigny, H; Roques, B

    1993-08-01

    Two DNA strand transfer reactions take place during reverse transcription of the retroviral genome. The first transfer, that of the minus-strand strong stop DNA from the 5' end of the viral RNA to the 3' end, has been studied in vitro with two RNAs mimicking the 5' and 3' regions of the HIV1 genome and with nucleocapsid protein, NCp7, and reverse transcriptase. The results show that NCp7 strongly activates the 5' to 3' DNA strand transfer during reverse transcription while a basic peptide resembling NCp7 is inactive. Activation of the first transfer by several NCp7 derived peptides and the influence of the terminal redundancies (R) present at the 5' and 3' ends of HIV1 RNA were also examined. The first transfer is optimal in the presence of intact NCp7 and necessitates R on both the 5' and 3' RNAs. Sequencing of full length viral DNA products reveals approximately 40% misincorporations at the first nucleotide beyond the transfer point. If such base misincorporations occur during proviral DNA synthesis with possible homologous recombinations it may well contribute to the high level of genetic variability of HIV.

  10. Tat proteins as novel thylakoid membrane anchors organize a biosynthetic pathway in chloroplasts and increase product yield 5-fold

    DEFF Research Database (Denmark)

    Henriques de Jesus, Maria Perestrello Ramos; Nielsen, Agnieszka Janina Zygadlo; Mellor, Silas Busck

    2017-01-01

    to their complex structures. Some of the crucial enzymes catalyzing their biosynthesis are the cytochromes P450 (P450s) situated in the endoplasmic reticulum (ER), powered by electron transfers from NADPH. Dhurrin is a cyanogenic glucoside and its biosynthesis involves a dynamic metabolon formed by two P450s....... Nevertheless, translocation of the pathway from the ER to the chloroplast creates other difficulties, such as the loss of metabolon formation and intermediate diversion into other metabolic pathways. We show here that co-localization of these enzymes in the thylakoid membrane leads to a significant increase...... in product formation, with a concomitant decrease in off-pathway intermediates. This was achieved by exchanging the membrane anchors of the dhurrin pathway enzymes to components of the Twin-arginine translocation pathway, TatB and TatC, which have self-assembly properties. Consequently, we show 5-fold...

  11. Intermolecular masking of the HIV-1 Rev NLS by the cellular protein HIC: Novel insights into the regulation of Rev nuclear import.

    LENUS (Irish Health Repository)

    Gu, Lili

    2011-03-14

    Abstract Background The HIV-1 regulatory protein Rev, which is essential for viral replication, mediates the nuclear export of unspliced viral transcripts. Rev nuclear function requires active nucleocytoplasmic shuttling, and Rev nuclear import is mediated by the recognition of its Nuclear Localisation Signal (NLS) by multiple import factors, which include transportin and importin β. However, it remains unclear which nuclear import pathway(s) predominate in vivo, and the cellular environment that modulates Rev nucleocytoplasmic shuttling remains to be characterised. Results In our study, we have identified the cellular protein HIC (Human I-mfa domain-Containing protein) as a novel interactor of HIV-1 Rev. We demonstrate that HIC selectively interferes with Rev NLS interaction with importin β and impedes its nuclear import and function, but does not affect Rev nuclear import mediated by transportin. Hence, the molecular determinants mediating Rev-NLS recognition by importin β and transportin appear to be distinct. Furthermore, we have employed HIC and M9 M, a peptide specifically designed to inhibit the transportin-mediated nuclear import pathway, to characterise Rev nuclear import pathways within different cellular environments. Remarkably, we could show that in 293T, HeLa, COS7, Jurkat, U937, THP-1 and CEM cells, Rev nuclear import is cell type specific and alternatively mediated by transportin or importin β, in a mutually exclusive fashion. Conclusions Rev cytoplasmic sequestration by HIC may represent a novel mechanism for the control of Rev function. These studies highlight that the multivalent nature of the Rev NLS for different import receptors enables Rev to adapt its nuclear trafficking strategy.

  12. Intermolecular masking of the HIV-1 Rev NLS by the cellular protein HIC: Novel insights into the regulation of Rev nuclear import

    Directory of Open Access Journals (Sweden)

    Sheehy Noreen

    2011-03-01

    Full Text Available Abstract Background The HIV-1 regulatory protein Rev, which is essential for viral replication, mediates the nuclear export of unspliced viral transcripts. Rev nuclear function requires active nucleocytoplasmic shuttling, and Rev nuclear import is mediated by the recognition of its Nuclear Localisation Signal (NLS by multiple import factors, which include transportin and importin β. However, it remains unclear which nuclear import pathway(s predominate in vivo, and the cellular environment that modulates Rev nucleocytoplasmic shuttling remains to be characterised. Results In our study, we have identified the cellular protein HIC (Human I-mfa domain-Containing protein as a novel interactor of HIV-1 Rev. We demonstrate that HIC selectively interferes with Rev NLS interaction with importin β and impedes its nuclear import and function, but does not affect Rev nuclear import mediated by transportin. Hence, the molecular determinants mediating Rev-NLS recognition by importin β and transportin appear to be distinct. Furthermore, we have employed HIC and M9 M, a peptide specifically designed to inhibit the transportin-mediated nuclear import pathway, to characterise Rev nuclear import pathways within different cellular environments. Remarkably, we could show that in 293T, HeLa, COS7, Jurkat, U937, THP-1 and CEM cells, Rev nuclear import is cell type specific and alternatively mediated by transportin or importin β, in a mutually exclusive fashion. Conclusions Rev cytoplasmic sequestration by HIC may represent a novel mechanism for the control of Rev function. These studies highlight that the multivalent nature of the Rev NLS for different import receptors enables Rev to adapt its nuclear trafficking strategy.

  13. HIV-1 Env DNA vaccine plus protein boost delivered by EP expands B- and T-cell responses and neutralizing phenotype in vivo.

    Directory of Open Access Journals (Sweden)

    Kar Muthumani

    Full Text Available An effective HIV vaccine will most likely require the induction of strong T-cell responses, broadly neutralizing antibodies (bNAbs, and the elicitation of antibody-dependent cellular cytotoxicity (ADCC. Previously, we demonstrated the induction of strong HIV/SIV cellular immune responses in macaques and humans using synthetic consensus DNA immunogens delivered via adaptive electroporation (EP. However, the ability of this improved DNA approach to prime for relevant antibody responses has not been previously studied. Here, we investigate the immunogenicity of consensus DNA constructs encoding gp140 sequences from HIV-1 subtypes A, B, C and D in a DNA prime-protein boost vaccine regimen. Mice and guinea pigs were primed with single- and multi-clade DNA via EP and boosted with recombinant gp120 protein. Sera were analyzed for gp120 binding and induction of neutralizing antibody activity. Immunization with recombinant Env protein alone induced low-titer binding antibodies with limited neutralization breath. In contrast, the synthetic DNA prime-protein boost protocol induced significantly higher antibody binding titers. Furthermore, sera from DNA prime-protein boost groups were able to neutralize a broader range of viruses in a panel of tier 1 clade B viruses as well as multiple tier 1 clade A and clade C viruses. Further investigation of synthetic DNA prime plus adaptive EP plus protein boost appears warranted.

  14. HIV-1 infection induces changes in expression of cellular splicing factors that regulate alternative viral splicing and virus production in macrophages

    Directory of Open Access Journals (Sweden)

    Purcell Damian FJ

    2008-02-01

    Full Text Available Abstract Background Macrophages are important targets and long-lived reservoirs of HIV-1, which are not cleared of infection by currently available treatments. In the primary monocyte-derived macrophage model of infection, replication is initially productive followed by a decline in virion output over ensuing weeks, coincident with a decrease in the levels of the essential viral transactivator protein Tat. We investigated two possible mechanisms in macrophages for regulation of viral replication, which appears to be primarily regulated at the level of tat mRNA: 1 differential mRNA stability, used by cells and some viruses for the rapid regulation of gene expression and 2 control of HIV-1 alternative splicing, which is essential for optimal viral replication. Results Following termination of transcription at increasing times after infection in macrophages, we found that tat mRNA did indeed decay more rapidly than rev or nef mRNA, but with similar kinetics throughout infection. In addition, tat mRNA decayed at least as rapidly in peripheral blood lymphocytes. Expression of cellular splicing factors in uninfected and infected macrophage cultures from the same donor showed an inverse pattern over time between enhancing factors (members of the SR family of RNA binding proteins and inhibitory factors (members of the hnRNP family. While levels of the SR protein SC35 were greatly up-regulated in the first week or two after infection, hnRNPs of the A/B and H groups were down-regulated. Around the peak of virus production in each culture, SC35 expression declined to levels in uninfected cells or lower, while the hnRNPs increased to control levels or above. We also found evidence for increased cytoplasmic expression of SC35 following long-term infection. Conclusion While no evidence of differential regulation of tat mRNA decay was found in macrophages following HIV-1 infection, changes in the balance of cellular splicing factors which regulate alternative

  15. Human endogenous retrovirus K Gag coassembles with HIV-1 Gag and reduces the release efficiency and infectivity of HIV-1.

    Science.gov (United States)

    Monde, Kazuaki; Contreras-Galindo, Rafael; Kaplan, Mark H; Markovitz, David M; Ono, Akira

    2012-10-01

    Human endogenous retroviruses (HERVs), which are remnants of ancestral retroviruses integrated into the human genome, are defective in viral replication. Because activation of HERV-K and coexpression of this virus with HIV-1 have been observed during HIV-1 infection, it is conceivable that HERV-K could affect HIV-1 replication, either by competition or by cooperation, in cells expressing both viruses. In this study, we found that the release efficiency of HIV-1 Gag was 3-fold reduced upon overexpression of HERV-K(CON) Gag. In addition, we observed that in cells expressing Gag proteins of both viruses, HERV-K(CON) Gag colocalized with HIV-1 Gag at the plasma membrane. Furthermore, HERV-K(CON) Gag was found to coassemble with HIV-1 Gag, as demonstrated by (i) processing of HERV-K(CON) Gag by HIV-1 protease in virions, (ii) coimmunoprecipitation of virion-associated HERV-K(CON) Gag with HIV-1 Gag, and (iii) rescue of a late-domain-defective HERV-K(CON) Gag by wild-type (WT) HIV-1 Gag. Myristylation-deficient HERV-K(CON) Gag localized to nuclei, suggesting cryptic nuclear trafficking of HERV-K Gag. Notably, unlike WT HERV-K(CON) Gag, HIV-1 Gag failed to rescue myristylation-deficient HERV-K(CON) Gag to the plasma membrane. Efficient colocalization and coassembly of HIV-1 Gag and HERV-K Gag also required nucleocapsid (NC). These results provide evidence that HIV-1 Gag heteromultimerizes with HERV-K Gag at the plasma membrane, presumably through NC-RNA interaction. Intriguingly, HERV-K Gag overexpression reduced not only HIV-1 release efficiency but also HIV-1 infectivity in a myristylation- and NC-dependent manner. Altogether, these results indicate that Gag proteins of endogenous retroviruses can coassemble with HIV-1 Gag and modulate the late phase of HIV-1 replication.

  16. PDGF-mediated protection of SH-SY5Y cells against Tat toxin involves regulation of extracellular glutamate and intracellular calcium

    International Nuclear Information System (INIS)

    Zhu Xuhui; Yao Honghong; Peng Fuwang; Callen, Shannon; Buch, Shilpa

    2009-01-01

    The human immunodeficiency virus (HIV-1) protein Tat has been implicated in mediating neuronal apoptosis, one of the hallmark features of HIV-associated dementia (HAD). Mitigation of the toxic effects of Tat could thus be a potential mechanism for reducing HIV toxicity in the brain. In this study we demonstrated that Tat-induced neurotoxicity was abolished by NMDA antagonist-MK801, suggesting the role of glutamate in this process. Furthermore, we also found that pretreatment of SH-SY5Y cells with PDGF exerted protection against Tat toxicity by decreasing extracellular glutamate levels. We also demonstrated that extracellular calcium chelator EGTA was able to abolish PDGF-mediated neuroprotection, thereby underscoring the role of calcium signaling in PDGF-mediated neuroprotection. We also showed that Erk signaling pathway was critical for PDGF-mediated protection of cells. Additionally, blocking calcium entry with EGTA resulted in suppression of PDGF-induced Erk activation. These findings thus underscore the role of PDGF-mediated calcium signaling and Erk phosphorylation in the protection of cells against HIV Tat toxicity.

  17. Structural determinants of HIV-1 nucleocapsid protein for cTAR DNA binding and destabilization, and correlation with inhibition of self-primed DNA synthesis.

    Science.gov (United States)

    Beltz, Hervé; Clauss, Céline; Piémont, Etienne; Ficheux, Damien; Gorelick, Robert J; Roques, Bernard; Gabus, Caroline; Darlix, Jean-Luc; de Rocquigny, Hugues; Mély, Yves

    2005-05-20

    The nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) is formed of two highly conserved CCHC zinc fingers flanked by small basic domains. NC is required for the two obligatory strand transfers in viral DNA synthesis through its nucleic acid chaperoning properties. The first DNA strand transfer relies on NC's ability to bind and destabilize the secondary structure of complementary transactivation response region (cTAR) DNA, to inhibit self-priming, and to promote the annealing of cTAR to TAR RNA. To further investigate NC chaperone properties, our aim was to identify by fluorescence spectroscopy and gel electrophoresis, the NC structural determinants for cTAR binding and destabilization, and for the inhibition of self-primed DNA synthesis on a model system using a series of NC mutants and HIV-1 reverse transcriptase. NC destabilization and self-priming inhibition properties were found to be supported by the two fingers in their proper context and the basic (29)RAPRKKG(35) linker. The strict requirement of the native proximal finger suggests that its hydrophobic platform (Val13, Phe16, Thr24 and Ala25) is crucial for binding, destabilization and inhibition of self-priming. In contrast, only partial folding of the distal finger is required, probably for presenting the Trp37 residue in an appropriate orientation. Also, Trp37 and the hydrophobic residues of the proximal finger appear to be essential for the propagation of the melting from the cTAR ends up to the middle of the stem. Finally, both N-terminal and C-terminal basic domains contribute to cTAR binding but not to its destabilization.

  18. HIV-1 Nef control of cell signalling molecules: multiple strategies

    Indian Academy of Sciences (India)

    HIV-1 has at its disposal numerous proteins encoded by its genome which provide the required arsenal to establish and maintain infection in its host for a considerable number of years. One of the most important and enigmatic of these proteins is Nef. The Nef protein of HIV-1 plays a fundamental role in the virus life cycle.

  19. Human CNS cultures exposed to HIV-1 gp120 reproduce dendritic injuries of HIV-1-associated dementia

    Directory of Open Access Journals (Sweden)

    Hammond Robert R

    2004-05-01

    Full Text Available Abstract HIV-1-associated dementia remains a common subacute to chronic central nervous system degeneration in adult and pediatric HIV-1 infected populations. A number of viral and host factors have been implicated including the HIV-1 120 kDa envelope glycoprotein (gp120. In human post-mortem studies using confocal scanning laser microscopy for microtubule-associated protein 2 and synaptophysin, neuronal dendritic pathology correlated with dementia. In the present study, primary human CNS cultures exposed to HIV-1 gp120 at 4 weeks in vitro suffered gliosis and dendritic damage analogous to that described in association with HIV-1-associated dementia.

  20. Production and purification of immunologically active core protein p24 from HIV-1 fused to ricin toxin B subunit in E. coli

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    Gómez-Lim Miguel A

    2009-02-01

    Full Text Available Abstract Background Gag protein from HIV-1 is a polyprotein of 55 kDa, which, during viral maturation, is cleaved to release matrix p17, core p24 and nucleocapsid proteins. The p24 antigen contains epitopes that prime helper CD4 T-cells, which have been demonstrated to be protective and it can elicit lymphocyte proliferation. Thus, p24 is likely to be an integral part of any multicomponent HIV vaccine. The availability of an optimal adjuvant and carrier to enhance antiviral responses may accelerate the development of a vaccine candidate against HIV. The aim of this study was to investigate the adjuvant-carrier properties of the B ricin subunit (RTB when fused to p24. Results A fusion between ricin toxin B subunit and p24 HIV (RTB/p24 was expressed in E. coli. Affinity chromatography was used for purification of p24 alone and RTB/p24 from cytosolic fractions. Biological activity of RTB/p24 was determined by ELISA and affinity chromatography using the artificial receptor glycoprotein asialofetuin. Both assays have demonstrated that RTB/p24 is able to interact with complex sugars, suggesting that the chimeric protein retains lectin activity. Also, RTB/p24 was demonstrated to be immunologically active in mice. Two weeks after intraperitoneal inoculation with RTB/p24 without an adjuvant, a strong anti-p24 immune response was detected. The levels of the antibodies were comparable to those found in mice immunized with p24 alone in the presence of Freund adjuvant. RTB/p24 inoculated intranasally in mice, also elicited significant immune responses to p24, although the response was not as strong as that obtained in mice immunized with p24 in the presence of the mucosal adjuvant cholera toxin. Conclusion In this work, we report the expression in E. coli of HIV-1 p24 fused to the subunit B of ricin toxin. The high levels of antibodies obtained after intranasal and intraperitoneal immunization of mice demonstrate the adjuvant-carrier properties of RTB when

  1. Extensive interactions between HIV TAT and TAF(II)250.

    Science.gov (United States)

    Weissman, J D; Hwang, J R; Singer, D S

    2001-03-09

    The HIV transactivator, Tat, has been shown to be capable of potent repression of transcription initiation. Repression is mediated by the C-terminal segment of Tat, which binds the TFIID component, TAF(II)250, although the site(s) of interaction were not defined previously. We now report that the interaction between Tat and TAF(II)250 is extensive and involves multiple contacts between the Tat protein and TAF(II)250. The C-terminal domain of Tat, which is necessary for repression of transcription initiation, binds to a segment of TAF(II)250 that encompasses its acetyl transferase (AT) domain (885-1034 amino acids (aa)). Surprisingly, the N-terminal segment of Tat, which contains its activation domains, also binds to TAF(II)250 and interacts with two discontinuous segments of TAF(II)250 located between 885 and 984 aa and 1120 and 1279 aa. Binding of Tat to the 885-984 aa segment of TAF(II)250 requires the cysteine-rich domain of Tat, but not the acidic or glutamine-rich domains. Binding by the N-terminal domain of Tat to the 1120-1279 aa TAF(II)250 segment does not involve the acidic, cysteine- or glutamine-rich domains. Repression of transcription initiation by Tat requires functional TAF(II)250. We now demonstrate that transcription of the HIV LTR does not depend on TAF(II)250 which may account for its resistance to Tat mediated repression.

  2. Transient nature of long-term nonprogression and broad virus-specific proliferative T-cell responses with sustained thymic output in HIV-1 controllers.

    Directory of Open Access Journals (Sweden)

    Samantha J Westrop

    Full Text Available HIV-1(+ individuals who, without therapy, conserve cellular anti-HIV-1 responses, present with high, stable CD4(+ T-cell numbers, and control viral replication, facilitate analysis of atypical viro-immunopathology. In the absence of universal definition, immune function in such HIV controllers remains an indication of non-progression.CD4 T-cell responses to a number of HIV-1 proteins and peptide pools were assessed by IFN-gamma ELISpot and lymphoproliferative assays in HIV controllers and chronic progressors. Thymic output was assessed by sjTRECs levels. Follow-up of 41 HIV-1(+ individuals originally identified as "Long-term non-progressors" in 1996 according to clinical criteria, and longitudinal analysis of two HIV controllers over 22 years, was also performed. HIV controllers exhibited substantial IFN-gamma producing and proliferative HIV-1-specific CD4 T-cell responses to both recombinant proteins and peptide pools of Tat, Rev, Nef, Gag and Env, demonstrating functional processing and presentation. Conversely, HIV-specific T-cell responses were limited to IFN-gamma production in chronic progressors. Additionally, thymic output was approximately 19 fold higher in HIV controllers than in age-matched chronic progressors. Follow-up of 41 HIV-1(+ patients identified as LTNP in 1996 revealed the transitory characteristics of this status. IFN-gamma production and proliferative T-cell function also declines in 2 HIV controllers over 22 years.Although increased thymic output and anti-HIV-1 T-cell responses are observed in HIV controllers compared to chronic progressors, the nature of nonprogressor/controller status appears to be transitory.

  3. The Ebola virus VP35 protein is a suppressor of RNA silencing.

    Directory of Open Access Journals (Sweden)

    Joost Haasnoot

    2007-06-01

    Full Text Available RNA silencing or interference (RNAi is a gene regulation mechanism in eukaryotes that controls cell differentiation and developmental processes via expression of microRNAs. RNAi also serves as an innate antiviral defence response in plants, nematodes, and insects. This antiviral response is triggered by virus-specific double-stranded RNA molecules (dsRNAs that are produced during infection. To overcome antiviral RNAi responses, many plant and insect viruses encode RNA silencing suppressors (RSSs that enable them to replicate at higher titers. Recently, several human viruses were shown to encode RSSs, suggesting that RNAi also serves as an innate defence response in mammals. Here, we demonstrate that the Ebola virus VP35 protein is a suppressor of RNAi in mammalian cells and that its RSS activity is functionally equivalent to that of the HIV-1 Tat protein. We show that VP35 can replace HIV-1 Tat and thereby support the replication of a Tat-minus HIV-1 variant. The VP35 dsRNA-binding domain is required for this RSS activity. Vaccinia virus E3L protein and influenza A virus NS1 protein are also capable of replacing the HIV-1 Tat RSS function. These findings support the hypothesis that RNAi is part of the innate antiviral response in mammalian cells. Moreover, the results indicate that RSSs play a critical role in mammalian virus replication.

  4. The Ixodes scapularis Salivary Protein, Salp15, Prevents the Association of HIV-1 gp120 and CD4

    OpenAIRE

    Juncadella, Ignacio J.; Garg, Renu; Bates, Tonya C.; Olivera, Elias R.; Anguita, Juan

    2007-01-01

    Ixodes scapularis salivary protein, Salp15, inhibits CD4+ T cell activation by binding to the most-extracellular domains of the CD4 molecule, potentially overlapping with the gp120-binding region. We now show that Salp15 inhibits the interaction of gp120 and CD4. Furthermore, Salp15 prevents syncytia formation between HL2/3 (a stable HeLa cell line expressing the envelope protein) and CD4-expressing cells. Salp15 prevented gp120-CD4 interaction at least partially through its direct interactio...

  5. Proteomic analysis of HIV-1 Nef cellular binding partners reveals a role for exocyst complex proteins in mediating enhancement of intercellular nanotube formation

    Directory of Open Access Journals (Sweden)

    Mukerji Joya

    2012-06-01

    Full Text Available Abstract Background HIV-1 Nef protein contributes to pathogenesis via multiple functions that include enhancement of viral replication and infectivity, alteration of intracellular trafficking, and modulation of cellular signaling pathways. Nef stimulates formation of tunneling nanotubes and virological synapses, and is transferred to bystander cells via these intercellular contacts and secreted microvesicles. Nef associates with and activates Pak2, a kinase that regulates T-cell signaling and actin cytoskeleton dynamics, but how Nef promotes nanotube formation is unknown. Results To identify Nef binding partners involved in Pak2-association dependent Nef functions, we employed tandem mass spectrometry analysis of Nef immunocomplexes from Jurkat cells expressing wild-type Nef or Nef mutants defective for the ability to associate with Pak2 (F85L, F89H, H191F and A72P, A75P in NL4-3. We report that wild-type, but not mutant Nef, was associated with 5 components of the exocyst complex (EXOC1, EXOC2, EXOC3, EXOC4, and EXOC6, an octameric complex that tethers vesicles at the plasma membrane, regulates polarized exocytosis, and recruits membranes and proteins required for nanotube formation. Additionally, Pak2 kinase was associated exclusively with wild-type Nef. Association of EXOC1, EXOC2, EXOC3, and EXOC4 with wild-type, but not mutant Nef, was verified by co-immunoprecipitation assays in Jurkat cells. Furthermore, shRNA-mediated depletion of EXOC2 in Jurkat cells abrogated Nef-mediated enhancement of nanotube formation. Using bioinformatic tools, we visualized protein interaction networks that reveal functional linkages between Nef, the exocyst complex, and the cellular endocytic and exocytic trafficking machinery. Conclusions Exocyst complex proteins are likely a key effector of Nef-mediated enhancement of nanotube formation, and possibly microvesicle secretion. Linkages revealed between Nef and the exocyst complex suggest a new paradigm of

  6. Endogenous MCM7 microRNA cluster as a novel platform to multiplex small interfering and nucleolar RNAs for combinational HIV-1 gene therapy.

    Science.gov (United States)

    Chung, Janet; Zhang, Jane; Li, Haitang; Ouellet, Dominique L; DiGiusto, David L; Rossi, John J

    2012-11-01

    Combinational therapy with small RNA inhibitory agents against multiple viral targets allows efficient inhibition of viral production by controlling gene expression at critical time points. Here we explore combinations of different classes of therapeutic anti-HIV-1 RNAs expressed from within the context of an intronic MCM7 (minichromosome maintenance complex component-7) platform that naturally harbors 3 microRNAs (miRNAs). We replaced the endogenous miRNAs with anti-HIV small RNAs, including small interfering RNAs (siRNAs) targeting HIV-1 tat and rev messages that function to induce post-transcriptional gene silencing by the RNA interference pathway, a nucleolar-localizing RNA ribozyme that targets the conserved U5 region of HIV-1 transcripts for degradation, and finally nucleolar trans-activation response (TAR) and Rev-binding element (RBE) RNA decoys designed to sequester HIV-1 Tat and Rev proteins inside the nucleolus. We demonstrate the versatility of the MCM7 platform in expressing and efficiently processing the siRNAs as miRNA mimics along with nucleolar small RNAs. Furthermore, three of the combinatorial constructs tested potently suppressed viral replication during a 1-month HIV challenge, with greater than 5-log inhibition compared with untransduced, HIV-1-infected CEM T lymphocytes. One of the most effective constructs contains an anti-HIV siRNA combined with a nucleolar-localizing U5 ribozyme and TAR decoy. This represents the first efficacious example of combining Drosha-processed siRNAs with small nucleolar ribonucleoprotein (snoRNP)-processed nucleolar RNA chimeras from a single intron platform for effective inhibition of viral replication. Moreover, we demonstrated enrichment/selection for cells expressing levels of the antiviral RNAs that provide optimal inhibition under the selective pressure of HIV. The combinations of si/snoRNAs represent a new paradigm for combinatorial RNA-based gene therapy applications.

  7. Enhancing T cell activation and antiviral protection by introducing the HIV-1 protein transduction domain into a DNA vaccine.

    Science.gov (United States)

    Leifert, J A; Lindencrona, J A; Charo, J; Whitton, J L

    2001-10-10

    Protein transduction domains (PTD), which can transport proteins or peptides across biological membranes, have been identified in several proteins of viral, invertebrate, and vertebrate origin. Here, we evaluate the immunological and biological consequences of including PTD in synthetic peptides and in DNA vaccines that contain CD8(+) T cell epitopes from lymphocytic choriomeningitis virus (LCMV). Synthetic PTD-peptides did not induce detectable CD8(+) T cell responses. However, fusion of an open reading frame encoding a PTD to an epitope minigene caused transfected tissue culture cells to stimulate epitope-specific T cells much more effectively. Kinetic studies indicated that the epitope reached the surface of transfected cells more rapidly and that the number of transfected cells needed to stimulate T cell responses was reduced by 35- to 50-fold when compared to cells transfected with a standard minigene plasmid. The mechanism underlying the effect of PTD linkage is not clear, but transit of the PTD-attached epitope from transfected cells to nontransfected cells (cross presentation) seemed to play, at most, a minimal role. Mice immunized once with the plasmid encoding the PTD-linked epitope showed a markedly accelerated CD8(+) T cell response and, unlike mice immunized with a standard plasmid, were completely protected against a normally lethal LCMV challenge administered only 8 days post-immunization.

  8. High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus

    Directory of Open Access Journals (Sweden)

    Bianco Linda

    2009-11-01

    Full Text Available Abstract Background In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein. In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in Nicotiana benthamiana, followed by a two-step affinity purification protocol of plant-derived Nef. Results The effect of three gene silencing viral suppressor proteins (P25 of Potato Virus X, P19 of either Artichoke Mottled Crinckle virus and Tomato Bushy Stunt virus on Nef transient expression yield was evaluated. The P19 protein of Artichoke Mottled Crinckle virus (AMCV-P19 gave the highest expression yield in vacuum co-agroinfiltration experiments reaching 1.3% of total soluble protein, a level almost three times higher than that previously reported in stable transgenic plants. The high yield observed in the co-agroinfiltrated plants was correlated to a remarkable decrease of Nef-specific small interfering RNAs (siRNAs indicating an effective modulation of RNA silencing mechanisms by AMCV-P19. Interestingly, we also showed that expression levels in top leaves of vacuum co-agroinfiltrated plants were noticeably reduced compared to bottom leaves. Moreover, purification of Nef from agroinfiltrated tissue was achieved by a two-step immobilized metal ion affinity chromatography protocol with yields of 250 ng/g of fresh tissue. Conclusion We demonstrated that expression level of HIV-1 Nef in plant can be improved using a transient expression system enhanced by the AMCV-P19 gene silencing suppressor

  9. Induction of human immunodeficiency virus (HIV-1 envelope specific cell-mediated immunity by a non-homologous synthetic peptide.

    Directory of Open Access Journals (Sweden)

    Ammar Achour

    2007-11-01

    Full Text Available Cell mediated immunity, including efficient CTL response, is required to prevent HIV-1 from cell-to-cell transmission. In previous investigations, we have shown that B1 peptide derived by Fourier transformation of HIV-1 primary structures and sharing no sequence homology with the parent proteins was able to generate antiserum which recognizes envelope and Tat proteins. Here we have investigated cellular immune response towards a novel non-homologous peptide, referred to as cA1 peptide.The 20 amino acid sequence of cA1 peptide was predicted using the notion of peptide hydropathic properties; the peptide is encoded by the complementary anti-sense DNA strand to the sense strand of previously described non-homologous A1 peptide. In this report we demonstrate that the cA1 peptide can be a target for major histocompatibility complex (MHC class I-restricted cytotoxic T lymphocytes in HIV-1-infected or envelope-immunized individuals. The cA1 peptide is recognized in association with different MHC class I allotypes and could prime in vitro CTLs, derived from gp160-immunized individuals capable to recognize virus variants.For the first time a theoretically designed immunogen involved in broad-based cell-immune memory activation is described. Our findings may thus contribute to the advance in vaccine research by describing a novel strategy to develop a synthetic AIDS vaccine.

  10. Site-selective probing of cTAR destabilization highlights the necessary plasticity of the HIV-1 nucleocapsid protein to chaperone the first strand transfer

    Science.gov (United States)

    Godet, Julien; Kenfack, Cyril; Przybilla, Frédéric; Richert, Ludovic; Duportail, Guy; Mély, Yves

    2013-01-01

    The HIV-1 nucleocapsid protein (NCp7) is a nucleic acid chaperone required during reverse transcription. During the first strand transfer, NCp7 is thought to destabilize cTAR, the (−)DNA copy of the TAR RNA hairpin, and subsequently direct the TAR/cTAR annealing through the zipping of their destabilized stem ends. To further characterize the destabilizing activity of NCp7, we locally probe the structure and dynamics of cTAR by steady-state and time resolved fluorescence spectroscopy. NC(11–55), a truncated NCp7 version corresponding to its zinc-finger domain, was found to bind all over the sequence and to preferentially destabilize the penultimate double-stranded segment in the lower part of the cTAR stem. This destabilization is achieved through zinc-finger–dependent binding of NC to the G10 and G50 residues. Sequence comparison further revealed that C•A mismatches close to the two G residues were critical for fine tuning the stability of the lower part of the cTAR stem and conferring to G10 and G50 the appropriate mobility and accessibility for specific recognition by NC. Our data also highlight the necessary plasticity of NCp7 to adapt to the sequence and structure variability of cTAR to chaperone its annealing with TAR through a specific pathway. PMID:23511968

  11. Nullbasic, a potent anti-HIV tat mutant, induces CRM1-dependent disruption of HIV rev trafficking.

    Directory of Open Access Journals (Sweden)

    Min-Hsuan Lin

    Full Text Available Nullbasic, a mutant of the HIV-1 Tat protein, has anti-HIV-1 activity through mechanisms that include inhibition of Rev function and redistribution of the HIV-1 Rev protein from the nucleolus to the nucleoplasm and cytoplasm. Here we investigate the mechanism of this effect for the first time, establishing that redistribution of Rev by Nullbasic is not due to direct interaction between the two proteins. Rather, Nullbasic affects subcellular localization of cellular proteins that regulate Rev trafficking. In particular, Nullbasic induced redistribution of exportin 1 (CRM1, nucleophosmin (B23 and nucleolin (C23 from the nucleolus to the nucleus when Rev was coexpressed, but never in its absence. Inhibition of the Rev:CRM1 interaction by leptomycin B or a non-interacting RevM10 mutant completely blocked redistribution of Rev by Nullbasic. Finally, Nullbasic did not inhibit importin β- or transportin 1-mediated nuclear import, suggesting that cytoplasmic accumulation of Rev was due to increased export by CRM1. Overall, our data support the conclusion that CRM1-dependent subcellular redistribution of Rev from the nucleolus by Nullbasic is not through general perturbation of either nuclear import or export. Rather, Nullbasic appears to interact with and disrupt specific components of a Rev trafficking complex required for its nucleocytoplasmic shuttling and, in particular, its nucleolar accumulation.

  12. Tat-dependent repression of human immunodeficiency virus type 1 long terminal repeat promoter activity by fusion of cellular transcription factors

    International Nuclear Information System (INIS)

    Zhao Cunyou; Chen Yali; Park, Jiyoung; Kim, Jae Bum; Tang Hong

    2004-01-01

    Transcription initiation from HIV-1 long terminal repeat (LTR) promoter requires the virally encoded transactivator, Tat, and several cellular co-factors to accomplish the Tat-dependent processive transcription elongation. Individual cellular transcription activators, LBP-1b and Oct-1, on the other hand, have been shown to inhibit LTR promoter activities probably via competitive binding against TFIID to the TATA-box in LTR promoter. To explore the genetic interference strategies against the viral replication, we took advantage of the existence of the bipartite DNA binding domains and the repression domains of LBP-1b and Oct-1 factors to generate a chimeric transcription repressor. Our results indicated that the fusion protein of LBP-1b and Oct-1 exhibited higher DNA binding affinity to the viral promoter than the individual factors, and little interference with the host cell gene expression due to its anticipated rare cognate DNA sites in the host cell genome. Moreover, the chimera exerted increased Tat-dependent repression of transcription initiation at the LTR promoter both in vitro and in vivo compared to LBP-1b, Oct-1 or combination of LBP-1b and Oct-1. These results might provide the lead in generating a therapeutic reagent useful to suppress HIV-1 replication

  13. Effects of integration and replication on transcription of the HIV-1 long terminal repeat

    NARCIS (Netherlands)

    Jeang, K. T.; Berkhout, B.; Dropulic, B.

    1993-01-01

    The activity of a promoter is influenced by chromosomal and cell cycle/replication context. We analyzed the influences of integration and replication on transcription of the human immunodeficiency virus (HIV)-1 long terminal repeat (LTR). We found that one requirement for Tat trans-activated

  14. Imperfect DNA mirror repeats in the gag gene of HIV-1 (HXB2 identify key functional domains and coincide with protein structural elements in each of the mature proteins

    Directory of Open Access Journals (Sweden)

    Lang Dorothy M

    2007-10-01

    Full Text Available Abstract Background A DNA mirror repeat is a sequence segment delimited on the basis of its containing a center of symmetry on a single strand, e.g. 5'-GCATGGTACG-3'. It is most frequently described in association with a functionally significant site in a genomic sequence, and its occurrence is regarded as noteworthy, if not unusual. However, imperfect mirror repeats (IMRs having ≥ 50% symmetry are common in the protein coding DNA of monomeric proteins and their distribution has been found to coincide with protein structural elements – helices, β sheets and turns. In this study, the distribution of IMRs is evaluated in a polyprotein – to determine whether IMRs may be related to the position or order of protein cleavage or other hierarchal aspects of protein function. The gag gene of HIV-1 [GenBank:K03455] was selected for the study because its protein motifs and structural components are well documented. Results There is a highly specific relationship between IMRs and structural and functional aspects of the Gag polyprotein. The five longest IMRs in the polyprotein translate a key functional segment in each of the five cleavage products. Throughout the protein, IMRs coincide with functionally significant segments of the protein. A detailed annotation of the protein, which combines structural, functional and IMR data illustrates these associations. There is a significant statistical correlation between the ends of IMRs and the ends of PSEs in each of the mature proteins. Weakly symmetric IMRs (≥ 33% are related to cleavage positions and processes. Conclusion The frequency and distribution of IMRs in HIV-1 Gag indicates that DNA symmetry is a fundamental property of protein coding DNA and that different levels of symmetry are associated with different functional aspects of the gene and its protein. The interaction between IMRs and protein structure and function is precise and interwoven over the entire length of the polyprotein. The

  15. HIV-1 Replication and the Cellular Eukaryotic Translation Apparatus

    Directory of Open Access Journals (Sweden)

    Santiago Guerrero

    2015-01-01

    Full Text Available Eukaryotic translation is a complex process composed of three main steps: initiation, elongation, and termination. During infections by RNA- and DNA-viruses, the eukaryotic translation machinery is used to assure optimal viral protein synthesis. Human immunodeficiency virus type I (HIV-1 uses several non-canonical pathways to translate its own proteins, such as leaky scanning, frameshifting, shunt, and cap-independent mechanisms. Moreover, HIV-1 modulates the host translation machinery by targeting key translation factors and overcomes different cellular obstacles that affect protein translation. In this review, we describe how HIV-1 proteins target several components of the eukaryotic translation machinery, which consequently improves viral translation and replication.

  16. Cold denaturation of the HIV-1 protease monomer

    DEFF Research Database (Denmark)

    Rösner, Heike Ilona; Caldarini, Martina; Prestel, Andreas

    2017-01-01

    The HIV-1-protease is a complex protein which in its active form adopts a homodimer dominated by -sheet structures. We have discovered a cold-denatured state of the monomeric subunit of HIV-1-protease which is populated above 0ºC and therefore directly accessible to various spectroscopic approac...

  17. An MHC-I cytoplasmic domain/HIV-1 Nef fusion protein binds directly to the mu subunit of the AP-1 endosomal coat complex.

    Directory of Open Access Journals (Sweden)

    Rajendra Kumar Singh

    2009-12-01

    Full Text Available The down-regulation of the major histocompatibility complex class I (MHC-I from the surface of infected cells by the Nef proteins of primate immunodeficiency viruses likely contributes to pathogenesis by providing evasion of cell-mediated immunity. HIV-1 Nef-induced down-regulation involves endosomal trafficking and a cooperative interaction between the cytoplasmic domain (CD of MHC-I, Nef, and the clathrin adaptor protein complex-1 (AP-1. The CD of MHC-I contains a key tyrosine within the sequence YSQA that is required for down-regulation by Nef, but this sequence does not conform to the canonical AP-binding tyrosine-based motif Yxxphi, which mediates binding to the medium (micro subunits of AP complexes. We previously proposed that Nef allows the MHC-I CD to bind the mu subunit of AP-1 (micro1 as if it contained a Yxxphimotif.Here, we show that a direct interaction between the MHC-I CD/Nef and micro1 plays a primary role in the down-regulation of MHC-I: GST pulldown assays using recombinant proteins indicated that most of the MHC-I CD and Nef residues that are required for the down-regulation in human cells contribute to direct interactions with a truncated version of micro1. Specifically, the tyrosine residue of the YSQA sequence in the MHC-I CD as well as Nef residues E62-65 and P78 each contributed to the interaction between MHC-I CD/Nef and micro1 in vitro, whereas Nef M20 had little to no role. Conversely, residues F172/D174 and V392/L395 of the binding pocket on micro1 for Yxxphi motifs were required for a robust interaction.These data indicate that the MHC-I cytoplasmic domain, Nef, and the C-terminal two thirds of the mu subunit of AP-1 are sufficient to constitute a biologically relevant interaction. The data also reveal an unexpected role for a hydrophobic pocket in micro1 for interaction with MHC-I CD/Nef.

  18. CTL epitope distribution patterns in the Gag and Nef proteins of HIV-1 from subtype A infected subjects in Kenya: Use of multiple peptide sets increases the detectable breadth of the CTL response

    Directory of Open Access Journals (Sweden)

    Birx Deborah L

    2006-04-01

    Full Text Available Abstract Background Subtype A is a major strain in the HIV-1 pandemic in eastern Europe, central Asia and in certain regions of east Africa, notably in rural Kenya. While considerable effort has been focused upon mapping and defining immunodominant CTL epitopes in HIV-1 subtype B and subtype C infections, few epitope mapping studies have focused upon subtype A. Results We have used the IFN-γ ELIspot assay and overlapping peptide pools to show that the pattern of CTL recognition of the Gag and Nef proteins in subtype A infection is similar to that seen in subtypes B and C. The p17 and p24 proteins of Gag and the central conserved region of Nef were targeted by CTL from HIV-1-infected Kenyans. Several epitope/HLA associations commonly seen in subtype B and C infection were also observed in subtype A infections. Notably, an immunodominant HLA-C restricted epitope (Gag 296–304; YL9 was observed, with 8/9 HLA-CW0304 subjects responding to this epitope. Screening the cohort with peptide sets representing subtypes A, C and D (the three most prevalent HIV-1 subtypes in east Africa, revealed that peptide sets based upon an homologous subtype (either isolate or consensus only marginally improved the capacity to detect CTL responses. While the different peptide sets detected a similar number of responses (particularly in the Gag protein, each set was capable of detecting unique responses not identified with the other peptide sets. Conclusion Hence, screening with multiple peptide sets representing different sequences, and by extension different epitope variants, can increase the detectable breadth of the HIV-1-specific CTL response. Interpreting the true extent of cross-reactivity may be hampered by the use of 15-mer peptides at a single concentration and a lack of knowledge of the sequence that primed any given CTL response. Therefore, reagent choice and knowledge of the exact sequences that prime CTL responses will be important factors in

  19. Immunization with Clinical HIV-1 Env Proteins Induces Broad Antibody Dependent Cellular Cytotoxicity-Mediating Antibodies in a Rabbit Vaccination Model

    DEFF Research Database (Denmark)

    Karlsson, Ingrid; Borggren, Marie; Jensen, Sanne Skov

    2018-01-01

    The induction of both neutralizing antibodies and non-neutralizing antibodies with effector functions, for example, antibody-dependent cellular cytotoxicity (ADCC), is desired in the search for effective vaccines against HIV-1. In the pursuit of novel immunogens capable of inducing an efficient a...

  20. HIV-1 splicing is controlled by local RNA structure and binding of splicing regulatory proteins at the major 5' splice site

    NARCIS (Netherlands)

    Mueller, Nancy; Berkhout, Ben; Das, Atze T.

    2015-01-01

    The 5' leader region of the human immunodeficiency virus 1 (HIV-1) RNA genome contains the major 5' splice site (ss) that is used in the production of the many spliced viral RNAs. This splice-donor (SD) region can fold into a stable stem-loop structure and the thermodynamic stability of this RNA

  1. Quantitative proteomic analysis of HIV-1 infected CD4+ T cells reveals an early host response in important biological pathways: Protein synthesis, cell proliferation, and T-cell activation

    Energy Technology Data Exchange (ETDEWEB)

    Navare, Arti T.; Sova, Pavel; Purdy, David E.; Weiss, Jeffrey M. [Department of Microbiology, University of Washington, Seattle, WA (United States); Wolf-Yadlin, Alejandro [Department of Genome Sciences, University of Washington, Seattle, WA (United States); Korth, Marcus J.; Chang, Stewart T.; Proll, Sean C. [Department of Microbiology, University of Washington, Seattle, WA (United States); Jahan, Tahmina A. [Proteomics Resource, UW Medicine at South Lake Union, Seattle, WA (United States); Krasnoselsky, Alexei L.; Palermo, Robert E. [Department of Microbiology, University of Washington, Seattle, WA (United States); Katze, Michael G., E-mail: honey@uw.edu [Department of Microbiology, University of Washington, Seattle, WA (United States); Washington National Primate Research Center, University of Washington, Seattle, WA (United States)

    2012-07-20

    Human immunodeficiency virus (HIV-1) depends upon host-encoded proteins to facilitate its replication while at the same time inhibiting critical components of innate and/or intrinsic immune response pathways. To characterize the host cell response on protein levels in CD4+ lymphoblastoid SUP-T1 cells after infection with HIV-1 strain LAI, we used mass spectrometry (MS)-based global quantitation with iTRAQ (isobaric tag for relative and absolute quantification). We found 266, 60 and 22 proteins differentially expressed (DE) (P-value{<=}0.05) at 4, 8, and 20 hours post-infection (hpi), respectively, compared to time-matched mock-infected samples. The majority of changes in protein abundance occurred at an early stage of infection well before the de novo production of viral proteins. Functional analyses of these DE proteins showed enrichment in several biological pathways including protein synthesis, cell proliferation, and T-cell activation. Importantly, these early changes before the time of robust viral production have not been described before.

  2. Novel host restriction factors implicated in HIV-1 replication.

    Science.gov (United States)

    Ghimire, Dibya; Rai, Madhu; Gaur, Ritu

    2018-04-01

    Human immunodeficiency virus-1 (HIV-1) is known to interact with multiple host cellular proteins during its replication in the target cell. While many of these host cellular proteins facilitate viral replication, a number of them are reported to inhibit HIV-1 replication at various stages of its life cycle. These host cellular proteins, which are known as restriction factors, constitute an integral part of the host's first line of defence against the viral pathogen. Since the discovery of apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G) as an HIV-1 restriction factor, several human proteins have been identified that exhibit anti-HIV-1 restriction. While each restriction factor employs a distinct mechanism of inhibition, the HIV-1 virus has equally evolved complex counter strategies to neutralize their inhibitory effect. APOBEC3G, tetherin, sterile alpha motif and histidine-aspartate domain 1 (SAMHD1), and trim-5α are some of the best known HIV-1 restriction factors that have been studied in great detail. Recently, six novel restriction factors were discovered that exhibit significant antiviral activity: endoplasmic reticulum α1,2-mannosidase I (ERManI), translocator protein (TSPO), guanylate-binding protein 5 (GBP5), serine incorporator (SERINC3/5) and zinc-finger antiviral protein (ZAP). The focus of this review is to discuss the antiviral mechanism of action of these six restriction factors and provide insights into the probable counter-evasion strategies employed by the HIV-1 virus. The recent discovery of new restriction factors substantiates the complex host-pathogen interactions occurring during HIV-1 pathogenesis and makes it imperative that further investigations are conducted to elucidate the molecular basis of HIV-1 replication.

  3. Artificial 64-Residue HIV-1 Enhancer-Binding Peptide Is a Potent Inhibitor of Viral Replication in HIV-1-Infected Cells.

    Science.gov (United States)

    Oufir, Mouhssin; Bisset, Leslie R; Hoffmann, Stefan R K; Xue, Gongda; Klauser, Stephan; Bergamaschi, Bianca; Gervaix, Alain; Böni, Jürg; Schüpbach, Jörg; Gutte, Bernd

    2011-01-01

    An artificial HIV-1 enhancer-binding peptide was extended by nine consecutive arginine residues at the C-terminus and by the nuclear localization signal of SV40 large T antigen at the N-terminus. The resulting synthetic 64-residue peptide was found to bind to the two enhancers of the HIV-1 long terminal repeat, cross the plasma membrane and the nuclear envelope of human cells, and suppress the HIV-1 enhancer-controlled expression of a green fluorescent protein reporter gene. Moreover, HIV-1 replication is inhibited by this peptide in HIV-1-infected CEM-GFP cells as revealed by HIV-1 p24 ELISA and real-time RT-PCR of HIV-1 RNA. Rapid uptake of this intracellular stable and inhibitory peptide into the cells implies that this peptide may have the potential to attenuate HIV-1 replication in vivo.

  4. Artificial 64-Residue HIV-1 Enhancer-Binding Peptide Is a Potent Inhibitor of Viral Replication in HIV-1-Infected Cells

    Directory of Open Access Journals (Sweden)

    Mouhssin Oufir

    2011-01-01

    Full Text Available An artificial HIV-1 enhancer-binding peptide was extended by nine consecutive arginine residues at the C-terminus and by the nuclear localization signal of SV40 large T antigen at the N-terminus. The resulting synthetic 64-residue peptide was found to bind to the two enhancers of the HIV-1 long terminal repeat, cross the plasma membrane and the nuclear envelope of human cells, and suppress the HIV-1 enhancer-controlled expression of a green fluorescent protein reporter gene. Moreover, HIV-1 replication is inhibited by this peptide in HIV-1-infected CEM-GFP cells as revealed by HIV-1 p24 ELISA and real-time RT-PCR of HIV-1 RNA. Rapid uptake of this intracellular stable and inhibitory peptide into the cells implies that this peptide may have the potential to attenuate HIV-1 replication in vivo.

  5. Hepatitis B virus (HBV)-specific T-cell responses to recombinant HBV core protein in patients with normal liver function and co-infected with chronic HBV and human immunodeficiency virus 1 (HIV-1)

    Science.gov (United States)

    2013-01-01

    Background Little is known about HBV-specific T-cell responses in chronic Hepatitis B patients (HBV) that are co-infected with Human immunodeficiency virus type 1 (HIV-1), especially those with normal alanine aminotransferase (ALT) levels. Methods Twenty-five patients with chronic HBV (11 hepatitis B e antigen [HBeAg]-positive, 14 HBeAg-negative) were enrolled in a cross-sectional study. A longitudinal study as also conducted in which follow-up was done at 3, 12, and 24 months, after acute HIV-1 infection, in 11 individuals who also had chronic HBV. Peripheral blood mononuclear cells were stimulated with recombinant HBV surface protein (S protein), core protein (C protein) or gag peptide. IFN-γ-secreting T cells were identified by ELISPOT assay. Results In the cross-sectional study, co-infected chronic HBV patients had lower C protein-specific T-cell responses compared with mono-infected individuals, though the difference was not significant. In co-infected, chronic HBV patients, the magnitude of C protein-specific T-cell responses was significantly greater in HBeAg-positive subjects compared to HBeAg-negative subjects (p = 0.011). C protein-specific T-cell responses were positively correlated with HBV viral load (rs = 0.40, p = 0.046). However, gag-specific T-cell responses were negatively correlated with HIV viral load (rs = −0.44, p = 0.026) and positively correlated with CD4+ count (rs = 0.46, p = 0.021). The results were different in mono-infected individuals. PBMCs from co-infected HBeAg-positive patients secreted more specific-IFN-γ in cultured supernatants compared with PBMCs from co-infected HBeAg-negative patients (p = 0.019). In the longitudinal study, S protein- and C protein-specific T-cell responses were decreased as the length of follow-up increased (p = 0.034, for S protein; p = 0.105, for C protein). Additionally, the S protein- and C protein-specific T-cell responses were significantly higher in HBe

  6. Distribution of HIV-1 resistance-conferring polymorphic alleles SDF ...

    Indian Academy of Sciences (India)

    Unknown

    involved with delay in disease progression. ... proteins in 525 healthy individuals without any history of HIV-1 infection from 11 diverse populations of ... in three populations (Yamani, Pathan and Kamma), all in low frequencies (i.e. 1% to 3%).

  7. Genetic architecture of HIV-1 genes circulating in north India & their functional implications.

    Science.gov (United States)

    Neogi, Ujjwal; Sood, Vikas; Ronsard, Larence; Singh, Jyotsna; Lata, Sneh; Ramachandran, V G; Das, S; Wanchu, Ajay; Banerjea, Akhil C

    2011-12-01

    This review presents data on genetic and functional analysis of some of the HIV-1 genes derived from HIV-1 infected individuals from north India (Delhi, Punjab and Chandigarh). We found evidence of novel B/C recombinants in HIV-1 LTR region showing relatedness to China/Myanmar with 3 copies of Nfκb sites; B/C/D mosaic genomes for HIV-1 Vpr and novel B/C Tat. We reported appearance of a complex recombinant form CRF_02AG of HIV-1 envelope sequences which is predominantly found in Central/Western Africa. Also one Indian HIV-1 envelope subtype C sequence suggested exclusive CXCR4 co-receptor usage. This extensive recombination, which is observed in about 10 per cent HIV-1 infected individuals in the Vpr genes, resulted in remarkably altered functions when compared with prototype subtype B Vpr. The Vpu C was found to be more potent in causing apoptosis when compared with Vpu B when analyzed for subG1 DNA content. The functional implications of these changes as well as in other genes of HIV-1 are discussed in detail with possible implications for subtype-specific pathogenesis highlighted.

  8. Antiviral Therapy by HIV-1 Broadly Neutralizing and Inhibitory Antibodies

    Directory of Open Access Journals (Sweden)

    Zhiqing Zhang

    2016-11-01

    Full Text Available Human immunodeficiency virus type 1 (HIV-1 infection causes acquired immune deficiency syndrome (AIDS, a global epidemic for more than three decades. HIV-1 replication is primarily controlled through antiretroviral therapy (ART but this treatment does not cure HIV-1 infection. Furthermore, there is increasing viral resistance to ART, and side effects associated with long-term therapy. Consequently, there is a need of alternative candidates for HIV-1 prevention and therapy. Recent advances have discovered multiple broadly neutralizing antibodies against HIV-1. In this review, we describe the key epitopes on the HIV-1 Env protein and the reciprocal broadly neutralizing antibodies, and discuss the ongoing clinical trials of broadly neutralizing and inhibitory antibody therapy as well as antibody combinations, bispecific antibodies, and methods that improve therapeutic efficacy by combining broadly neutralizing antibodies (bNAbs with latency reversing agents. Compared with ART, HIV-1 therapeutics that incorporate these broadly neutralizing and inhibitory antibodies offer the advantage of decreasing virus load and clearing infected cells, which is a promising prospect in HIV-1 prevention and treatment.

  9. Structural Study of a New HIV-1 Entry Inhibitor and Interaction with the HIV-1 Fusion Peptide in Dodecylphosphocholine Micelles.

    Science.gov (United States)

    Pérez, Yolanda; Gómara, Maria José; Yuste, Eloísa; Gómez-Gutierrez, Patricia; Pérez, Juan Jesús; Haro, Isabel

    2017-08-25

    Previous studies support the hypothesis that the envelope GB virus C (GBV-C) E1 protein interferes the HIV-1 entry and that a peptide, derived from the region 139-156 of this protein, has been defined as a novel HIV-1 entry inhibitor. In this work, we firstly focus on the characterization of the structural features of this peptide, which are determinant for its anti-HIV-1 activity and secondly, on the study of its interaction with the proposed viral target (i.e., the HIV-1 fusion peptide). We report the structure of the peptide determined by NMR spectroscopy in dodecylphosphocholine (DPC) micelles solved by using restrained molecular dynamics calculations. The acquisition of different NMR experiments in DPC micelles (i.e., peptide-peptide titration, diffusion NMR spectroscopy, and addition of paramagnetic relaxation agents) allows a proposal of an inhibition mechanism. We conclude that a 18-mer peptide from the non-pathogenic E1 GBV-C protein, with a helix-turn-helix structure inhibits HIV-1 by binding to the HIV-1 fusion peptide at the membrane level, thereby interfering with those domains in the HIV-1, which are critical for stabilizing the six-helix bundle formation in a membranous environment. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Sequence similarity between the erythrocyte binding domain 1 of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals binding residues for the Duffy Antigen Receptor for Chemokines

    OpenAIRE

    Bolton, Michael J; Garry, Robert F

    2011-01-01

    Abstract Background The surface glycoprotein (SU, gp120) of the human immunodeficiency virus (HIV) must bind to a chemokine receptor, CCR5 or CXCR4, to invade CD4+ cells. Plasmodium vivax uses the Duffy Binding Protein (DBP) to bind the Duffy Antigen Receptor for Chemokines (DARC) and invade reticulocytes. Results Variable loop 3 (V3) of HIV-1 SU and domain 1 of the Plasmodium vivax DBP share a sequence similarity. The site of amino acid sequence similarity was necessary, but not sufficient, ...

  11. Sequence similarity between the erythrocyte binding domain 1 of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals binding residues for the Duffy Antigen Receptor for Chemokines

    Directory of Open Access Journals (Sweden)

    Garry Robert F

    2011-01-01

    Full Text Available Abstract Background The surface glycoprotein (SU, gp120 of the human immunodeficiency virus (HIV must bind to a chemokine receptor, CCR5 or CXCR4, to invade CD4+ cells. Plasmodium vivax uses the Duffy Binding Protein (DBP to bind the Duffy Antigen Receptor for Chemokines (DARC and invade reticulocytes. Results Variable loop 3 (V3 of HIV-1 SU and domain 1 of the Plasmodium vivax DBP share a sequence similarity. The site of amino acid sequence similarity was necessary, but not sufficient, for DARC binding and contained a consensus heparin binding site essential for DARC binding. Both HIV-1 and P. vivax can be blocked from binding to their chemokine receptors by the chemokine, RANTES and its analog AOP-RANTES. Site directed mutagenesis of the heparin binding motif in members of the DBP family, the P. knowlesi alpha, beta and gamma proteins abrogated their binding to erythrocytes. Positively charged residues within domain 1 are required for binding of P. vivax and P. knowlesi erythrocyte binding proteins. Conclusion A heparin binding site motif in members of the DBP family may form part of a conserved erythrocyte receptor binding pocket.

  12. Molecular Basis for Drug Resistance in HIV-1 Protease

    Directory of Open Access Journals (Sweden)

    Celia A. Schiffer

    2010-11-01

    Full Text Available HIV-1 protease is one of the major antiviral targets in the treatment of patients infected with HIV-1. The nine FDA approved HIV-1 protease inhibitors were developed with extensive use of structure-based drug design, thus the atomic details of how the inhibitors bind are well characterized. From this structural understanding the molecular basis for drug resistance in HIV-1 protease can be elucidated. Selected mutations in response to therapy and diversity between clades in HIV-1 protease have altered the shape of the active site, potentially altered the dynamics and even altered the sequence of the cleavage sites in the Gag polyprotein. All of these interdependent changes act in synergy to confer drug resistance while simultaneously maintaining the fitness of the virus. New strategies, such as incorporation of the substrate envelope constraint to design robust inhibitors that incorporate details of HIV-1 protease’s function and decrease the probability of drug resistance, are necessary to continue to effectively target this key protein in HIV-1 life cycle.

  13. Extracellular histones identified in crocodile blood inhibit in-vitro HIV-1 infection.

    Science.gov (United States)

    Kozlowski, Hannah N; Lai, Eric T L; Havugimana, Pierre C; White, Carl; Emili, Andrew; Sakac, Darinka; Binnington, Beth; Neschadim, Anton; McCarthy, Stephen D S; Branch, Donald R

    2016-08-24

    It has been reported that crocodile blood contains potent antibacterial and antiviral properties. However, its effects on HIV-1 infection remain unknown. We obtained blood from saltwater crocodiles to examine whether serum or plasma could inhibit HIV-1 infection. We purified plasma fractions then used liquid chromatography-mass spectrometry to identify the inhibitory protein factor(s). We then analyzed the ability of recombinant proteins to recapitulate HIV-1 inhibition and determine their mechanism of action. Crocodylus porosus plasma was tested for inhibition of Jurkat T-cell HIV-1 infection. Inhibitor(s) were purified by reverse-phase chromatography then identified by protein liquid chromatography-mass spectrometry. Anti-HIV-1 activity of purified plasma or recombinant proteins were measured by p24 enzyme-linked immunosorbent assay and luciferase readouts, and mechanism of action was determined by measuring HIV-1 RNA, cDNA and transcription (using 1G5 cells). Crocodile plasma contains potent inhibitors of HIV-1IIIB infection, which were identified as histones. Recombinant human histones H1 and H2A significantly reduced HIV-1JR-FL infection (IC50 of 0.79 and 0.45 μmol/l, respectively), whereas H4 enhanced JR-FL luciferase activity. The inhibitory effects of crocodile plasma, recombinant H1 or recombinant H2A on HIV-1 infection were during or post-viral transcription. Circulating histones in crocodile blood, possibly released by neutrophil extracellular traps, are significant inhibitors of HIV-1 infection in-vitro. Extracellular recombinant histones have different effects on HIV-1 transcription and protein expression and are downregulated in HIV-1 patients. Circulating histones may be a novel resistance factor during HIV-1 infection, and peptide versions should be explored as future HIV-1 therapeutics that modulate viral transcription.

  14. Morphogenesis of the infectious HIV-1 virion

    Directory of Open Access Journals (Sweden)

    Jun-Ichi eSakuragi

    2011-12-01

    Full Text Available The virion of HIV-1 is spherical and viral glycoprotein spikes (gp120, gp41 protrude from its envelope. The characteristic cone-shaped core exists within the virion, caging the ribonucleoprotein (RNP complex, which is comprised of viral RNA, nucleocapsid (NC and viral enzymes. The HIV-1 virion is budded and released from the infected cell as an immature donut-shaped particle. During or immediately after release, viral protease (PR is activated and subsequently processes the viral structural protein Gag. Through this maturation process, virions acquire infectivity, but its mechanism and transition of morphology largely remain unclear. Recent technological advances in experimental devices and techniques have made it possible to closely dissect the viral production site on the cell, the exterior – or even the interior – of an individual virion, and many new aspects on virion morphology and maturation. In this manuscript, I review the morphogenesis of HIV-1 virions. I focus on several studies, including some of our recent findings, which examined virion formation and/or maturation processes. The story of novel compound, which inhibits virion maturation, and the importance of maturation research are also discussed.

  15. Modification of a loop sequence between α-helices 6 and 7 of virus capsid (CA protein in a human immunodeficiency virus type 1 (HIV-1 derivative that has simian immunodeficiency virus (SIVmac239 vif and CA α-helices 4 and 5 loop improves replication in cynomolgus monkey cells

    Directory of Open Access Journals (Sweden)

    Adachi Akio

    2009-08-01

    Full Text Available Abstract Background Human immunodeficiency virus type 1 (HIV-1 productively infects only humans and chimpanzees but not cynomolgus or rhesus monkeys while simian immunodeficiency virus isolated from macaque (SIVmac readily establishes infection in those monkeys. Several HIV-1 and SIVmac chimeric viruses have been constructed in order to develop an animal model for HIV-1 infection. Construction of an HIV-1 derivative which contains sequences of a SIVmac239 loop between α-helices 4 and 5 (L4/5 of capsid protein (CA and the entire SIVmac239 vif gene was previously reported. Although this chimeric virus could grow in cynomolgus monkey cells, it did so much more slowly than did SIVmac. It was also reported that intrinsic TRIM5α restricts the post-entry step of HIV-1 replication in rhesus and cynomolgus monkey cells, and we previously demonstrated that a single amino acid in a loop between α-helices 6 and 7 (L6/7 of HIV type 2 (HIV-2 CA determines the susceptibility of HIV-2 to cynomolgus monkey TRIM5α. Results In the study presented here, we replaced L6/7 of HIV-1 CA in addition to L4/5 and vif with the corresponding segments of SIVmac. The resultant HIV-1 derivatives showed enhanced replication capability in established T cell lines as well as in CD8+ cell-depleted primary peripheral blood mononuclear cells from cynomolgus monkey. Compared with the wild type HIV-1 particles, the viral particles produced from a chimeric HIV-1 genome with those two SIVmac loops were less able to saturate the intrinsic restriction in rhesus monkey cells. Conclusion We have succeeded in making the replication of simian-tropic HIV-1 in cynomolgus monkey cells more efficient by introducing into HIV-1 the L6/7 CA loop from SIVmac. It would be of interest to determine whether HIV-1 derivatives with SIVmac CA L4/5 and L6/7 can establish infection of cynomolgus monkeys in vivo.

  16. Intragenic HIV-1 env sequences that enhance gag expression

    International Nuclear Information System (INIS)

    Suptawiwat, Ornpreya; Sutthent, Ruengpung; Lee, T.-H.; Auewarakul, Prasert

    2003-01-01

    Expression of HIV-1 genes is regulated at multiple levels including the complex RNA splicing and transport mechanisms. Multiple cis-acting elements involved in these regulations have been previously identified in various regions of HIV-1 genome. Here we show that another cis-acting element was present in HIV-1 env region. This element enhanced the expression of Gag when inserted together with Rev response element (RRE) into a truncated HIV-1 genome in the presence of Rev. The enhancing activity was mapped to a 263-bp fragment in the gp41 region downstream to RRE. RNA analysis showed that it might function by promoting RNA stability and Rev-dependent RNA export. The enhancement was specific to Rev-dependent expression, since it did not enhance Gag expression driven by Sam68, a cellular protein that has been shown to be able to substitute for Rev in RNA export function

  17. The latest evidence for possible HIV-1 curative strategies.

    Science.gov (United States)

    Pham, Hanh Thi; Mesplède, Thibault

    2018-01-01

    Human immunodeficiency virus type 1 (HIV-1) infection remains a major health issue worldwide. In developed countries, antiretroviral therapy has extended its reach from treatment of people living with HIV-1 to post-exposure prophylaxis, treatment as prevention, and, more recently, pre-exposure prophylaxis. These healthcare strategies offer the epidemiological tools to curve the epidemic in rich settings and will be concomitantly implemented in developing countries. One of the remaining challenges is to identify an efficacious curative strategy. This review manuscript will focus on some of the current curative strategies aiming at providing a sterilizing or functional cure to HIV-1-positive individuals. These include the following: early treatment initiation in post-treatment controllers as a long-term HIV-1 remission strategy, latency reversal, gene editing with or without stem cell transplantation, and antibodies against either the viral envelope protein or the host integrin α4β7.

  18. Preliminary X-ray diffraction analysis of YcdB from Escherichia coli: a novel haem-containing and Tat-secreted periplasmic protein with a potential role in iron transport

    International Nuclear Information System (INIS)

    Cartron, Michaël L.; Mitchell, Sue A.; Woodhall, Mark R.; Andrews, Simon C.; Watson, Kimberly A.

    2006-01-01

    The crystallization and structure determination of the apo form of a novel haem-containing Tat substrate, YcdB from E. coli, has been solved to 2.0 Å resolution. The preliminary structure shows similarity to other haem-dependent peroxidases, despite low sequence homology. YcdB is a periplasmic haem-containing protein from Escherichia coli that has a potential role in iron transport. It is currently the only reported haem-containing Tat-secreted substrate. Here, the overexpression, purification, crystallization and structure determination at 2.0 Å resolution are reported for the apo form of the protein. The apo-YcdB structure resembles those of members of the haem-dependent peroxidase family and thus confirms that YcdB is also a member of this family. Haem-soaking experiments with preformed apo-YcdB crystals have been optimized to successfully generate haem-containing YcdB crystals that diffract to 2.9 Å. Completion of model building and structure refinement are under way

  19. Discovery of natural mouse serum derived HIV-1 entry inhibitor(s).

    Science.gov (United States)

    Wei, M; Chen, Y; Xi, J; Ru, S; Ji, M; Zhang, D; Fang, Q; Tang, B

    Among rationally designed human immunodeficiency virus 1 (HIV-1) inhibitors, diverse natural factors have showed as potent anti-HIV activity in human blood. We have discovered that the boiled supernatant of healthy mouse serum could suppress HIV-1 entry, and exhibited reduced inhibitory activity after trypsin digestion. Further analysis demonstrated that only the fraction containing 10-25 K proteins could inhibit HIV-1 mediated cell-cell fusion. These results suggest that the 10-25 K protein(s) is novel natural HIV-1 entry inhibitor(s). Our findings provide important information about novel natural HIV entry inhibitors in mouse serum.

  20. HIV-1 and the macrophage

    NARCIS (Netherlands)

    Bol, Sebastiaan M.; Cobos-Jimenez, Viviana; Kootstra, Neeltje A.; van 't Wout, Angelique B.

    2011-01-01

    Macrophages and CD4(+) T cells are natural target cells for HIV-1, and both cell types contribute to the establishment of the viral reservoir that is responsible for continuous residual virus replication during antiretroviral therapy and viral load rebound upon treatment interruption. Scientific

  1. Developing strategies for HIV-1 eradication

    Science.gov (United States)

    Durand, Christine M.; Blankson, Joel N.; Siliciano, Robert F.

    2014-01-01

    Highly active antiretroviral therapy (HAART) suppresses HIV-1 replication, transforming the outlook for infected patients. However, reservoirs of replication-competent forms of the virus persist during HAART, and when treatment is stopped, high rates of HIV-1 replication return. Recent insights into HIV-1 latency, as well as a report that HIV-1 infection was eradicated in one individual, have renewed interest in finding a cure for HIV-1 infection. Strategies for HIV-1 eradication include gene therapy and hematopoietic stem cell transplantation, stimulating host immunity to control HIV-1 replication, and targeting latent HIV-1 in resting memory CD4+ T cells. Future efforts should aim to provide better understanding of how to reconstitute the CD4+ T cell compartment with genetically engineered cells, exert immune control over HIV-1 replication, and identify and eliminate all viral reservoirs. PMID:22867874

  2. Identifying HIV-1 dual infections

    Directory of Open Access Journals (Sweden)

    Cornelissen Marion

    2007-09-01

    Full Text Available Abstract Transmission of human immunodeficiency virus (HIV is no exception to the phenomenon that a second, productive infection with another strain of the same virus is feasible. Experiments with RNA viruses have suggested that both coinfections (simultaneous infection with two strains of a virus and superinfections (second infection after a specific immune response to the first infecting strain has developed can result in increased fitness of the viral population. Concerns about dual infections with HIV are increasing. First, the frequent detection of superinfections seems to indicate that it will be difficult to develop a prophylactic vaccine. Second, HIV-1 superinfections have been associated with accelerated disease progression, although this is not true for all persons. In fact, superinfections have even been detected in persons controlling their HIV infections without antiretroviral therapy. Third, dual infections can give rise to recombinant viruses, which are increasingly found in the HIV-1 epidemic. Recombinants could have increased fitness over the parental strains, as in vitro models suggest, and could exhibit increased pathogenicity. Multiple drug resistant (MDR strains could recombine to produce a pan-resistant, transmittable virus. We will describe in this review what is presently known about super- and re-infection among ambient viral infections, as well as the first cases of HIV-1 superinfection, including HIV-1 triple infections. The clinical implications, the impact of the immune system, and the effect of anti-retroviral therapy will be covered, as will as the timing of HIV superinfection. The methods used to detect HIV-1 dual infections will be discussed in detail. To increase the likelihood of detecting a dual HIV-1 infection, pre-selection of patients can be done by serotyping, heteroduplex mobility assays (HMA, counting the degenerate base codes in the HIV-1 genotyping sequence, or surveying unexpected increases in the

  3. Induction of a protein-targeted catalytic response in autoimmune prone mice: antibody-mediated cleavage of HIV-1 glycoprotein GP120.

    Science.gov (United States)

    Ponomarenko, Natalia A; Vorobiev, Ivan I; Alexandrova, Elena S; Reshetnyak, Andrew V; Telegin, Georgy B; Khaidukov, Sergey V; Avalle, Bérangère; Karavanov, Alexander; Morse, Herbert C; Thomas, Daniel; Friboulet, Alain; Gabibov, Alexander G

    2006-01-10

    We have induced a polyclonal IgG that degrades the HIV-1 surface antigen, glycoprotein gp120, by taking advantage of the susceptibility of SJL mice to a peptide-induced autoimmune disorder, experimental autoimmune encephalomyelitis (EAE). Specific pathogen-free SJL mice were immunized with structural fragments of gp120, fused in-frame with encephalitogenic peptide MBP(85-101). It has resulted in a pronounced disease-associated immune response against antigens. A dramatic increase of gp120 degradation level by purified polyclonal IgG from immunized versus nonimmunized mice has been demonstrated by a newly developed fluorescence-based assay. This activity was inhibited by anti-mouse immunoglobulin antibodies as well as by Ser- and His-reactive covalent inhibitors. A dominant proteolysis site in recombinant gp120 incubated with purified polyclonal IgG from immunized mice was shown by SDS-PAGE. The SELDI-based mass spectrometry revealed that these antibodies exhibited significant specificity toward the Pro484-Leu485 peptide bond. The sequence surrounding this site is present in nearly half of the HIV-I variants. This novel strategy can be generalized for creating a catalytic vaccine against viral pathogens.

  4. Development of Th1 Imprints to rBCG Expressing a Foreign Protein: Implications for Vaccination against HIV-1 and Diverse Influenza Strains

    Directory of Open Access Journals (Sweden)

    Carl Power

    2010-01-01

    Full Text Available We demonstrate here that immunizing naïve mice with low numbers of recombinant Bacille Calmette-Guérin (rBCG expressing β-galactosidase (β-gal generates predominant Th1 responses to both BCG and β-gal whereas infection with high numbers generates a mixed Th1/Th2 response to both BCG and β-gal. Furthermore, the Th1 response to both BCG and β-gal is stable when mice, pre-exposed to low numbers of rBCG, are challenged four months later with high numbers of rBCG. Thus the Th1/Th2 phenotypes of the immune responses to β-gal and to BCG are “coherently” regulated. Such rBCG vectors, encoding antigens of pathogens preferentially susceptible to cell-mediated attack, may be useful in vaccinating against such pathogens. We discuss vaccination strategies employing rBCG vectors that are designed to provide protection against diverse influenza strains or numerous variants of HIV-1 and consider what further experiments are essential to explore the possibility of realizing such strategies.

  5. Enrichment of intersubtype HIV-1 recombinants in a dual infection system using HIV-1 strain-specific siRNAs

    Science.gov (United States)

    2011-01-01

    env regions suggest that other mechanisms are at play. Conclusion These findings show that siRNAs can be used as an efficient in vitro tool for enriching recombinants, to facilitate further study on mechanisms of intersubytpe HIV-1 recombination, and to generate replication-competent intersubtype recombinant proteins with a breadth in HIV-1 diversity for future vaccine studies. PMID:21232148

  6. DBR1 siRNA inhibition of HIV-1 replication

    Directory of Open Access Journals (Sweden)

    Naidu Yathi

    2005-10-01

    Full Text Available Abstract Background HIV-1 and all retroviruses are related to retroelements of simpler organisms such as the yeast Ty elements. Recent work has suggested that the yeast retroelement Ty1 replicates via an unexpected RNA lariat intermediate in cDNA synthesis. The putative genomic RNA lariat intermediate is formed by a 2'-5' phosphodiester bond, like that found in pre-mRNA intron lariats and it facilitates the minus-strand template switch during cDNA synthesis. We hypothesized that HIV-1 might also form a genomic RNA lariat and therefore that siRNA-mediated inhibition of expression of the human RNA lariat de-branching enzyme (DBR1 expression would specifically inhibit HIV-1 replication. Results We designed three short interfering RNA (siRNA molecules targeting DBR1, which were capable of reducing DBR1 mRNA expression by 80% and did not significantly affect cell viability. We assessed HIV-1 replication in the presence of DBR1 siRNA and found that DBR1 knockdown led to decreases in viral cDNA and protein production. These effects could be reversed by cotransfection of a DBR1 cDNA indicating that the inhibition of HIV-1 replication was a specific effect of DBR1 underexpression. Conclusion These data suggest that DBR1 function may be needed to debranch a putative HIV-1 genomic RNA lariat prior to completion of reverse transcription.

  7. Cerebrospinal fluid HIV-1 RNA levels in asymptomatic patients with early stage chronic HIV-1 infection: support for the hypothesis of local virus replication.

    Science.gov (United States)

    García, F; Niebla, G; Romeu, J; Vidal, C; Plana, M; Ortega, M; Ruiz, L; Gallart, T; Clotet, B; Miró, J M; Pumarola, T; Gatell, J M

    1999-08-20

    To assess HIV-1 RNA levels in cerebrospinal fluid (CSF) and their potential correlation with plasma viral load and central nervous system (CNS) HIV-1 infection markers in stable asymptomatic patients with a CD4 T cell count >500x10(6) cells/l. Consecutive patients screened for two trials were eligible for lumbar puncture assessment. At day 0, simultaneous samples of CSF and plasma were obtained and levels of total proteins, albumin, IgG, antibodies against HIV-1 p24 antigen, HIV-1 RNA (using the polymerase chain technique) and white cells were measured. The integrity of the blood-brain barrier was preserved (albumin index > or =7) in 59 out of 70 patients (84%). Intrathecal production of antibodies against HIV-1 p24 antigen was demonstrated in 55 out of 70 individuals (78%). Viral load in CSF was significantly lower than plasma values (3.13+/-0.95 versus 4.53+/-0.53, P = 0.0001). HIV-1 RNA was not detected in CSF in only three of the 70 patients (4%). Overall, there was a significant correlation between plasma and CSF HIV-1 RNA levels (r = 0.43, P = 0.0001); however, in 29 patients (41%) there were significant differences (>1.5 log10 copies/ml) between the viral loads in plasma and CSF. In the multivariate analysis, a high level of protein and white cells in CSF, but not the HIV-1 RNA plasma level, were factors independently associated with a higher level of HIV-1 RNA in CSF (P = 0.0001). HIV-1 RNA can be detected almost always in CSF of asymptomatic patients in early stages of HIV-1 infection including those with a preserved integrity of the blood-brain barrier. The important discrepancies between plasma and CSF viral load, and the independent association between CSF abnormalities and CSF viral load, support the hypothesis of local production of HIV-1.

  8. Cyclophilin B enhances HIV-1 infection

    Energy Technology Data Exchange (ETDEWEB)

    DeBoer, Jason; Madson, Christian J. [Department of Medical Microbiology and Immunology, Creighton University, Omaha, NE (United States); Belshan, Michael, E-mail: michaelbelshan@creighton.edu [Department of Medical Microbiology and Immunology, Creighton University, Omaha, NE (United States); The Nebraska Center for Virology, University of Nebraska, Lincoln, NE (United States)

    2016-02-15

    Cyclophilin B (CypB) is a member of the immunophilin family and intracellular chaperone. It predominantly localizes to the ER, but also contains a nuclear localization signal and is secreted from cells. CypB has been shown to interact with the Gag protein of human immunodeficiency type 1 (HIV-1). Several proteomic and genetic studies identified it as a potential factor involved in HIV replication. Herein, we show that over-expression of CypB enhances HIV infection by increasing nuclear import of viral DNA. This enhancement was unaffected by cyclosporine treatment and requires the N-terminus of the protein. The N-terminus contains an ER leader sequence, putative nuclear localization signal, and is required for secretion. Deletion of the N-terminus resulted in mislocalization from the ER and suppression of HIV infection. Passive transfer experiments showed that secreted CypB did not impact HIV infection. Combined, these experiments show that intracellular CypB modulates a pathway of HIV nuclear import. - Highlights: • CypB has been identified in several proteomic studies of HIV-1 infection. • CypB expression is upregulated in activated and infected T-cells. • Over-expression of CypB enhances HIV nuclear import and infection. • The N-terminus of CypB is necessary for these effects.

  9. Cyclophilin B enhances HIV-1 infection

    International Nuclear Information System (INIS)

    DeBoer, Jason; Madson, Christian J.; Belshan, Michael

    2016-01-01

    Cyclophilin B (CypB) is a member of the immunophilin family and intracellular chaperone. It predominantly localizes to the ER, but also contains a nuclear localization signal and is secreted from cells. CypB has been shown to interact with the Gag protein of human immunodeficiency type 1 (HIV-1). Several proteomic and genetic studies identified it as a potential factor involved in HIV replication. Herein, we show that over-expression of CypB enhances HIV infection by increasing nuclear import of viral DNA. This enhancement was unaffected by cyclosporine treatment and requires the N-terminus of the protein. The N-terminus contains an ER leader sequence, putative nuclear localization signal, and is required for secretion. Deletion of the N-terminus resulted in mislocalization from the ER and suppression of HIV infection. Passive transfer experiments showed that secreted CypB did not impact HIV infection. Combined, these experiments show that intracellular CypB modulates a pathway of HIV nuclear import. - Highlights: • CypB has been identified in several proteomic studies of HIV-1 infection. • CypB expression is upregulated in activated and infected T-cells. • Over-expression of CypB enhances HIV nuclear import and infection. • The N-terminus of CypB is necessary for these effects.

  10. Relaxed evolution in the tyrosine aminotransferase gene tat in old world fruit bats (Chiroptera: Pteropodidae).

    Science.gov (United States)

    Shen, Bin; Fang, Tao; Yang, Tianxiao; Jones, Gareth; Irwin, David M; Zhang, Shuyi

    2014-01-01

    Frugivorous and nectarivorous bats fuel their metabolism mostly by using carbohydrates and allocate the restricted amounts of ingested proteins mainly for anabolic protein syntheses rather than for catabolic energy production. Thus, it is possible that genes involved in protein (amino acid) catabolism may have undergone relaxed evolution in these fruit- and nectar-eating bats. The tyrosine aminotransferase (TAT, encoded by the Tat gene) is the rate-limiting enzyme in the tyrosine catabolic pathway. To test whether the Tat gene has undergone relaxed evolution in the fruit- and nectar-eating bats, we obtained the Tat coding region from 20 bat species including four Old World fruit bats (Pteropodidae) and two New World fruit bats (Phyllostomidae). Phylogenetic reconstructions revealed a gene tree in which all echolocating bats (including the New World fruit bats) formed a monophyletic group. The phylogenetic conflict appears to stem from accelerated TAT protein sequence evolution in the Old World fruit bats. Our molecular evolutionary analyses confirmed a change in the selection pressure acting on Tat, which was likely caused by a relaxation of the evolutionary constraints on the Tat gene in the Old World fruit bats. Hepatic TAT activity assays showed that TAT activities in species of the Old World fruit bats are significantly lower than those of insectivorous bats and omnivorous mice, which was not caused by a change in TAT protein levels in the liver. Our study provides unambiguous evidence that the Tat gene has undergone relaxed evolution in the Old World fruit bats in response to changes in their metabolism due to the evolution of their special diet.

  11. Relaxed evolution in the tyrosine aminotransferase gene tat in old world fruit bats (Chiroptera: Pteropodidae.

    Directory of Open Access Journals (Sweden)

    Bin Shen

    Full Text Available Frugivorous and nectarivorous bats fuel their metabolism mostly by using carbohydrates and allocate the restricted amounts of ingested proteins mainly for anabolic protein syntheses rather than for catabolic energy production. Thus, it is possible that genes involved in protein (amino acid catabolism may have undergone relaxed evolution in these fruit- and nectar-eating bats. The tyrosine aminotransferase (TAT, encoded by the Tat gene is the rate-limiting enzyme in the tyrosine catabolic pathway. To test whether the Tat gene has undergone relaxed evolution in the fruit- and nectar-eating bats, we obtained the Tat coding region from 20 bat species including four Old World fruit bats (Pteropodidae and two New World fruit bats (Phyllostomidae. Phylogenetic reconstructions revealed a gene tree in which all echolocating bats (including the New World fruit bats formed a monophyletic group. The phylogenetic conflict appears to stem from accelerated TAT protein sequence evolution in the Old World fruit bats. Our molecular evolutionary analyses confirmed a change in the selection pressure acting on Tat, which was likely caused by a relaxation of the evolutionary constraints on the Tat gene in the Old World fruit bats. Hepatic TAT activity assays showed that TAT activities in species of the Old World fruit bats are significantly lower than those of insectivorous bats and omnivorous mice, which was not caused by a change in TAT protein levels in the liver. Our study provides unambiguous evidence that the Tat gene has undergone relaxed evolution in the Old World fruit bats in response to changes in their metabolism due to the evolution of their special diet.

  12. Curcumin derivatives as HIV-1 protease inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Sui, Z.; Li, J.; Craik, C.S.; Ortiz de Montellano, P.R. [Univ. of California, San Francisco, CA (United States)

    1993-12-31

    Curcumin, a non-toxic natural compound from Curcuma longa, has been found to be an HIV-1 protease inhibitor. Some of its derivatives were synthesized and their inhibitory activity against the HIV-1 protease was tested. Curcumin analogues containing boron enhanced the inhibitory activity. At least of the the synthesized compounds irreversibly inhibits the HIV-1 protease.

  13. Cellular and molecular mechanisms of HIV-1 integration targeting.

    Science.gov (United States)

    Engelman, Alan N; Singh, Parmit K

    2018-07-01

    Integration is central to HIV-1 replication and helps mold the reservoir of cells that persists in AIDS patients. HIV-1 interacts with specific cellular factors to target integration to interior regions of transcriptionally active genes within gene-dense regions of chromatin. The viral capsid interacts with several proteins that are additionally implicated in virus nuclear import, including cleavage and polyadenylation specificity factor 6, to suppress integration into heterochromatin. The viral integrase protein interacts with transcriptional co-activator lens epithelium-derived growth factor p75 to principally position integration within gene bodies. The integrase additionally senses target DNA distortion and nucleotide sequence to help fine-tune the specific phosphodiester bonds that are cleaved at integration sites. Research into virus-host interactions that underlie HIV-1 integration targeting has aided the development of a novel class of integrase inhibitors and may help to improve the safety of viral-based gene therapy vectors.

  14. Stable assembly of HIV-1 export complexes occurs cotranscriptionally

    DEFF Research Database (Denmark)

    Nawroth, Isabel; Mueller, Florian; Basyuk, Eugenia

    2014-01-01

    The HIV-1 Rev protein mediates export of unspliced and singly spliced viral transcripts by binding to the Rev response element (RRE) and recruiting the cellular export factor CRM1. Here, we investigated the recruitment of Rev to the transcription sites of HIV-1 reporters that splice either post......- or cotranscriptionally. In both cases, we observed that Rev localized to the transcription sites of the reporters and recruited CRM1. Rev and CRM1 remained at the reporter transcription sites when cells were treated with the splicing inhibitor Spliceostatin A (SSA), showing that the proteins associate with RNA prior...... to or during early spliceosome assembly. Fluorescence recovery after photobleaching (FRAP) revealed that Rev and CRM1 have similar kinetics as the HIV-1 RNA, indicating that Rev, CRM1, and RRE-containing RNAs are released from the site of transcription in one single export complex. These results suggest...

  15. A single gp120 residue can affect HIV-1 tropism in macaques.

    Directory of Open Access Journals (Sweden)

    Gregory Q Del Prete

    2017-09-01

    Full Text Available Species-dependent variation in proteins that aid or limit virus replication determines the ability of lentiviruses to jump between host species. Identifying and overcoming these differences facilitates the development of animal models for HIV-1, including models based on chimeric SIVs that express HIV-1 envelope (Env glycoproteins, (SHIVs and simian-tropic HIV-1 (stHIV strains. Here, we demonstrate that the inherently poor ability of most HIV-1 Env proteins to use macaque CD4 as a receptor is improved during adaptation by virus passage in macaques. We identify a single amino acid, A281, in HIV-1 Env that consistently changes during adaptation in macaques and affects the ability of HIV-1 Env to use macaque CD4. Importantly, mutations at A281 do not markedly affect HIV-1 Env neutralization properties. Our findings should facilitate the design of HIV-1 Env proteins for use in non-human primate models and thus expedite the development of clinically relevant reagents for testing interventions against HIV-1.

  16. Striking HIV-1 Entry by Targeting HIV-1 gp41. But, Where Should We Target?

    Directory of Open Access Journals (Sweden)

    Cátia Teixeira

    Full Text Available HIV-1 gp41 facilitates the viral fusion through a conformational switch involving the association of three C-terminal helices along the conserved hydrophobic grooves of three N-terminal helices coiled-coil. The control of these structural rearrangements is thought to be central to HIV-1 entry and, therefore, different strategies of intervention are being developed. Herewith, we describe a procedure to simulate the folding of an HIV-1 gp41 simplified model. This procedure is based on the construction of plausible conformational pathways, which describe protein transition between non-fusogenic and fusogenic conformations. The calculation of the paths started with 100 molecular dynamics simulations of the non-fusogenic conformation, which were found to converge to different intermediate states. Those presenting defined criteria were selected for separate targeted molecular dynamics simulations, subjected to a force constant imposing a movement towards the gp41 fusogenic conformation. Despite significant diversity, a preferred sequence of events emerged when the simulations were analyzed in terms of the formation, breakage and evolution of the contacts. We pointed out 29 residues as the most relevant for the movement of gp41; also, 2696 possible interactions were reduced to only 48 major interactions, which reveals the efficiency of the method. The analysis of the evolution of the main interactions lead to the detection of four main behaviors for those contacts: stable, increasing, decreasing and repulsive interactions. Altogether, these results suggest a specific small cavity of the HIV-1 gp41 hydrophobic groove as the preferred target to small molecules.

  17. Macrophage Resistance to HIV-1 Infection Is Enhanced by the Neuropeptides VIP and PACAP

    Science.gov (United States)

    Temerozo, Jairo R.; Joaquim, Rafael; Regis, Eduardo G.; Savino, Wilson; Bou-Habib, Dumith Chequer

    2013-01-01

    It is well established that host factors can modulate HIV-1 replication in macrophages, critical cells in the pathogenesis of HIV-1 infection due to their ability to continuously produce virus. The neuropeptides VIP and PACAP induce well-characterized effects on macrophages through binding to the G protein-coupled receptors VPAC1, VPAC2 and PAC1, but their influence on HIV-1 production by these cells has not been established. Here, we describe that VIP and PACAP reduce macrophage production of HIV-1, acting in a synergistic or additive manner to decrease viral growth. Using receptor antagonists, we detected that the HIV-1 inhibition promoted by VIP is dependent on its ligation to VPAC1/2, whereas PACAP decreases HIV-1 growth via activation of the VPAC1/2 and PAC1 receptors. Specific agonists of VPAC2 or PAC1 decrease macrophage production of HIV-1, whereas sole activation of VPAC1 enhances viral growth. However, the combination of specific agonists mimicking the receptor preference of the natural neuropeptides reproduces the ability of VIP and PACAP to increase macrophage resistance to HIV-1 replication. VIP and PACAP up-regulated macrophage secretion of the β-chemokines CCL3 and CCL5 and the cytokine IL-10, whose neutralization reversed the neuropeptide-induced inhibition of HIV-1 replication. Our results suggest that VIP and PACAP and the receptors VPAC2 and PAC1 could be used as targets for developing alternative therapeutic strategies for HIV-1 infection. PMID:23818986

  18. A European multicientre study on the comparison of HIV-1 viral loads between VERIS HIV-1 Assay and Roche COBAS® TAQMAN® HIV-1 test, Abbott RealTime HIV-1 Assay, and Siemens VERSANT HIV-1 Assay.

    Science.gov (United States)

    Braun, Patrick; Delgado, Rafael; Drago, Monica; Fanti, Diana; Fleury, Hervé; Hofmann, Jörg; Izopet, Jacques; Kühn, Sebastian; Lombardi, Alessandra; Mancon, Alessandro; Marcos, Mª Angeles; Mileto, Davide; Sauné, Karine; O'Shea, Siobhan; Pérez-Rivilla, Alfredo; Ramble, John; Trimoulet, Pascale; Vila, Jordi; Whittaker, Duncan; Artus, Alain; Rhodes, Daniel

    2017-07-01

    Viral load monitoring is essential for patients under treatment for HIV. Beckman Coulter has developed the VERIS HIV-1 Assay for use on the novel, automated DxN VERIS Molecular Diagnostics System. ¥ OBJECTIVES: Evaluation of the clinical performance of the new quantitative VERIS HIV-1 Assay at multiple EU laboratories. Method comparison with the VERIS HIV-1 Assay was performed with 415 specimens at 5 sites tested with COBAS ® AmpliPrep/COBAS ® TaqMan ® HIV-1 Test, v2.0, 169 specimens at 3 sites tested with RealTime HIV-1 Assay, and 202 specimens from 2 sites tested with VERSANT HIV-1 Assay. Patient monitoring sample results from 4 sites were also compared. Bland-Altman analysis showed the average bias between VERIS HIV-1 Assay and COBAS HIV-1 Test, RealTime HIV-1 Assay, and VERSANT HIV-1 Assay to be 0.28, 0.39, and 0.61 log 10 cp/mL, respectively. Bias at low end levels below 1000cp/mL showed predicted bias to be <0.3 log 10 cp/mL for VERIS HIV-1 Assay versus COBAS HIV-1 Test and RealTime HIV-1 Assay, and <0.5 log 10 cp/mL versus VERSANT HIV-1 Assay. Analysis on 174 specimens tested with the 0.175mL volume VERIS HIV-1 Assay and COBAS HIV-1 Test showed average bias of 0.39 log 10 cp/mL. Patient monitoring results using VERIS HIV-1 Assay demonstrated similar viral load trends over time to all comparators. The VERIS HIV-1 Assay for use on the DxN VERIS System demonstrated comparable clinical performance to COBAS ® HIV-1 Test, RealTime HIV-1 Assay, and VERSANT HIV-1 Assay. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Neurofilament light chain protein as a marker of neuronal injury: review of its use in HIV-1 infection and reference values for HIV-negative controls

    NARCIS (Netherlands)

    Yilmaz, Aylin; Blennow, Kaj; Hagberg, Lars; Nilsson, Staffan; Price, Richard W.; Schouten, Judith; Spudich, Serena; Underwood, Jonathan; Zetterberg, Henrik; Gisslén, Magnus

    2017-01-01

    Introduction: Several CSF biomarkers of neuronal injury have been studied in people living with HIV. At this time, the most useful is the light subunit of the neurofilament protein (NFL). This major structural component of myelinated axons is essential to maintain axonal caliber and to facilitate

  20. Dynamics of HIV-1 RNA Near the Plasma Membrane during Virus Assembly.

    Science.gov (United States)

    Sardo, Luca; Hatch, Steven C; Chen, Jianbo; Nikolaitchik, Olga; Burdick, Ryan C; Chen, De; Westlake, Christopher J; Lockett, Stephen; Pathak, Vinay K; Hu, Wei-Shau

    2015-11-01

    To increase our understanding of the events that lead to HIV-1 genome packaging, we examined the dynamics of viral RNA and Gag-RNA interactions near the plasma membrane by using total internal reflection fluorescence microscopy. We labeled HIV-1 RNA with a photoconvertible Eos protein via an RNA-binding protein that recognizes stem-loop sequences engineered into the viral genome. Near-UV light exposure causes an irreversible structural change in Eos and alters its emitted fluorescence from green to red. We studied the dynamics of HIV-1 RNA by photoconverting Eos near the plasma membrane, and we monitored the population of photoconverted red-Eos-labeled RNA signals over time. We found that in the absence of Gag, most of the HIV-1 RNAs stayed near the plasma membrane transiently, for a few minutes. The presence of Gag significantly increased the time that RNAs stayed near the plasma membrane: most of the RNAs were still detected after 30 min. We then quantified the proportion of HIV-1 RNAs near the plasma membrane that were packaged into assembling viral complexes. By tagging Gag with blue fluorescent protein, we observed that only a portion, ∼13 to 34%, of the HIV-1 RNAs that reached the membrane were recruited into assembling particles in an hour, and the frequency of HIV-1 RNA packaging varied with the Gag expression level. Our studies reveal the HIV-1 RNA dynamics on the plasma membrane and the efficiency of RNA recruitment and provide insights into the events leading to the generation of infectious HIV-1 virions. Nascent HIV-1 particles assemble on plasma membranes. During the assembly process, HIV-1 RNA genomes must be encapsidated into viral complexes to generate infectious particles. To gain insights into the RNA packaging and virus assembly mechanisms, we labeled and monitored the HIV-1 RNA signals near the plasma membrane. Our results showed that most of the HIV-1 RNAs stayed near the plasma membrane for only a few minutes in the absence of Gag, whereas

  1. Human cellular restriction factors that target HIV-1 replication

    Directory of Open Access Journals (Sweden)

    Jeang Kuan-Teh

    2009-09-01

    Full Text Available Abstract Recent findings have highlighted roles played by innate cellular factors in restricting intracellular viral replication. In this review, we discuss in brief the activities of apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G, bone marrow stromal cell antigen 2 (BST-2, cyclophilin A, tripartite motif protein 5 alpha (Trim5α, and cellular microRNAs as examples of host restriction factors that target HIV-1. We point to countermeasures encoded by HIV-1 for moderating the potency of these cellular restriction functions.

  2. Calcitonin Gene-Related Peptide Induces HIV-1 Proteasomal Degradation in Mucosal Langerhans Cells.

    Science.gov (United States)

    Bomsel, Morgane; Ganor, Yonatan

    2017-12-01

    to CD4 + T cells, the principal HIV-1 targets. We previously found that the neuroimmune dialogue between LCs and peripheral neurons, innervating mucosal epithelia, significantly inhibits trans -infection via the action of the secreted neuropeptide CGRP on LCs. In this study, we investigated whether CGRP-induced inhibition of trans -infection is linked to CGRP-controlled HIV-1 degradation in LCs. We show that in untreated LCs, HIV-1 is functionally degraded in endolysosomes. In sharp contrast, we reveal that in CGRP-treated LCs, HIV-1 is diverted toward and degraded via another cytosolic protein degradative pathway, namely, the proteasome. These results establish that CGRP regulates HIV-1 degradation in LCs. As CGRP contributes to the sexual response and present within mucosal epithelia, HIV-1 proteasomal degradation in LCs might predominate in vivo and should be enhanced clinically. Copyright © 2017 American Society for Microbiology.

  3. The HIV-1 Rev/RRE system is required for HIV-1 5' UTR cis elements to augment encapsidation of heterologous RNA into HIV-1 viral particles

    Directory of Open Access Journals (Sweden)

    Ma Hong

    2011-06-01

    Full Text Available Abstract Background The process of HIV-1 genomic RNA (gRNA encapsidation is governed by a number of viral encoded components, most notably the Gag protein and gRNA cis elements in the canonical packaging signal (ψ. Also implicated in encapsidation are cis determinants in the R, U5, and PBS (primer binding site from the 5' untranslated region (UTR. Although conventionally associated with nuclear export of HIV-1 RNA, there is a burgeoning role for the Rev/RRE in the encapsidation process. Pleiotropic effects exhibited by these cis and trans viral components may confound the ability to examine their independent, and combined, impact on encapsidation of RNA into HIV-1 viral particles in their innate viral context. We systematically reconstructed the HIV-1 packaging system in the context of a heterologous murine leukemia virus (MLV vector RNA to elucidate a mechanism in which the Rev/RRE system is central to achieving efficient and specific encapsidation into HIV-1 viral particles. Results We show for the first time that the Rev/RRE system can augment RNA encapsidation independent of all cis elements from the 5' UTR (R, U5, PBS, and ψ. Incorporation of all the 5' UTR cis elements did not enhance RNA encapsidation in the absence of the Rev/RRE system. In fact, we demonstrate that the Rev/RRE system is required for specific and efficient encapsidation commonly associated with the canonical packaging signal. The mechanism of Rev/RRE-mediated encapsidation is not a general phenomenon, since the combination of the Rev/RRE system and 5' UTR cis elements did not enhance encapsidation into MLV-derived viral particles. Lastly, we show that heterologous MLV RNAs conform to transduction properties commonly associated with HIV-1 viral particles, including in vivo transduction of non-dividing cells (i.e. mouse neurons; however, the cDNA forms are episomes predominantly in the 1-LTR circle form. Conclusions Premised on encapsidation of a heterologous RNA into

  4. Back to the future: revisiting HIV-1 lethal mutagenesis

    Science.gov (United States)

    Dapp, Michael J.; Patterson, Steven E.; Mansky, Louis M.

    2012-01-01

    The concept of eliminating HIV-1 infectivity by elevating the viral mutation rate was first proposed over a decade ago, even though the general concept had been conceived earlier for RNA viruses. Lethal mutagenesis was originally viewed as a novel chemotherapeutic approach for treating HIV-1 infection in which use of a viral mutagen would over multiple rounds of replication lead to the lethal accumulation of mutations, rendering the virus population non infectious – known as the slow mutation accumulation model. There have been limitations in obtaining good efficacy data with drug leads, leaving some doubt into clinical translation. More recent studies of the APOBEC3 proteins as well as new progress in the use of nucleoside analogs for inducing lethal mutagenesis have helped to refocus attention on rapid induction of HIV-1 lethal mutagenesis in a single or limited number of replication cycles leading to a rapid mutation accumulation model. PMID:23195922

  5. Glycosylation in HIV-1 envelope glycoprotein and its biological implications

    KAUST Repository

    Ho, Yung Shwen

    2013-08-01

    Glycosylation of HIV-1 envelope proteins (Env gp120/gp41) plays a vital role in viral evasion from the host immune response, which occurs through the masking of key neutralization epitopes and the presentation of the Env glycosylation as \\'self\\' to the host immune system. Env glycosylation is generally conserved, yet its continual evolution plays an important role in modulating viral infectivity and Env immunogenicity. Thus, it is believed that Env glycosylation, which is a vital part of the HIV-1 architecture, also controls intra- and inter-clade genetic variations. Discerning intra- and inter-clade glycosylation variations could therefore yield important information for understanding the molecular and biological differences between HIV clades and may assist in effectively designing Env-based immunogens and in clearly understanding HIV vaccines. This review provides an in-depth perspective of various aspects of Env glycosylation in the context of HIV-1 pathogenesis. © 2013 Future Medicine Ltd.

  6. Codon optimization of the HIV-1 vpu and vif genes stabilizes their mRNA and allows for highly efficient Rev-independent expression

    International Nuclear Information System (INIS)

    Nguyen, Kim-Lien; Llano, Manuel; Akari, Hirofumi; Miyagi, Eri; Poeschla, Eric M.; Strebel, Klaus; Bour, Stephan

    2004-01-01

    Two HIV-1 accessory proteins, Vpu and Vif, are notoriously difficult to express autonomously in the absence of the viral Tat and Rev proteins. We examined whether the codon bias observed in the vpu and vif genes relative to highly expressed human genes contributes to the Rev dependence and low expression level outside the context of the viral genome. The entire vpu gene as well as the 5' half of the vif gene were codon optimized and the resulting open reading frames (ORFs) (vphu and hvif, respectively) were cloned in autonomous expression vectors under the transcriptional control of the CMV promoter. Codon optimization efficiently removed the expression block observed in the native genes and allowed high levels of Rev- and Tat-independent expression of Vpu and Vif. Most of the higher protein levels detected are accounted for by enhanced steady-state levels of the mRNA encoding the optimized species. Nuclear run-on experiments show for the first time that codon optimization has no effect on the rate of transcriptional initiation or elongation of the vphu mRNA. Instead, optimization of the vpu gene was found to stabilize the vphu mRNA in the nucleus and enhance its export to the cytoplasm. This was achieved by allowing the optimized mRNA to use a new CRM1-independent nuclear export pathway. This work provides a better understanding of the molecular mechanisms underlying the process of codon optimization and introduces novel tools to study the biological functions of the Vpu and Vif proteins independently of other viral proteins

  7. DMPD: HIV-1 infection and regulation of Tat function in macrophages. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available (.html) CSML File (.csml) Open .csml file with CIOPlayer Open .csml file with CIOPlayer - ※CIO Playerのご利用上の注意 Open .csml file with CIO Open .csml file with CIO - ※CIOのご利用上の注意 ...

  8. In vitro assessment of TAT — Ciliary Neurotrophic Factor therapeutic potential for peripheral nerve regeneration

    International Nuclear Information System (INIS)

    Barbon, Silvia; Stocco, Elena; Negro, Alessandro; Dalzoppo, Daniele; Borgio, Luca; Rajendran, Senthilkumar; Grandi, Francesca; Porzionato, Andrea; Macchi, Veronica; De Caro, Raffaele

    2016-01-01

    In regenerative neurobiology, Ciliary Neurotrophic Factor (CNTF) is raising high interest as a multifunctional neurocytokine, playing a key role in the regeneration of injured peripheral nerves. Despite its promising trophic and regulatory activity, its clinical application is limited by the onset of severe side effects, due to the lack of efficient intracellular trafficking after administration. In this study, recombinant CNTF linked to the transactivator transduction domain (TAT) was investigated in vitro and found to be an optimized fusion protein which preserves neurotrophic activity, besides enhancing cellular uptake for therapeutic advantage. Moreover, a compelling protein delivery method was defined, in the future perspective of improving nerve regeneration strategies. Following determination of TAT-CNTF molecular weight and concentration, its specific effect on neural SH-SY5Y and PC12 cultures was assessed. Cell proliferation assay demonstrated that the fusion protein triggers PC12 cell growth within 6 h of stimulation. At the same time, the activation of signal transduction pathway and enhancement of cellular trafficking were found to be accomplished in both neural cell lines after specific treatment with TAT-CNTF. Finally, the recombinant growth factor was successfully loaded on oxidized polyvinyl alcohol (PVA) scaffolds, and more efficiently released when polymer oxidation rate increased. Taken together, our results highlight that the TAT domain addiction to the protein sequence preserves CNTF specific neurotrophic activity in vitro, besides improving cellular uptake. Moreover, oxidized PVA could represent an ideal biomaterial for the development of nerve conduits loaded with the fusion protein to be delivered to the site of nerve injury. - Highlights: • TAT-CNTF is an optimized fusion protein that preserves neurotrophic activity. • In neural cell lines, TAT-CNTF triggers the activation of signal transduction. • Fast cellular uptake of TAT-CNTF was

  9. In vitro assessment of TAT — Ciliary Neurotrophic Factor therapeutic potential for peripheral nerve regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Barbon, Silvia, E-mail: silvia.barbon@yahoo.it [Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Via Marzolo 5, 35131 Padua (Italy); Foundation for Biology and Regenerative Medicine, Tissue Engineering and Signaling (TES) ONLUS, Via De Sanctis 10, Caselle di Selvazzano Dentro, 35030 Padua (Italy); Stocco, Elena, E-mail: elena.stocco@gmail.com [Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Via Marzolo 5, 35131 Padua (Italy); Foundation for Biology and Regenerative Medicine, Tissue Engineering and Signaling (TES) ONLUS, Via De Sanctis 10, Caselle di Selvazzano Dentro, 35030 Padua (Italy); Negro, Alessandro, E-mail: alessandro.negro@unipd.it [Department of Biomedical Sciences, University of Padova, Via Colombo 3, 35121 Padua (Italy); Dalzoppo, Daniele, E-mail: daniele.dalzoppo@unipd.it [Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Via Marzolo 5, 35131 Padua (Italy); Borgio, Luca, E-mail: borgio.luca@gmail.com [Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Via Marzolo 5, 35131 Padua (Italy); Rajendran, Senthilkumar, E-mail: senthilstem@gmail.com [Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Via Marzolo 5, 35131 Padua (Italy); Grandi, Francesca, E-mail: francesca.grandi7825@gmail.com [Department of Women' s and Children' s Health, Pediatric Surgery, University of Padua, Via Giustiniani 3, 35121 Padua (Italy); Porzionato, Andrea, E-mail: andrea.porzionato@unipd.it [Section of Human Anatomy, Department of Molecular Medicine, University of Padua, Via Gabelli 65, 35121 Padua (Italy); Macchi, Veronica, E-mail: veronica.macchi@unipd.it [Section of Human Anatomy, Department of Molecular Medicine, University of Padua, Via Gabelli 65, 35121 Padua (Italy); De Caro, Raffaele, E-mail: raffaele.decaro@unipd.it [Section of Human Anatomy, Department of Molecular Medicine, University of Padua, Via Gabelli 65, 35121 Padua (Italy); and others

    2016-10-15

    In regenerative neurobiology, Ciliary Neurotrophic Factor (CNTF) is raising high interest as a multifunctional neurocytokine, playing a key role in the regeneration of injured peripheral nerves. Despite its promising trophic and regulatory activity, its clinical application is limited by the onset of severe side effects, due to the lack of efficient intracellular trafficking after administration. In this study, recombinant CNTF linked to the transactivator transduction domain (TAT) was investigated in vitro and found to be an optimized fusion protein which preserves neurotrophic activity, besides enhancing cellular uptake for therapeutic advantage. Moreover, a compelling protein delivery method was defined, in the future perspective of improving nerve regeneration strategies. Following determination of TAT-CNTF molecular weight and concentration, its specific effect on neural SH-SY5Y and PC12 cultures was assessed. Cell proliferation assay demonstrated that the fusion protein triggers PC12 cell growth within 6 h of stimulation. At the same time, the activation of signal transduction pathway and enhancement of cellular trafficking were found to be accomplished in both neural cell lines after specific treatment with TAT-CNTF. Finally, the recombinant growth factor was successfully loaded on oxidized polyvinyl alcohol (PVA) scaffolds, and more efficiently released when polymer oxidation rate increased. Taken together, our results highlight that the TAT domain addiction to the protein sequence preserves CNTF specific neurotrophic activity in vitro, besides improving cellular uptake. Moreover, oxidized PVA could represent an ideal biomaterial for the development of nerve conduits loaded with the fusion protein to be delivered to the site of nerve injury. - Highlights: • TAT-CNTF is an optimized fusion protein that preserves neurotrophic activity. • In neural cell lines, TAT-CNTF triggers the activation of signal transduction. • Fast cellular uptake of TAT-CNTF was

  10. Exploring Transduction Mechanisms of Protein Transduction Domains (PTDs in Living Cells Utilizing Single-Quantum Dot Tracking (SQT Technology

    Directory of Open Access Journals (Sweden)

    Yasuhiro Suzuki

    2012-01-01

    Full Text Available Specific protein domains known as protein transduction domains (PTDs can permeate cell membranes and deliver proteins or bioactive materials into living cells. Various approaches have been applied for improving their transduction efficacy. It is, therefore, crucial to clarify the entry mechanisms and to identify the rate-limiting steps. Because of technical limitations for imaging PTD behavior on cells with conventional fluorescent-dyes, how PTDs enter the cells has been a topic of much debate. Utilizing quantum dots (QDs, we recently tracked the behavior of PTD that was derived from HIV-1 Tat (TatP in living cells at the single-molecule level with 7-nm special precision. In this review article, we initially summarize the controversy on TatP entry mechanisms; thereafter, we will focus on our recent findings on single-TatP-QD tracking (SQT, to identify the major sequential steps of intracellular delivery in living cells and to discuss how SQT can easily provide direct information on TatP entry mechanisms. As a primer for SQT study, we also discuss the latest findings on single particle tracking of various molecules on the plasma membrane. Finally, we discuss the problems of QDs and the challenges for the future in utilizing currently available QD probes for SQT. In conclusion, direct identification of the rate-limiting steps of PTD entry with SQT should dramatically improve the methods for enhancing transduction efficiency.

  11. Molecular dynamics and MM/GBSA-integrated protocol probing the correlation between biological activities and binding free energies of HIV-1 TAR RNA inhibitors.

    Science.gov (United States)

    Peddi, Saikiran Reddy; Sivan, Sree Kanth; Manga, Vijjulatha

    2018-02-01

    The interaction of HIV-1 transactivator protein Tat with its cognate transactivation response (TAR) RNA has emerged as a promising target for developing antiviral compounds and treating HIV infection, since it is a crucial step for efficient transcription and replication. In the present study, molecular dynamics (MD) simulations and MM/GBSA calculations have been performed on a series of neamine derivatives in order to estimate appropriate MD simulation time for acceptable correlation between ΔG bind and experimental pIC 50 values. Initially, all inhibitors were docked into the active site of HIV-1 TAR RNA. Later to explore various conformations and examine the docking results, MD simulations were carried out. Finally, binding free energies were calculated using MM/GBSA method and were correlated with experimental pIC 50 values at different time scales (0-1 to 0-10 ns). From this study, it is clear that in case of neamine derivatives as simulation time increased the correlation between binding free energy and experimental pIC 50 values increased correspondingly. Therefore, the binding energies which can be interpreted at longer simulation times can be used to predict the bioactivity of new neamine derivatives. Moreover, in this work, we have identified some plausible critical nucleotide interactions with neamine derivatives that are responsible for potent inhibitory activity. Furthermore, we also provide some insights into a new class of oxadiazole-based back bone cyclic peptides designed by incorporating the structural features of neamine derivatives. On the whole, this approach can provide a valuable guidance for designing new potent inhibitors and modify the existing compounds targeting HIV-1 TAR RNA.

  12. Functional substitution by TAT-utrophin in dystrophin-deficient mice.

    Directory of Open Access Journals (Sweden)

    Kevin J Sonnemann

    2009-05-01

    Full Text Available The loss of dystrophin compromises muscle cell membrane stability and causes Duchenne muscular dystrophy and/or various forms of cardiomyopathy. Increased expression of the dystrophin homolog utrophin by gene delivery or pharmacologic up-regulation has been demonstrated to restore membrane integrity and improve the phenotype in the dystrophin-deficient mdx mouse. However, the lack of a viable therapy in humans predicates the need to explore alternative methods to combat dystrophin deficiency. We investigated whether systemic administration of recombinant full-length utrophin (Utr or DeltaR4-21 "micro" utrophin (muUtr protein modified with the cell-penetrating TAT protein transduction domain could attenuate the phenotype of mdx mice.Recombinant TAT-Utr and TAT-muUtr proteins were expressed using the baculovirus system and purified using FLAG-affinity chromatography. Age-matched mdx mice received six twice-weekly intraperitoneal injections of either recombinant protein or PBS. Three days after the final injection, mice were analyzed for several phenotypic parameters of dystrophin deficiency. Injected TAT-muUtr transduced all tissues examined, integrated with members of the dystrophin complex, reduced serum levels of creatine kinase (11,290+/-920 U versus 5,950+/-1,120 U; PBS versus TAT, the prevalence of muscle degeneration/regeneration (54%+/-5% versus 37%+/-4% of centrally nucleated fibers; PBS versus TAT, the susceptibility to eccentric contraction-induced force drop (72%+/-5% versus 40%+/-8% drop; PBS versus TAT, and increased specific force production (9.7+/-1.1 N/cm(2 versus 12.8+/-0.9 N/cm(2; PBS versus TAT.These results are, to our knowledge, the first to establish the efficacy and feasibility of TAT-utrophin-based constructs as a novel direct protein-replacement therapy for the treatment of skeletal and cardiac muscle diseases caused by loss of dystrophin.

  13. HIV-1 Latency in Monocytes/Macrophages

    Directory of Open Access Journals (Sweden)

    Amit Kumar

    2014-04-01

    Full Text Available Human immunodeficiency virus type 1 (HIV-1 targets CD4+ T cells and cells of the monocyte/macrophage lineage. HIV pathogenesis is characterized by the depletion of T lymphocytes and by the presence of a population of cells in which latency has been established called the HIV-1 reservoir. Highly active antiretroviral therapy (HAART has significantly improved the life of HIV-1 infected patients. However, complete eradication of HIV-1 from infected individuals is not possible without targeting latent sources of infection. HIV-1 establishes latent infection in resting CD4+ T cells and findings indicate that latency can also be established in the cells of monocyte/macrophage lineage. Monocyte/macrophage lineage includes among others, monocytes, macrophages and brain resident macrophages. These cells are relatively more resistant to apoptosis induced by HIV-1, thus are important stable hideouts of the virus. Much effort has been made in the direction of eliminating HIV-1 resting CD4+ T-cell reservoirs. However, it is impossible to achieve a cure for HIV-1 without considering these neglected latent reservoirs, the cells of monocyte/macrophage lineage. In this review we will describe our current understanding of the mechanism of latency in monocyte/macrophage lineage and how such cells can be specifically eliminated from the infected host.

  14. Neuroinflammation and Behavior in HIV-1 Transgenic Rats Exposed to Chronic Adolescent Stress.

    Science.gov (United States)

    Rowson, Sydney A; Harrell, Constance S; Bekhbat, Mandakh; Gangavelli, Apoorva; Wu, Matthew J; Kelly, Sean D; Reddy, Renuka; Neigh, Gretchen N

    2016-01-01

    Highly active antiretroviral therapy (HAART) has improved prognosis for people living with HIV (PLWH) and dramatically reduced the incidence of AIDS. However, even when viral load is controlled, PLWH develop psychiatric and neurological disorders more frequently than those living without HIV. Adolescents with HIV are particularly susceptible to the development of psychiatric illnesses and neurocognitive impairments. While both psychiatric and neurocognitive disorders have been found to be exacerbated by stress, the extent to which chronic stress and HIV-1 viral proteins interact to impact behavior and relevant neuroinflammatory processes is unknown. Determination of the individual contributions of stress and HIV to neuropsychiatric disorders is heavily confounded in humans. In order to isolate the influence of HIV-1 proteins and chronic stress on behavior and neuroinflammation, we employed the HIV-1 transgenic (Tg) rat model, which expresses HIV-1 proteins with a gag and pol deletion, allowing for viral protein expression without viral replication. This Tg line has been characterized as a model of HAART-controlled HIV-1 infection due to the lack of viral replication but continued presence of HIV-1 proteins. We exposed male and female adolescent HIV-1 Tg rats to a mixed-modality chronic stress paradigm consisting of isolation, social defeat and restraint, and assessed behavior, cerebral vascularization, and neuroinflammatory endpoints. Stress, sex, and presence of the HIV-1 transgene impacted weight gain in adolescent rats. Female HIV-1 Tg rats showed decreases in central tendency during the light cycle in the open field regardless of stress exposure. Both male and female HIV-1 Tg rats exhibited decreased investigative behavior in the novel object recognition task, but no memory impairments. Adolescent stress had no effect on the tested behaviors. Microglia in female HIV-1 Tg rats exhibited a hyper-ramified structure, and gene expression of complement factor B was

  15. Effects of human SAMHD1 polymorphisms on HIV-1 susceptibility

    International Nuclear Information System (INIS)

    White, Tommy E.; Brandariz-Nuñez, Alberto; Valle-Casuso, Jose Carlos; Knowlton, Caitlin; Kim, Baek; Sawyer, Sara L.; Diaz-Griffero, Felipe

    2014-01-01

    SAMHD1 is a human restriction factor that prevents efficient infection of macrophages, dendritic cells and resting CD4+ T cells by HIV-1. Here we explored the antiviral activity and biochemical properties of human SAMHD1 polymorphisms. Our studies focused on human SAMHD1 polymorphisms that were previously identified as evolving under positive selection for rapid amino acid replacement during primate speciation. The different human SAMHD1 polymorphisms were tested for their ability to block HIV-1, HIV-2 and equine infectious anemia virus (EIAV). All studied SAMHD1 variants block HIV-1, HIV-2 and EIAV infection when compared to wild type. We found that these variants did not lose their ability to oligomerize or to bind RNA. Furthermore, all tested variants were susceptible to degradation by Vpx, and localized to the nuclear compartment. We tested the ability of human SAMHD1 polymorphisms to decrease the dNTP cellular levels. In agreement, none of the different SAMHD1 variants lost their ability to reduce cellular levels of dNTPs. Finally, we found that none of the tested human SAMHD1 polymorphisms affected the ability of the protein to block LINE-1 retrotransposition. - Highlights: • Human SAMHD1 single-nucleotide polymorphisms block HIV-1 and HIV-2 infection. • SAMHD1 polymorphisms do not affect its ability to block LINE-1 retrotransposition. • SAMHD1 polymorphisms decrease the cellular levels of dNTPs

  16. Effects of human SAMHD1 polymorphisms on HIV-1 susceptibility

    Energy Technology Data Exchange (ETDEWEB)

    White, Tommy E.; Brandariz-Nuñez, Alberto; Valle-Casuso, Jose Carlos [Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, 1301 Morris Park – Price Center 501, New York, NY 10461 (United States); Knowlton, Caitlin; Kim, Baek [Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642 (United States); Sawyer, Sara L. [Department of Molecular Biosciences, University of Texas at Austin, Austin, TX 78712 (United States); Diaz-Griffero, Felipe, E-mail: Felipe.Diaz-Griffero@einstein.yu.edu [Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, 1301 Morris Park – Price Center 501, New York, NY 10461 (United States)

    2014-07-15

    SAMHD1 is a human restriction factor that prevents efficient infection of macrophages, dendritic cells and resting CD4+ T cells by HIV-1. Here we explored the antiviral activity and biochemical properties of human SAMHD1 polymorphisms. Our studies focused on human SAMHD1 polymorphisms that were previously identified as evolving under positive selection for rapid amino acid replacement during primate speciation. The different human SAMHD1 polymorphisms were tested for their ability to block HIV-1, HIV-2 and equine infectious anemia virus (EIAV). All studied SAMHD1 variants block HIV-1, HIV-2 and EIAV infection when compared to wild type. We found that these variants did not lose their ability to oligomerize or to bind RNA. Furthermore, all tested variants were susceptible to degradation by Vpx, and localized to the nuclear compartment. We tested the ability of human SAMHD1 polymorphisms to decrease the dNTP cellular levels. In agreement, none of the different SAMHD1 variants lost their ability to reduce cellular levels of dNTPs. Finally, we found that none of the tested human SAMHD1 polymorphisms affected the ability of the protein to block LINE-1 retrotransposition. - Highlights: • Human SAMHD1 single-nucleotide polymorphisms block HIV-1 and HIV-2 infection. • SAMHD1 polymorphisms do not affect its ability to block LINE-1 retrotransposition. • SAMHD1 polymorphisms decrease the cellular levels of dNTPs.

  17. Clustered epitopes within the Gag-Pol fusion protein DNA vaccine enhance immune responses and protection against challenge with recombinant vaccinia viruses expressing HIV-1 Gag and Pol antigens

    International Nuclear Information System (INIS)

    Bolesta, Elizabeth; Gzyl, Jaroslaw; Wierzbicki, Andrzej; Kmieciak, Dariusz; Kowalczyk, Aleksandra; Kaneko, Yutaro; Srinivasan, Alagarsamy; Kozbor, Danuta

    2005-01-01

    We have generated a codon-optimized hGagp17p24-Polp51 plasmid DNA expressing the human immunodeficiency virus type 1 (HIV-1) Gag-Pol fusion protein that consists of clusters of highly conserved cytotoxic T lymphocyte (CTL) epitopes presented by multiple MHC class I alleles. In the hGagp17p24-Polp51 construct, the ribosomal frameshift site had been deleted together with the potentially immunosuppressive Gag nucleocapsid (p15) as well as Pol protease (p10) and integrase (p31). Analyses of the magnitude and breadth of cellular responses demonstrated that immunization of HLA-A2/K b transgenic mice with the hGagp17p24-Polp51 construct induced 2- to 5-fold higher CD8 + T-cell responses to Gag p17-, p24-, and Pol reverse transcriptase (RT)-specific CTL epitopes than the full-length hGag-PolΔFsΔPr counterpart. The increases were correlated with higher protection against challenge with recombinant vaccinia viruses (rVVs) expressing gag and pol gene products. Consistent with the profile of Gag- and Pol-specific CD8 + T cell responses, an elevated level of type 1 cytokine production was noted in p24- and RT-stimulated splenocyte cultures established from hGagp17p24-Polp51-immunized mice compared to responses induced with the hGag-PolΔFsΔPr vaccine. Sera of mice immunized with the hGagp17p24-Polp51 vaccine also exhibited an increased titer of p24- and RT-specific IgG2 antibody responses. The results from our studies provide insights into approaches for boosting the breadth of Gag- and Pol-specific immune responses

  18. Accumulation of MxB/Mx2-resistant HIV-1 Capsid Variants During Expansion of the HIV-1 Epidemic in Human Populations.

    Science.gov (United States)

    Wei, Wei; Guo, Haoran; Ma, Min; Markham, Richard; Yu, Xiao-Fang

    2016-06-01

    Recent studies have identified human myxovirus resistance protein 2 (MxB or Mx2) as an interferon induced inhibitor of HIV-1 replication. However, whether HIV-1 can overcome MxB restriction without compromise of viral fitness has been undefined. Here, we have discovered that naturally occurring capsid (CA) variants can render HIV-1 resistant to the activity of MxB without losing viral infectivity or the ability to escape from interferon induction. Moreover, these MxB resistant HIV-1 variants do not lose MxB recognition. Surprisingly, MxB resistant CA variants are most commonly found in the Clade C HIV-1 that is the most rapidly expanding Clade throughout the world. Accumulation of MxB resistant mutations is also observed during HIV-1 spreading in human populations. These findings support a potential role for MxB as a selective force during HIV-1 transmission and evolution. Copyright © 2016. Published by Elsevier B.V.

  19. Molecular epidemiology of HIV-1 among the HIV infected people of Manipur, Northeastern India: Emergence of unique recombinant forms.

    Science.gov (United States)

    Sharma, Adhikarimayum Lakhikumar; Singh, Thiyam Ramsing; Devi, Khuraijam Ranjana; Singh, Lisam Shanjukumar

    2017-06-01

    According to the Joint National Programme on HIV/AIDS (UNAIDS), the northeastern region of India has the highest HIV prevalence in the country. This study was conducted to determine the current HIV-1 molecular epidemiology of Manipur, a state in northeast India. Blood samples from HIV-1 seropositive subjects were collected between June 2011 and February 2014. The partial regions of HIV-1 genes; pol and tat-vpu-env were independently amplified, sequenced, analyzed, and genotyped. Based on all sequences generated from 110 samples using pol and/or tat-vpu-env gene, the overall HIV-1 genotypes distribution of Manipur was as follows: 65.45% (72/110) subtype C, 32.73% (36/110) unique recombinant forms (URFs), and 1.82% (2/110) subtype B. The distribution of HIV-1 genotypes among the risk groups was: heterosexual: 58.33% (35/60) subtype C, 38.33% (23/60) URFs, and 3.34% (2/60) subtype B; intravenous drug users (IDUs): 85.36% (35/41) subtype C, 9.76% (4/41) URFs, and 4.88% (2/41) subtype B; mother to child (MTC): 50% (3/6) URFs and 50% (3/6) subtype C and blood transfusion: 100% (3/3) subtype C. The findings for the first time revealed the emergence of URFs of HIV-1 in Manipur which is predominant among the sexual and MTC risk groups as compared to IDUs. Taking together, this study illustrated that Manipur is the "recombinant hotspot of HIV" of India. The results will provide the clinical importance for continuous monitoring of HIV-infections in order to design appropriate prevention measures to limit the spread of new HIV infections. © 2016 Wiley Periodicals, Inc.

  20. Cyclophilin B enhances HIV-1 infection.

    Science.gov (United States)

    DeBoer, Jason; Madson, Christian J; Belshan, Michael

    2016-02-01

    Cyclophilin B (CypB) is a member of the immunophilin family and intracellular chaperone. It predominantly localizes to the ER, but also contains a nuclear localization signal and is secreted from cells. CypB has been shown to interact with the Gag protein of human immunodeficiency type 1 (HIV-1). Several proteomic and genetic studies identified it as a potential factor involved in HIV replication. Herein, we show that over-expression of CypB enhances HIV infection by increasing nuclear import of viral DNA. This enhancement was unaffected by cyclosporine treatment and requires the N-terminus of the protein. The N-terminus contains an ER leader sequence, putative nuclear localization signal, and is required for secretion. Deletion of the N-terminus resulted in mislocalization from the ER and suppression of HIV infection. Passive transfer experiments showed that secreted CypB did not impact HIV infection. Combined, these experiments show that intracellular CypB modulates a pathway of HIV nuclear import. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Direct and dynamic detection of HIV-1 in living cells.

    Directory of Open Access Journals (Sweden)

    Jonas Helma

    Full Text Available In basic and applied HIV research, reliable detection of viral components is crucial to monitor progression of infection. While it is routine to detect structural viral proteins in vitro for diagnostic purposes, it previously remained impossible to directly and dynamically visualize HIV in living cells without genetic modification of the virus. Here, we describe a novel fluorescent biosensor to dynamically trace HIV-1 morphogenesis in living cells. We generated a camelid single domain antibody that specifically binds the HIV-1 capsid protein (CA at subnanomolar affinity and fused it to fluorescent proteins. The resulting fluorescent chromobody specifically recognizes the CA-harbouring HIV-1 Gag precursor protein in living cells and is applicable in various advanced light microscopy systems. Confocal live cell microscopy and super-resolution microscopy allowed detection and dynamic tracing of individual virion assemblies at the plasma membrane. The analysis of subcellular binding kinetics showed cytoplasmic antigen recognition and incorporation into virion assembly sites. Finally, we demonstrate the use of this new reporter in automated image analysis, providing a robust tool for cell-based HIV research.

  2. Cytoplasmic Dynein Promotes HIV-1 Uncoating

    Directory of Open Access Journals (Sweden)

    Paulina Pawlica

    2014-11-01

    Full Text Available Retroviral capsid (CA cores undergo uncoating during their retrograde transport (toward the nucleus, and/or after reaching the nuclear membrane. However, whether HIV-1 CA core uncoating is dependent upon its transport is not understood. There is some evidence that HIV-1 cores retrograde transport involves cytoplasmic dynein complexes translocating on microtubules. Here we investigate the role of dynein-dependent transport in HIV-1 uncoating. To interfere with dynein function, we depleted dynein heavy chain (DHC using RNA interference, and we over-expressed p50/dynamitin. In immunofluorescence microscopy experiments, DHC depletion caused an accumulation of CA foci in HIV-1 infected cells. Using a biochemical assay to monitor HIV-1 CA core disassembly in infected cells, we observed an increase in amounts of intact (pelletable CA cores upon DHC depletion or p50 over-expression. Results from these two complementary assays suggest that inhibiting dynein-mediated transport interferes with HIV-1 uncoating in infected cells, indicating the existence of a functional link between HIV-1 transport and uncoating.

  3. Compartmentalization of the gut viral reservoir in HIV-1 infected patients

    Directory of Open Access Journals (Sweden)

    Grant Tannika

    2007-12-01

    Full Text Available Abstract Background Recently there has been an increasing interest and appreciation for the gut as both a viral reservoir as well as an important host-pathogen interface in human immunodefiency virus type 1 (HIV-1 infection. The gut associated lymphoid tissue (GALT is the largest lymphoid organ infected by HIV-1. In this study we examined if different HIV-1 quasispecies are found in different parts of the gut of HIV-1 infected individuals. Results Gut biopsies (esophagus, stomach, duodenum and colorectum were obtained from eight HIV-1 infected preHAART (highly active antiretroviral therapy patients. HIV-1 Nef and Reverse transcriptase (RT encoding sequences were obtained through nested PCR amplification from DNA isolated from the gut biopsy tissues. The PCR fragments were cloned and sequenced. The resulting sequences were subjected to various phylogenetic analyses. Expression of the nef gene and viral RNA in the different gut tissues was determined using real-time RT-PCR. Phylogenetic analysis of the Nef protein-encoding region revealed compartmentalization of viral replication in the gut within patients. Viral diversity in both the Nef and RT encoding region varied in different parts of the gut. Moreover, increased nef gene expression (p Conclusion Our results indicated that different HIV-1 quasispecies populate different parts of the gut, and that viral replication in the gut is compartmentalized. These observations underscore the importance of the gut as a host-pathogen interface in HIV-1 infection.

  4. Differential effect of CLK SR Kinases on HIV-1 gene expression: potential novel targets for therapy

    Directory of Open Access Journals (Sweden)

    Dobson Wendy

    2011-06-01

    Full Text Available Abstract Background RNA processing plays a critical role in the replication of HIV-1, regulated in part through the action of host SR proteins. To explore the impact of modulating SR protein activity on virus replication, the effect of increasing or inhibiting the activity of the Cdc2-like kinase (CLK family of SR protein kinases on HIV-1 expression and RNA processing was examined. Results Despite their high homology, increasing individual CLK expression had distinct effects on HIV-1, CLK1 enhancing Gag production while CLK2 inhibited the virus. Parallel studies on the anti-HIV-1 activity of CLK inhibitors revealed a similar discrepant effect on HIV-1 expression. TG003, an inhibitor of CLK1, 2 and 4, had no effect on viral Gag synthesis while chlorhexidine, a CLK2, 3 and 4 inhibitor, blocked virus production. Chlorhexidine treatment altered viral RNA processing, decreasing levels of unspliced and single spliced viral RNAs, and reduced Rev accumulation. Subsequent experiments in the context of HIV-1 replication in PBMCs confirmed the capacity of chlorhexidine to suppress virus replication. Conclusions Together, these findings establish that HIV-1 RNA processing can be targeted to suppress virus replication as demonstrated by manipulating individual CLK function and identified chlorhexidine as a lead compound in the development of novel anti-viral therapies.

  5. Immune defence against HIV-1 infection in HIV-1-exposed seronegative persons.

    Science.gov (United States)

    Schmechel, S C; Russell, N; Hladik, F; Lang, J; Wilson, A; Ha, R; Desbien, A; McElrath, M J

    2001-11-01

    Rare individuals who are repeatedly exposed to HIV-1 through unprotected sexual contact fail to acquire HIV-1 infection. These persons represent a unique study population to evaluate mechanisms by which HIV-1 replication is either prevented or controlled. We followed longitudinally a group of healthy HIV-1 seronegative persons each reporting repeated high-risk sexual activities with their HIV-1-infected partner at enrollment. The volunteers were primarily (90%) male homosexuals, maintaining high risk activities with their known infected partner (45%) or multiple other partners (61%). We evaluated the quantity and specificity of HIV-1-specific T cells in 31 exposed seronegatives (ES) using a IFN-gamma ELISPOT assay to enumerate T cells recognizing epitopes within HIV-1 Env, Gag, Pol and Nef. PBMC from only three of the 31 volunteers demonstrated ex vivo HIV-1-specific IFN-gamma secretion, in contrast to nearly 30% exhibiting cytolytic responses in previous studies. These findings suggest that if T cell responses in ES are induced by HIV-1 exposure, the frequency is at low levels in most of them, and below the level of detection using the ELISPOT assay. Alternative approaches to improve the sensitivity of detection may include use of dendritic cells as antigen-presenting cells in the ex vivo assay and more careful definition of the risk behavior and extent of HIV-1 exposure in conjunction with the evaluation of T cell responses.

  6. The C-terminal sequence of IFITM1 regulates its anti-HIV-1 activity.

    Directory of Open Access Journals (Sweden)

    Rui Jia

    Full Text Available The interferon-inducible transmembrane (IFITM proteins inhibit a wide range of viruses. We previously reported the inhibition of human immunodeficiency virus type 1 (HIV-1 strain BH10 by human IFITM1, 2 and 3. It is unknown whether other HIV-1 strains are similarly inhibited by IFITMs and whether there exists viral countermeasure to overcome IFITM inhibition. We report here that the HIV-1 NL4-3 strain (HIV-1NL4-3 is not restricted by IFITM1 and its viral envelope glycoprotein is partly responsible for this insensitivity. However, HIV-1NL4-3 is profoundly inhibited by an IFITM1 mutant, known as Δ(117-125, which is deleted of 9 amino acids at the C-terminus. In contrast to the wild type IFITM1, which does not affect HIV-1 entry, the Δ(117-125 mutant diminishes HIV-1NL4-3 entry by 3-fold. This inhibition correlates with the predominant localization of Δ(117-125 to the plasma membrane where HIV-1 entry occurs. In spite of strong conservation of IFITM1 among most species, mouse IFITM1 is 19 amino acids shorter at its C-terminus as compared to human IFITM1 and, like the human IFITM1 mutant Δ(117-125, mouse IFITM1 also inhibits HIV-1 entry. This is the first report illustrating the role of viral envelope protein in overcoming IFITM1 restriction. The results also demonstrate the importance of the C-terminal region of IFITM1 in modulating the antiviral function through controlling protein subcellular localization.

  7. NFAT5 regulates HIV-1 in primary monocytes via a highly conserved long terminal repeat site.

    Directory of Open Access Journals (Sweden)

    Shahin Ranjbar

    2006-12-01

    Full Text Available To replicate, HIV-1 capitalizes on endogenous cellular activation pathways resulting in recruitment of key host transcription factors to its viral enhancer. RNA interference has been a powerful tool for blocking key checkpoints in HIV-1 entry into cells. Here we apply RNA interference to HIV-1 transcription in primary macrophages, a major reservoir of the virus, and specifically target the transcription factor NFAT5 (nuclear factor of activated T cells 5, which is the most evolutionarily divergent NFAT protein. By molecularly cloning and sequencing isolates from multiple viral subtypes, and performing DNase I footprinting, electrophoretic mobility shift, and promoter mutagenesis transfection assays, we demonstrate that NFAT5 functionally interacts with a specific enhancer binding site conserved in HIV-1, HIV-2, and multiple simian immunodeficiency viruses. Using small interfering RNA to ablate expression of endogenous NFAT5 protein, we show that the replication of three major HIV-1 viral subtypes (B, C, and E is dependent upon NFAT5 in human primary differentiated macrophages. Our results define a novel host factor-viral enhancer interaction that reveals a new regulatory role for NFAT5 and defines a functional DNA motif conserved across HIV-1 subtypes and representative simian immunodeficiency viruses. Inhibition of the NFAT5-LTR interaction may thus present a novel therapeutic target to suppress HIV-1 replication and progression of AIDS.

  8. Large-scale functional purification of recombinant HIV-1 capsid.

    Directory of Open Access Journals (Sweden)

    Magdeleine Hung

    Full Text Available During human immunodeficiency virus type-1 (HIV-1 virion maturation, capsid proteins undergo a major rearrangement to form a conical core that protects the viral nucleoprotein complexes. Mutations in the capsid sequence that alter the stability of the capsid core are deleterious to viral infectivity and replication. Recently, capsid assembly has become an attractive target for the development of a new generation of anti-retroviral agents. Drug screening efforts and subsequent structural and mechanistic studies require gram quantities of active, homogeneous and pure protein. Conventional means of laboratory purification of Escherichia coli expressed recombinant capsid protein rely on column chromatography steps that are not amenable to large-scale production. Here we present a function-based purification of wild-type and quadruple mutant capsid proteins, which relies on the inherent propensity of capsid protein to polymerize and depolymerize. This method does not require the packing of sizable chromatography columns and can generate double-digit gram quantities of functionally and biochemically well-behaved proteins with greater than 98% purity. We have used the purified capsid protein to characterize two known assembly inhibitors in our in-house developed polymerization assay and to measure their binding affinities. Our capsid purification procedure provides a robust method for purifying large quantities of a key protein in the HIV-1 life cycle, facilitating identification of the next generation anti-HIV agents.

  9. Identification of dual-tropic HIV-1 using evolved neural networks.

    Science.gov (United States)

    Fogel, Gary B; Lamers, Susanna L; Liu, Enoch S; Salemi, Marco; McGrath, Michael S

    2015-11-01

    Blocking the binding of the envelope HIV-1 protein to immune cells is a popular concept for development of anti-HIV therapeutics. R5 HIV-1 binds CCR5, X4 HIV-1 binds CXCR4, and dual-tropic HIV-1 can bind either coreceptor for cellular entry. R5 viruses are associated with early infection and over time can evolve to X4 viruses that are associated with immune failure. Dual-tropic HIV-1 is less studied; however, it represents functional antigenic intermediates during the transition of R5 to X4 viruses. Viral tropism is linked partly to the HIV-1 envelope V3 domain, where the amino acid sequence helps dictate the receptor a particular virus will target; however, using V3 sequence information to identify dual-tropic HIV-1 isolates has remained difficult. Our goal in this study was to elucidate features of dual-tropic HIV-1 isolates that assist in the biological understanding of dual-tropism and develop an approach for their detection. Over 1559 HIV-1 subtype B sequences with known tropisms were analyzed. Each sequence was represented by 73 structural, biochemical and regional features. These features were provided to an evolved neural network classifier and evaluated using balanced and unbalanced data sets. The study resolved R5X4 viruses from R5 with an accuracy of 81.8% and from X4 with an accuracy of 78.8%. The approach also identified a set of V3 features (hydrophobicity, structural and polarity) that are associated with tropism transitions. The ability to distinguish R5X4 isolates will improve computational tropism decisions for R5 vs. X4 and assist in HIV-1 research and drug development efforts. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  10. HIV-1 transmission linkage in an HIV-1 prevention clinical trial

    Energy Technology Data Exchange (ETDEWEB)

    Leitner, Thomas [Los Alamos National Laboratory; Campbell, Mary S [UNIV OF WASHINGTON; Mullins, James I [UNIV OF WASHINGTON; Hughes, James P [UNIV OF WASHINGTON; Wong, Kim G [UNIV OF WASHINGTON; Raugi, Dana N [UNIV OF WASHINGTON; Scrensen, Stefanie [UNIV OF WASHINGTON

    2009-01-01

    HIV-1 sequencing has been used extensively in epidemiologic and forensic studies to investigate patterns of HIV-1 transmission. However, the criteria for establishing genetic linkage between HIV-1 strains in HIV-1 prevention trials have not been formalized. The Partners in Prevention HSV/HIV Transmission Study (ClinicaITrials.gov NCT00194519) enrolled 3408 HIV-1 serodiscordant heterosexual African couples to determine the efficacy of genital herpes suppression with acyclovir in reducing HIV-1 transmission. The trial analysis required laboratory confirmation of HIV-1 linkage between enrolled partners in couples in which seroconversion occurred. Here we describe the process and results from HIV-1 sequencing studies used to perform transmission linkage determination in this clinical trial. Consensus Sanger sequencing of env (C2-V3-C3) and gag (p17-p24) genes was performed on plasma HIV-1 RNA from both partners within 3 months of seroconversion; env single molecule or pyrosequencing was also performed in some cases. For linkage, we required monophyletic clustering between HIV-1 sequences in the transmitting and seroconverting partners, and developed a Bayesian algorithm using genetic distances to evaluate the posterior probability of linkage of participants sequences. Adjudicators classified transmissions as linked, unlinked, or indeterminate. Among 151 seroconversion events, we found 108 (71.5%) linked, 40 (26.5%) unlinked, and 3 (2.0%) to have indeterminate transmissions. Nine (8.3%) were linked by consensus gag sequencing only and 8 (7.4%) required deep sequencing of env. In this first use of HIV-1 sequencing to establish endpoints in a large clinical trial, more than one-fourth of transmissions were unlinked to the enrolled partner, illustrating the relevance of these methods in the design of future HIV-1 prevention trials in serodiscordant couples. A hierarchy of sequencing techniques, analysis methods, and expert adjudication contributed to the linkage

  11. Discovery of a small molecule agonist of phosphatidylinositol 3-kinase p110α that reactivates latent HIV-1.

    Directory of Open Access Journals (Sweden)

    Geneviève Doyon

    Full Text Available Combination antiretroviral therapy (cART can effectively suppress HIV-1 replication, but the latent viral reservoir in resting memory CD4(+ T cells is impervious to cART and represents a major barrier to curing HIV-1 infection. Reactivation of latent HIV-1 represents a possible strategy for elimination of this reservoir. In this study we describe the discovery of 1,2,9,10-tetramethoxy-7H-dibenzo[de,g]quinolin-7-one (57704 which reactivates latent HIV-1 in several cell-line models of latency (J89GFP, U1 and ACH-2. 57704 also increased HIV-1 expression in 3 of 4 CD8(+-depleted blood mononuclear cell preparations isolated from HIV-1-infected individuals on suppressive cART. In contrast, vorinostat increased HIV-1 expression in only 1 of the 4 donors tested. Importantly, 57704 does not induce global T cell activation. Mechanistic studies revealed that 57704 reactivates latent HIV-1 via the phosphatidylinositol 3-kinase (PI3K/protein kinase B (Akt signaling pathway. 57704 was found to be an agonist of PI3K with specificity to the p110α isoform, but not the p110β, δ or γ isoforms. Taken together, our work suggests that 57704 could serve as a scaffold for the development of more potent activators of latent HIV-1. Furthermore, it highlights the involvement of the PI3K/Akt pathway in the maintenance of HIV-1 latency.

  12. The Presence and Anti-HIV-1 Function of Tenascin C in Breast Milk and Genital Fluids.

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    Robin G Mansour

    Full Text Available Tenascin-C (TNC is a newly identified innate HIV-1-neutralizing protein present in breast milk, yet its presence and potential HIV-inhibitory function in other mucosal fluids is unknown. In this study, we identified TNC as a component of semen and cervical fluid of HIV-1-infected and uninfected individuals, although it is present at a significantly lower concentration and frequency compared to that of colostrum and mature breast milk, potentially due to genital fluid protease degradation. However, TNC was able to neutralize HIV-1 after exposure to low pH, suggesting that TNC could be active at low pH in the vaginal compartment. As mucosal fluids are complex and contain a number of proteins known to interact with the HIV-1 envelope, we further studied the relationship between the concentration of TNC and neutralizing activity in breast milk. The amount of TNC correlated only weakly with the overall innate HIV-1-neutralizing activity of breast milk of uninfected women and negatively correlated with neutralizing activity in milk of HIV-1 infected women, indicating that the amount of TNC in mucosal fluids is not adequate to impede HIV-1 transmission. Moreover, the presence of polyclonal IgG from milk of HIV-1 infected women, but not other HIV-1 envelope-binding milk proteins or monoclonal antibodies, blocked the neutralizing activity of TNC. Finally, as exogenous administration of TNC would be necessary for it to mediate measurable HIV-1 neutralizing activity in mucosal compartments, we established that recombinantly produced TNC has neutralizing activity against transmitted/founder HIV-1 strains that mimic that of purified TNC. Thus, we conclude that endogenous TNC concentration in mucosal fluids is likely inadequate to block HIV-1 transmission to uninfected individuals.

  13. HIV-1 Nef in Macrophage-Mediated Disease Pathogenesis

    Science.gov (United States)

    Lamers, Susanna L.; Fogel, Gary B.; Singer, Elyse J.; Salemi, Marco; Nolan, David J.; Huysentruyt, Leanne C.; McGrath, Michael S.

    2013-01-01

    Combined anti-retroviral therapy (cART) has significantly reduced the number of AIDS-associated illnesses and changed the course of HIV-1 disease in developed countries. Despite the ability of cART to maintain high CD4+ T-cell counts, a number of macrophage-mediated diseases can still occur in HIV-infected subjects. These diseases include lymphoma, metabolic diseases, and HIV-associated neurological disorders. Within macrophages, the HIV-1 regulatory protein “Nef” can modulate surface receptors, interact with signaling pathways, and promote specific environments that contribute to each of these pathologies. Moreover, genetic variation in Nef may also guide the macrophage response. Herein, we review findings relating to the Nef–macrophage interaction and how this relationship contributes to disease pathogenesis. PMID:23215766

  14. Inhibition of HIV-1 by curcumin A, a novel curcumin analog

    Science.gov (United States)

    Kumari, Namita; Kulkarni, Amol A; Lin, Xionghao; McLean, Charlee; Ammosova, Tatiana; Ivanov, Andrey; Hipolito, Maria; Nekhai, Sergei; Nwulia, Evaristus

    2015-01-01

    Despite the remarkable success of combination antiretroviral therapy at curtailing HIV progression, emergence of drug-resistant viruses, chronic low-grade inflammation, and adverse effects of combination antiretroviral therapy treatments, including metabolic disorders collectively present the impetus for development of newer and safer antiretroviral drugs. Curcumin, a phytochemical compound, was previously reported to have some in vitro anti-HIV and anti-inflammatory activities, but poor bioavailability has limited its clinical utility. To circumvent the bioavailability problem, we derivatized curcumin to sustain retro-aldol decomposition at physiological pH. The lead compound derived, curcumin A, showed increased stability, especially in murine serum where it was stable for up to 25 hours, as compared to curcumin that only had a half-life of 10 hours. Both curcumin and curcumin A showed similar inhibition of one round of HIV-1 infection in cultured lymphoblastoid (also called CEM) T cells (IC50=0.7 μM). But in primary peripheral blood mononuclear cells, curcumin A inhibited HIV-1 more potently (IC50=2 μM) compared to curcumin (IC50=12 μM). Analysis of specific steps of HIV-1 replication showed that curcumin A inhibited HIV-1 reverse transcription, but had no effect on HIV-1 long terminal repeat basal or Tat-induced transcription, or NF-κB-driven transcription at low concentrations that affected reverse transcription. Finally, we showed curcumin A induced expression of HO-1 and decreased cell cycle progression of T cells. Our findings thus indicate that altering the core structure of curcumin could yield more stable compounds with potent antiretroviral and anti-inflammatory activities. PMID:26366056

  15. Construction of Nef-positive doxycycline-dependent HIV-1 variants using bicistronic expression elements

    Energy Technology Data Exchange (ETDEWEB)

    Velden, Yme U. van der; Kleibeuker, Wendy; Harwig, Alex; Klaver, Bep; Siteur-van Rijnstra, Esther; Frankin, Esmay; Berkhout, Ben; Das, Atze T., E-mail: a.t.das@amc.uva.nl

    2016-01-15

    Conditionally replicating HIV-1 variants that can be switched on and off at will are attractive tools for HIV research. We previously developed a genetically modified HIV-1 variant that replicates exclusively when doxycycline (dox) is administered. The nef gene in this HIV-rtTA variant was replaced with the gene encoding the dox-dependent rtTA transcriptional activator. Because loss of Nef expression compromises virus replication in primary cells and precludes studies on Nef function, we tested different approaches to restore Nef production in HIV-rtTA. Strategies that involved translation via an EMCV or synthetic internal ribosome entry site (IRES) failed because these elements were incompatible with efficient virus replication. Fusion protein approaches with the FMDV 2A peptide and human ubiquitin were successful and resulted in genetically-stable Nef-expressing HIV-rtTA strains that replicate more efficiently in primary T-cells and human immune system (HIS) mice than Nef-deficient variants, thus confirming the positive effect of Nef on in vivo virus replication. - Highlights: • Different approaches to encode additional proteins in the HIV-1 genome were tested. • IRES translation elements are incompatible with efficient HIV-1 replication. • Ubiquitin and 2A fusion protein approaches allow efficient HIV-1 replication. • Doxycycline-controlled HIV-1 variants that encode all viral proteins were developed. • Nef stimulates HIV-rtTA replication in primary cells and human immune system mice.

  16. Nef enhances HIV-1 infectivity via association with the virus assembly complex

    International Nuclear Information System (INIS)

    Qi Mingli; Aiken, Christopher

    2008-01-01

    The HIV-1 accessory protein Nef enhances virus infectivity by facilitating an early post-entry step of infection. Nef acts in the virus producer cell, leading to a beneficial modification to HIV-1 particles. Nef itself is incorporated into HIV-1 particles, where it is cleaved by the viral protease during virion maturation. To probe the role of virion-associated Nef in HIV-1 infection, we generated a fusion protein consisting of the host protein cyclophilin A (CypA) linked to the amino terminus of Nef. The resulting CypA-Nef protein enhanced the infectivity of Nef-defective HIV-1 particles and was specifically incorporated into the virions via association with Gag during particle assembly. Pharmacologic or genetic inhibition of CypA-Nef binding to Gag prevented incorporation of CypA-Nef into virions and inhibited infectivity enhancement. Our results indicate that infectivity enhancement by Nef requires its association with a component of the assembling HIV-1 particle

  17. TAT improves in vitro transportation of fortilin through midgut and into hemocytes of white shrimp Litopenaeus vannamei

    Science.gov (United States)

    Zhou, Yi; Zhang, Wenbing; Mai, Kangsen; Xu, Wei; Zhang, Yanjiao; Ai, Qinghui; Wang, Xiaojie

    2012-06-01

    Fortilin is a multifunctional protein implicated in many important cellular processes. Since injection of Pm-fortilin reduces shrimp mortality caused by white spot syndrome virus (WSSV), there is potential application of fortilin in shrimp culture. In the present study, in order to improve trans-membrane transportation efficiency, the protein transduction domain of the transactivator of transcription (TAT) peptide was fused to fortilin. The Pichia pastoris yeast expression system, which is widely accepted in animal feeds, was used for production of recombinant fusion protein. Green fluorescence protein (GFP) was selected as a reporter because of its intrinsic visible fluorescence. The fortilin, TAT and GFP fusion protein were constructed. Their trans-membrane transportation efficiency and effects on immune response of shrimp were analyzed in vitro. Results showed that TAT peptide improved in vitro uptake of fortilin into the hemocytes and midgut of Litopenaeus vannamei. The phenoloxidase (PO) activity of hemocytes incubated with GFP-Fortilin or GFP-Fortilin-TAT was significantly increased compared with that in the control without expressed fortilin. The PO activity of hemocytes incubated with 200 μg mL-1 GFP-Fortilin-TAT was significantly higher than that in the group with the same concentration of GFP-Fortilin. Hemocytes incubated with GFP-Fortilin-TAT at all concentrations showed significantly higher nitric oxide synthase (NOS) activity than those in the control or in the GFP-Fortilin treatment. The present in vitro study indicated that TAT fusion protein improved the immune effect of fortilin.

  18. Structure-based virtual screening toward the discovery of novel inhibitors for impeding the protein-protein interaction between HIV-1 integrase and human lens epithelium-derived growth factor (LEDGF/p75).

    Science.gov (United States)

    Panwar, Umesh; Singh, Sanjeev Kumar

    2017-10-23

    HIV-1 integrase is a unique promising component of the viral replication cycle, catalyzing the integration of reverse transcribed viral cDNA into the host cell genome. Generally, IN activity requires both viral as well as a cellular co-factor in the processing replication cycle. Among them, the human lens epithelium-derived growth factor (LEDGF/p75) represented as promising cellular co-factor which supports the viral replication by tethering IN to the chromatin. Due to its major importance in the early steps of HIV replication, the interaction between IN and LEDGF/p75 has become a pleasing target for anti-HIV drug discovery. The present study involves the finding of novel inhibitor based on the information of dimeric CCD of IN in complex with known inhibitor, which were carried out by applying a structure-based virtual screening concept with molecular docking. Additionally, Free binding energy, ADME properties, PAINS analysis, Density Functional Theory, and Enrichment Calculations were performed on selected compounds for getting a best lead molecule. On the basis of these analyses, the current study proposes top 3 compounds: Enamine-Z742267384, Maybridge-HTS02400, and Specs-AE-848/37125099 with acceptable pharmacological properties and enhanced binding affinity to inhibit the interaction between IN and LEDGF/p75. Furthermore, Simulation studies were carried out on these molecules to expose their dynamics behavior and stability. We expect that the findings obtained here could be future therapeutic agents and may provide an outline for the experimental studies to stimulate the innovative strategy for research community.

  19. Inhibition of human immunodeficiency virus type 1 (HIV-1) nuclear import via Vpr-Importin α interactions as a novel HIV-1 therapy

    International Nuclear Information System (INIS)

    Suzuki, Tatsunori; Yamamoto, Norio; Nonaka, Mizuho; Hashimoto, Yoshie; Matsuda, Go; Takeshima, Shin-nosuke; Matsuyama, Megumi; Igarashi, Tatsuhiko; Miura, Tomoyuki; Tanaka, Rie; Kato, Shingo; Aida, Yoko

    2009-01-01

    The development of multidrug-resistant viruses compromises the efficacy of anti-human immunodeficiency virus (HIV) therapy and limits treatment options. Therefore, new targets that can be used to develop novel antiviral agents need to be identified. One such target is the interaction between Vpr, one of the accessory gene products of HIV-1 and Importin α, which is crucial, not only for the nuclear import of Vpr, but also for HIV-1 replication in macrophages. We have identified a potential parent compound, hematoxylin, which suppresses Vpr-Importin α interaction, thereby inhibiting HIV-1 replication in a Vpr-dependent manner. Analysis by real-time PCR demonstrated that hematoxylin specifically inhibited nuclear import step of pre-integration complex. Thus, hematoxylin is a new anti-HIV-1 inhibitor that targets the nuclear import of HIV-1 via the Vpr-Importin α interaction, suggesting that a specific inhibitor of the interaction between viral protein and the cellular factor may provide a new strategy for HIV-1 therapy.

  20. Semen Bacterial Concentrations and HIV-1 RNA Shedding Among HIV-1–Seropositive Kenyan Men

    Science.gov (United States)

    Srinivasan, Sujatha; Huang, Dandi; Ko, Daisy L.; Sanders, Eduard J.; Peshu, Norbert M.; Krieger, John N.; Muller, Charles H.; Coombs, Robert W.; Fredricks, David N.; Graham, Susan M.

    2017-01-01

    Introduction: HIV-1 is transmitted through semen from men to their sexual partners. Genital infections can increase HIV-1 RNA shedding in semen, but shedding also occurs in the absence of typical pathogens. We hypothesized that higher bacterial concentrations in semen would be associated with higher HIV-1 RNA levels. Methods: We analyzed semen samples from 42 HIV-1–seropositive Kenyan men using quantitative polymerase chain reaction (PCR) to assess bacterial concentrations and real-time PCR to measure HIV-1 RNA levels. Generalized estimation equations were used to evaluate associations between these 2 measures. Broad-range 16S rRNA gene PCR with pyrosequencing was performed on a subset of 13 samples to assess bacterial community composition. Results: Bacteria were detected in 96.6% of 88 samples by quantitative PCR. Semen bacterial concentration and HIV-1 RNA levels were correlated 0.30 (P = 0.01). The association between bacterial concentration and HIV-1 RNA detection was not significant after adjustment for antiretroviral therapy (ART) (adjusted odds ratio: 1.27, 95% CI: 0.84 to 1.91). Factors associated with semen bacterial concentration included insertive anal sex (adjusted beta 0.92, 95% CI: 0.12 to 1.73) and ART use (adjusted beta: −0.77, 95% CI: −1.50 to 0.04). Among 13 samples with pyrosequencing data, Corynebacterium spp., Staphylococcus spp., and Streptococcus spp. were most frequently detected. Conclusion: Most of these HIV-1–infected men had bacteria in their semen. ART use was associated with undetectable semen HIV-1 RNA and lower semen bacterial concentrations, whereas insertive anal sex was associated with higher bacterial concentrations. Additional studies evaluating the relationship between semen bacteria, inflammation, mucosal immunity, and HIV-1 shedding are needed to understand implications for HIV-1 transmission. PMID:27861240

  1. Semen Bacterial Concentrations and HIV-1 RNA Shedding Among HIV-1-Seropositive Kenyan Men.

    Science.gov (United States)

    Korhonen, Christine J; Srinivasan, Sujatha; Huang, Dandi; Ko, Daisy L; Sanders, Eduard J; Peshu, Norbert M; Krieger, John N; Muller, Charles H; Coombs, Robert W; Fredricks, David N; Graham, Susan M

    2017-03-01

    HIV-1 is transmitted through semen from men to their sexual partners. Genital infections can increase HIV-1 RNA shedding in semen, but shedding also occurs in the absence of typical pathogens. We hypothesized that higher bacterial concentrations in semen would be associated with higher HIV-1 RNA levels. We analyzed semen samples from 42 HIV-1-seropositive Kenyan men using quantitative polymerase chain reaction (PCR) to assess bacterial concentrations and real-time PCR to measure HIV-1 RNA levels. Generalized estimation equations were used to evaluate associations between these 2 measures. Broad-range 16S rRNA gene PCR with pyrosequencing was performed on a subset of 13 samples to assess bacterial community composition. Bacteria were detected in 96.6% of 88 samples by quantitative PCR. Semen bacterial concentration and HIV-1 RNA levels were correlated 0.30 (P = 0.01). The association between bacterial concentration and HIV-1 RNA detection was not significant after adjustment for antiretroviral therapy (ART) (adjusted odds ratio: 1.27, 95% CI: 0.84 to 1.91). Factors associated with semen bacterial concentration included insertive anal sex (adjusted beta 0.92, 95% CI: 0.12 to 1.73) and ART use (adjusted beta: -0.77, 95% CI: -1.50 to 0.04). Among 13 samples with pyrosequencing data, Corynebacterium spp., Staphylococcus spp., and Streptococcus spp. were most frequently detected. Most of these HIV-1-infected men had bacteria in their semen. ART use was associated with undetectable semen HIV-1 RNA and lower semen bacterial concentrations, whereas insertive anal sex was associated with higher bacterial concentrations. Additional studies evaluating the relationship between semen bacteria, inflammation, mucosal immunity, and HIV-1 shedding are needed to understand implications for HIV-1 transmission.

  2. Maturation Pathways of Cross-Reactive HIV-1 Neutralizing Antibodies

    Directory of Open Access Journals (Sweden)

    Dimiter S. Dimitrov

    2009-11-01

    Full Text Available Several human monoclonal antibodies (hmAbs and antibody fragments, including the best characterized in terms of structure-function b12 and Fab X5, exhibit relatively potent and broad HIV-1 neutralizing activity. However, the elicitation of b12 or b12-like antibodies in vivo by vaccine immunogens based on the HIV-1 envelope glycoprotein (Env has not been successful. B12 is highly divergent from the closest corresponding germline antibody while X5 is less divergent. We have hypothesized that the relatively high degree of specific somatic hypermutations may preclude binding of the HIV-1 envelope glycoprotein (Env to closest germline antibodies, and that identifying antibodies that are intermediates in the pathways to maturation could help design novel vaccine immunogens to guide the immune system for their enhanced elicitation. In support of this hypothesis we have previously found that a germline-like b12 (monovalent and bivalent scFv as an Fc fusion protein or IgG lacks measurable binding to an Env as measured by ELISA with a sensitivity in the μM range [1]; here we present evidence confirming and expanding these findings for a panel of Envs. In contrast, a germline-like scFv X5 bound Env with high (nM affinity. To begin to explore the maturation pathways of these antibodies we identified several possible b12 intermediate antibodies and tested their neutralizing activity. These intermediate antibodies neutralized only some HIV-1 isolates and with relatively weak potency. In contrast, germline-like scFv X5 neutralized a subset of the tested HIV-1 isolates with comparable efficiencies to that of the mature X5. These results could help explain the relatively high immunogenicity of the coreceptor binding site on gp120 and the abundance of CD4-induced (CD4i antibodies in HIV-1-infected patients (X5 is a CD4i antibody as well as the maturation pathway of X5. They also can help identify antigens that can bind specifically to b12 germline and

  3. Creation of chimeric human/rabbit APOBEC1 with HIV-1 restriction and DNA mutation activities

    Science.gov (United States)

    Ikeda, Terumasa; Ong, Eugene Boon Beng; Watanabe, Nobumoto; Sakaguchi, Nobuo; Maeda, Kazuhiko; Koito, Atsushi

    2016-01-01

    APOBEC1 (A1) proteins from lagomorphs and rodents have deaminase-dependent restriction activity against HIV-1, whereas human A1 exerts a negligible effect. To investigate these differences in the restriction of HIV-1 by A1 proteins, a series of chimeric proteins combining rabbit and human A1s was constructed. Homology models of the A1s indicated that their activities derive from functional domains that likely act in tandem through a dimeric interface. The C-terminal region containing the leucine-rich motif and the dimerization domains of rabbit A1 is important for its anti-HIV-1 activity. The A1 chimeras with strong anti-HIV-1 activity were incorporated into virions more efficiently than those without anti-HIV-1 activity, and exhibited potent DNA-mutator activity. Therefore, the C-terminal region of rabbit A1 is involved in both its packaging into the HIV-1 virion and its deamination activity against both viral cDNA and genomic RNA. This study identifies the novel molecular mechanism underlying the target specificity of A1.

  4. μ-opioid modulation of HIV-1 coreceptor expressionand HIV-1 replication

    International Nuclear Information System (INIS)

    Steele, Amber D.; Henderson, Earl E.; Rogers, Thomas J.

    2003-01-01

    A substantial proportion of HIV-1-infected individuals are intravenous drug users (IVDUs) who abuse opiates. Opioids induce a number of immunomodulatory effects that may directly influence HIV-1 disease progression. In the present report, we have investigated the effect of opioids on the expression of the major HIV-1 coreceptors CXCR4 and CCR5. For these studies we have focused on opiates which are ligands for the μ-opioid receptor. Our results show that DAMGO, a selective μ-opioid agonist, increases CXCR4 and CCR5 expression in both CD3 + lymphoblasts and CD14 + monocytes three- to fivefold. Furthermore, DAMGO-induced elevation of HIV-1 coreceptor expression translates into enhanced replication of both X4 and R5 viral strains of HIV-1. We have confirmed the role of the μ-opioid receptor based on the ability of a μ-opioid receptor-selective antagonist to block the effects of DAMGO. We have also found that morphine enhances CXCR4 and CCR5 expression and subsequently increases both X4 and R5 HIV-1 infection. We suggest that the capacity of μ-opioids to increase HIV-1 coreceptor expression and replication may promote viral binding, trafficking of HIV-1-infected cells, and enhanced disease progression

  5. Influenza vaccination of HIV-1-positive and HIV-1-negative former intravenous drug users.

    Science.gov (United States)

    Amendola, A; Boschini, A; Colzani, D; Anselmi, G; Oltolina, A; Zucconi, R; Begnini, M; Besana, S; Tanzi, E; Zanetti, A R

    2001-12-01

    The immunogenicity of an anti-influenza vaccine was assessed in 409 former intravenous drug user volunteers and its effect on the levels of HIV-1 RNA, proviral DNA and on CD4+ lymphocyte counts in a subset HIV-1-positive subjects was measured. HIV-1-positive individuals (n = 72) were divided into three groups on the basis of their CD4+ lymphocyte counts, while the 337 HIV-1-negative participants were allocated into group four. Haemagglutination inhibiting (HI) responses varied from 45.8 to 70% in the HIV-1-positive subjects and were significantly higher in group four (80.7% responses to the H1N1 strain, 81.6% to the H3N2 strain, and 83% to the B strain). The percentage of subjects with HI protective antibody titres (> or = 1:40) increased significantly after vaccination, especially in HIV-1 uninfected subjects. Immunization caused no significant changes in CD4+ counts and in neither plasma HIV-1 RNA nor proviral DNA levels. Therefore, vaccination against influenza may benefit persons infected by HIV-1. Copyright 2001 Wiley-Liss, Inc.

  6. Acyclovir and Transmission of HIV-1 from Persons Infected with HIV-1 and HSV-2

    Science.gov (United States)

    Celum, Connie; Wald, Anna; Lingappa, Jairam R.; Magaret, Amalia S.; Wang, Richard S.; Mugo, Nelly; Mujugira, Andrew; Baeten, Jared M.; Mullins, James I.; Hughes, James P.; Bukusi, Elizabeth A.; Cohen, Craig R.; Katabira, Elly; Ronald, Allan; Kiarie, James; Farquhar, Carey; Stewart, Grace John; Makhema, Joseph; Essex, Myron; Were, Edwin; Fife, Kenneth H.; de Bruyn, Guy; Gray, Glenda E.; McIntyre, James A.; Manongi, Rachel; Kapiga, Saidi; Coetzee, David; Allen, Susan; Inambao, Mubiana; Kayitenkore, Kayitesi; Karita, Etienne; Kanweka, William; Delany, Sinead; Rees, Helen; Vwalika, Bellington; Stevens, Wendy; Campbell, Mary S.; Thomas, Katherine K.; Coombs, Robert W.; Morrow, Rhoda; Whittington, William L.H.; McElrath, M. Juliana; Barnes, Linda; Ridzon, Renee; Corey, Lawrence

    2010-01-01

    BACKGROUND Most persons who are infected with human immunodeficiency virus type 1 (HIV-1) are also infected with herpes simplex virus type 2 (HSV-2), which is frequently reactivated and is associated with increased plasma and genital levels of HIV-1. Therapy to suppress HSV-2 reduces the frequency of reactivation of HSV-2 as well as HIV-1 levels, suggesting that suppression of HSV-2 may reduce the risk of transmission of HIV-1. METHODS We conducted a randomized, placebo-controlled trial of suppressive therapy for HSV-2 (acyclovir at a dose of 400 mg orally twice daily) in couples in which only one of the partners was seropositive for HIV-1 (CD4 count, ≥250 cells per cubic millimeter) and that partner was also infected with HSV-2 and was not taking antiretroviral therapy at the time of enrollment. The primary end point was transmission of HIV-1 to the partner who was not initially infected with HIV-1; linkage of transmissions was assessed by means of genetic sequencing of viruses. RESULTS A total of 3408 couples were enrolled at 14 sites in Africa. Of the partners who were infected with HIV-1, 68% were women, and the baseline median CD4 count was 462 cells per cubic millimeter. Of 132 HIV-1 seroconversions that occurred after randomization (an incidence of 2.7 per 100 person-years), 84 were linked within couples by viral sequencing: 41 in the acyclovir group and 43 in the placebo group (hazard ratio with acyclovir, 0.92, 95% confidence interval [CI], 0.60 to 1.41; P = 0.69). Suppression with acyclovir reduced the mean plasma concentration of HIV-1 by 0.25 log10 copies per milliliter (95% CI, 0.22 to 0.29; P<0.001) and the occurrence of HSV-2–positive genital ulcers by 73% (risk ratio, 0.27; 95% CI, 0.20 to 0.36; P<0.001). A total of 92% of the partners infected with HIV-1 and 84% of the partners not infected with HIV-1 remained in the study for 24 months. The level of adherence to the dispensed study drug was 96%. No serious adverse events related to acyclovir

  7. Multimerized CHR-derived peptides as HIV-1 fusion inhibitors.

    Science.gov (United States)

    Nomura, Wataru; Hashimoto, Chie; Suzuki, Takaharu; Ohashi, Nami; Fujino, Masayuki; Murakami, Tsutomu; Yamamoto, Naoki; Tamamura, Hirokazu

    2013-08-01

    To date, several HIV-1 fusion inhibitors based on the carboxy-terminal leucine/isoleucine heptad repeat (CHR) region of an HIV-1 envelope protein gp41 have been discovered. We have shown that a synthetic peptide mimetic of a trimer form of the CHR-derived peptide C34 has potent inhibitory activity against the HIV-1 fusion mechanism, compared to a monomer C34 peptide. The present study revealed that a dimeric form of C34 is evidently structurally critical for fusion inhibitors, and that the activity of multimerized CHR-derived peptides in fusion inhibition is affected by the properties of the unit peptides C34, SC34EK, and T20. The fluorescence-based study suggested that the N36-interactive sites of the C34 trimer, including hydrophobic residues, are exposed outside the trimer and that trimerization of C34 caused a remarkable increase in fusion inhibitory activity. The present results could be useful in the design of fusion inhibitors against viral infections which proceed via membrane fusion with host cells. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

  8. The dual action of poly(ADP-ribose polymerase -1 (PARP-1 inhibition in HIV-1 infection: HIV-1 LTR inhibition and diminution in Rho GTPase activity

    Directory of Open Access Journals (Sweden)

    Slava eRom

    2015-08-01

    Full Text Available The transcription of HIV-1 (HIV is regulated by complex mechanisms involving various cellular factors and virus-encoded transactivators. Poly(ADP-ribose polymerase 1 (PARP-1 inhibition has emerged recently as a potent anti-inflammatory tool, since PARP-1 is involved in the regulation of some genes through its interaction with various transcription factors. We propose a novel approach to diminish HIV replication via PARP-1 inhibition using human primary monocyte-derived macrophages (MDM as an in vitro model system. PARP-1 inhibitors were able to reduce HIV replication in MDM by 60-80% after 7 days infection. Long Terminal Repeat (LTR acts as a switch in virus replication and can be triggered by several agents such as: Tat, tumor necrosis factor α (TNFα, and phorbol 12-myristate 13-acetate (PMA. Overexpression of Tat in MDM transfected with an LTR reporter plasmid led to a 4.2-fold increase in LTR activation; PARP inhibition resulted in 70% reduction of LTR activity. LTR activity, which increased 3-fold after PMA or TNFα treatment, was reduced by PARP inhibition (by 85-95%. MDM treated with PARP inhibitors showed 90% reduction in NFκB activity (known to mediate PMA- and TNFα-induced HIV LTR activation. Cytoskeleton rearrangements are important in effective HIV-1 infection. PARP inactivation reduced actin cytoskeleton rearrangements by affecting Rho GTPase machinery. These findings suggest that HIV replication in MDM could be suppressed by PARP inhibition via NFκB suppression, diminution of LTR activation and its effects on the cytoskeleton. PARP appears to be essential for HIV replication and its inhibition may provide a potent approach to treatment of HIV infection.

  9. Interleukin-7 facilitates HIV-1 transmission to cervico-vaginal tissue ex vivo.

    Directory of Open Access Journals (Sweden)

    Andrea Introini

    2013-02-01

    Full Text Available The majority of HIV-1 infections in women occur through vaginal intercourse, in which virus-containing semen is deposited on the cervico-vaginal mucosa. Semen is more than a mere carrier of HIV-1, since it contains many biological factors, in particular cytokines, that may affect HIV-1 transmission. The concentration of interleukin (IL-7, one of the most prominent cytokines in semen of healthy individuals, is further increased in semen of HIV-1-infected men. Here, we investigated the potential role of IL-7 in HIV-1 vaginal transmission in an ex vivo system of human cervico-vaginal tissue. We simulated an in vivo situation by depositing HIV-1 on cervico-vaginal tissue in combination with IL-7 at concentrations comparable with those measured in semen of HIV-1-infected individuals. We found that IL-7 significantly enhanced virus replication in ex vivo infected cervico-vaginal tissue. Similarly, we observed an enhancement of HIV-1 replication in lymphoid tissue explants. Analysis of T cells isolated from infected tissues showed that IL-7 reduced CD4⁺ T cell depletion preventing apoptosis, as shown by the decrease in the number of cells expressing the apoptotic marker APO2.7 and the increase in the expression of the anti-apoptotic protein B-cell lymphoma (Bcl-2. Also, IL-7 increased the fraction of cycling CD4⁺ T cells, as evidenced by staining for the nuclear factor Ki-67. High levels of seminal IL-7 in vivo may be relevant to the survival of the founder pool of HIV-1-infected cells in the cervico-vaginal mucosa at the initial stage of infection, promoting local expansion and dissemination of HIV infection.

  10. Contribution of nonneutralizing vaccine-elicited antibody activities to improved protective efficacy in rhesus macaques immunized with Tat/Env compared with multigenic vaccines.

    Science.gov (United States)

    Florese, Ruth H; Demberg, Thorsten; Xiao, Peng; Kuller, LaRene; Larsen, Kay; Summers, L Ebonita; Venzon, David; Cafaro, Aurelio; Ensoli, Barbara; Robert-Guroff, Marjorie

    2009-03-15

    Previously, chronic-phase protection against SHIV(89.6P) challenge was significantly greater in macaques primed with replicating adenovirus type 5 host range mutant (Ad5hr) recombinants encoding HIVtat and env and boosted with Tat and Env protein compared with macaques primed with multigenic adenovirus recombinants (HIVtat, HIVenv, SIVgag, SIVnef) and boosted with Tat, Env, and Nef proteins. The greater protection was correlated with Tat- and Env-binding Abs. Because the macaques lacked SHIV(89.6P)-neutralizing activity prechallenge, we investigated whether Ab-dependent cellular cytotoxicity (ADCC) and Ab-dependent cell-mediated viral inhibition (ADCVI) might exert a protective effect. We clearly show that Tat can serve as an ADCC target, although the Tat-specific activity elicited did not correlate with better protection. However, Env-specific ADCC activity was consistently higher in the Tat/Env group, with sustained cell killing postchallenge exhibited at higher levels (p vaccine regimens.

  11. HIV-1 Reservoir Association with Immune Activation

    Directory of Open Access Journals (Sweden)

    Alejandro Vallejo

    2015-09-01

    Full Text Available In this issue of EBioMedicine, Ruggiero and colleagues describe immune activation biomarkers associated with the size of the HIV reservoir in a carefully designed cross-sectional study. The cohort consists of a homogeneous sample of HIV-1-infected patients with long-term plasma HIV-1 RNA suppression under antiretroviral treatment (ART. It is crucial to explore the potential utility of biomarkers that are easier (less labor intensive, less expensive to measure than integrated HIV DNA load, in order to quickly and accurately quantify cellular reservoirs of HIV.

  12. Interactions Between HIV-1 Gag and Viral RNA Genome Enhance Virion Assembly

    DEFF Research Database (Denmark)

    Dilley, Kari A; Nikolaitchik, Olga A; Galli, Andrea

    2017-01-01

    between Gag and viral RNA are required for the enhancement of particle production. Taken together, these studies are consistent with our previous hypothesis that specific dimeric viral RNA:Gag interactions are the nucleation event of infectious virion assembly, ensuring that one RNA dimer is packaged......Most HIV-1 virions contain two copies of full-length viral RNA, indicating that genome packaging is efficient and tightly regulated. However, the structural protein Gag is the only component required for the assembly of noninfectious virus-like particles and the viral RNA is dispensable...... in this process. The mechanism that allows HIV-1 to achieve such high efficiency of genome packaging when a packageable viral RNA is not required for virus assembly is currently unknown. In this report, we examined the role of HIV-1 RNA in virus assembly and found that packageable HIV-1 RNA enhances particle...

  13. Plasma membrane is the site of productive HIV-1 particle assembly.

    Directory of Open Access Journals (Sweden)

    Nolwenn Jouvenet

    2006-12-01

    Full Text Available Recently proposed models that have gained wide acceptance posit that HIV-1 virion morphogenesis is initiated by targeting the major structural protein (Gag to late endosomal membranes. Thereafter, late endosome-based secretory pathways are thought to deliver Gag or assembled virions to the plasma membrane (PM and extracellular milieu. We present several findings that are inconsistent with this model. Specifically, we demonstrate that HIV-1 Gag is delivered to the PM, and virions are efficiently released into the extracellular medium, when late endosome motility is abolished. Furthermore, we show that HIV-1 virions are efficiently released when assembly is rationally targeted to the PM, but not when targeted to late endosomes. Recently synthesized Gag first accumulates and assembles at the PM, but a proportion is subsequently internalized via endocytosis or phagocytosis, thus accounting for observations of endosomal localization. We conclude that HIV-1 assembly is initiated and completed at the PM, and not at endosomal membranes.

  14. Maturation of the viral core enhances the fusion of HIV-1 particles with primary human T cells and monocyte-derived macrophages

    International Nuclear Information System (INIS)

    Jiang Jiyang; Aiken, Christopher

    2006-01-01

    HIV-1 infection requires fusion of viral and cellular membranes in a reaction catalyzed by the viral envelope proteins gp120 and gp41. We recently reported that efficient HIV-1 particle fusion with target cells is linked to maturation of the viral core by an activity of the gp41 cytoplasmic domain. Here, we show that maturation enhances the fusion of a variety of recombinant viruses bearing primary and laboratory-adapted Env proteins with primary human CD4 + T cells. Overall, HIV-1 fusion was more dependent on maturation for viruses bearing X4-tropic envelope proteins than for R5-tropic viruses. Fusion of HIV-1 with monocyte-derived macrophages was also dependent on particle maturation. We conclude that the ability to couple fusion to particle maturation is a common feature of HIV-1 Env proteins and may play an important role during HIV-1 replication in vivo

  15. Correlation of Naturally Occurring HIV-1 Resistance to DEB025 with Capsid Amino Acid Polymorphisms

    Directory of Open Access Journals (Sweden)

    Brigitte Rosenwirth

    2013-03-01

    Full Text Available DEB025 (alisporivir is a synthetic cyclosporine with inhibitory activity against human immunodeficiency virus type-1 (HIV-1 and hepatitis C virus (HCV. It binds to cyclophilin A (CypA and blocks essential functions of CypA in the viral replication cycles of both viruses. DEB025 inhibits clinical HIV-1 isolates in vitro and decreases HIV-1 virus load in the majority of patients. HIV-1 isolates being naturally resistant to DEB025 have been detected in vitro and in nonresponder patients. By sequence analysis of their capsid protein (CA region, two amino acid polymorphisms that correlated with DEB025 resistance were identified: H87Q and I91N, both located in the CypA-binding loop of the CA protein of HIV-1. The H87Q change was by far more abundant than I91N. Additional polymorphisms in the CypA-binding loop (positions 86, 91 and 96, as well as in the N-terminal loop of CA were detected in resistant isolates and are assumed to contribute to the degree of resistance. These amino acid changes may modulate the conformation of the CypA-binding loop of CA in such a way that binding and/or isomerase function of CypA are no longer necessary for virus replication. The resistant HIV-1 isolates thus are CypA-independent.

  16. Identification of a methylated oligoribonucleotide as a potent inhibitor of HIV-1 reverse transcription complex.

    Science.gov (United States)

    Grigorov, Boyan; Bocquin, Anne; Gabus, Caroline; Avilov, Sergey; Mély, Yves; Agopian, Audrey; Divita, Gilles; Gottikh, Marina; Witvrouw, Myriam; Darlix, Jean-Luc

    2011-07-01

    Upon HIV-1 infection of a target cell, the viral reverse transcriptase (RT) copies the genomic RNA to synthesize the viral DNA. The genomic RNA is within the incoming HIV-1 core where it is coated by molecules of nucleocapsid (NC) protein that chaperones the reverse transcription process. Indeed, the RT chaperoning properties of NC extend from the initiation of cDNA synthesis to completion of the viral DNA. New and effective drugs against HIV-1 continue to be required, which prompted us to search for compounds aimed at inhibiting NC protein. Here, we report that the NC chaperoning activity is extensively inhibited in vitro by small methylated oligoribonucleotides (mODN). These mODNs were delivered intracellularly using a cell-penetrating-peptide and found to impede HIV-1 replication in primary human cells at nanomolar concentrations. Extensive analysis showed that viral cDNA synthesis was severely impaired by mODNs. Partially resistant viruses with mutations in NC and RT emerged after months of passaging in cell culture. A HIV-1 molecular clone (NL4.3) bearing these mutations was found to replicate at high concentrations of mODN, albeit with a reduced fitness. Small, methylated ODNs such as mODN-11 appear to be a new type of highly potent inhibitor of HIV-1.

  17. An efficient procedure for the expression and purification of HIV-1 protease from inclusion bodies.

    Science.gov (United States)

    Nguyen, Hong-Loan Thi; Nguyen, Thuy Thi; Vu, Quy Thi; Le, Hang Thi; Pham, Yen; Trinh, Phuong Le; Bui, Thuan Phuong; Phan, Tuan-Nghia

    2015-12-01

    Several studies have focused on HIV-1 protease for developing drugs for treating AIDS. Recombinant HIV-1 protease is used to screen new drugs from synthetic compounds or natural substances. However, large-scale expression and purification of this enzyme is difficult mainly because of its low expression and solubility. In this study, we constructed 9 recombinant plasmids containing a sequence encoding HIV-1 protease along with different fusion tags and examined the expression of the enzyme from these plasmids. Of the 9 plasmids, pET32a(+) plasmid containing the HIV-1 protease-encoding sequence along with sequences encoding an autocleavage site GTVSFNF at the N-terminus and TEV plus 6× His tag at the C-terminus showed the highest expression of the enzyme and was selected for further analysis. The recombinant protein was isolated from inclusion bodies by using 2 tandem Q- and Ni-Sepharose columns. SDS-PAGE of the obtained HIV-1 protease produced a single band of approximately 13 kDa. The enzyme was recovered efficiently (4 mg protein/L of cell culture) and had high specific activity of 1190 nmol min(-1) mg(-1) at an optimal pH of 4.7 and optimal temperature of 37 °C. This procedure for expressing and purifying HIV-1 protease is now being scaled up to produce the enzyme on a large scale for its application. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Developing a Dynamic Pharmacophore Model for HIV-1 Integrase

    International Nuclear Information System (INIS)

    Carlson, Heather A.; Masukawa, Keven M.; Rubins, Kathleen; Bushman, Frederic; Jorgensen, William L.; Lins, Roberto; Briggs, James; Mccammon, Andy

    2000-01-01

    We present the first receptor-based pharmacophore model for HIV-1 integrase. The development of ''dynamic'' pharmacophore models is a new method that accounts for the inherent flexibility of the active site and aims to reduce the entropic penalties associated with binding a ligand. Furthermore, this new drug discovery method overcomes the limitation of an incomplete crystal structure of the target protein. A molecular dynamics (MD) simulation describes the flexibility of the uncomplexed protein. Many conformational models of the protein are saved from the MD simulations and used in a series of multi-unit search for interacting conformers (MUSIC) simulations. MUSIC is a multiple-copy minimization method, available in the BOSS program; it is used to determine binding regions for probe molecules containing functional groups that complement the active site. All protein conformations from the MD are overlaid, and conserved binding regions for the probe molecules are identified. Those conserved binding regions define the dynamic pharmacophore model. Here, the dynamic model is compared to known inhibitors of the integrase as well as a three-point, ligand-based pharmacophore model from the literature. Also, a ''static'' pharmacophore model was determined in the standard fashion, using a single crystal structure. Inhibitors thought to bind in the active site of HIV-1 integrase fit the dynamic model but not the static model. Finally, we have identified a set of compounds from the Available Chemicals Directory that fit the dynamic pharmacophore model, and experimental testing of the compounds has confirmed several new inhibitors

  19. The effect of CD4 receptor downregulation and its downstream signaling molecules on HIV-1 latency

    International Nuclear Information System (INIS)

    Kim, Kyung-Chang; Kim, Hyeon Guk; Roh, Tae-Young; Park, Jihwan; Jung, Kyung-Min; Lee, Joo-Shil; Choi, Sang-Yun; Kim, Sung Soon; Choi, Byeong-Sun

    2011-01-01

    Research highlights: → CD4 receptors were downregulated on the surface of HIV-1 latently infected cells. → CD4 downstream signaling molecules were suppressed in HIV-1 latently infected cells. → HIV-1 progeny can be reactivated by induction of T-cell activation signal molecules. → H3K4me3 and H3K9ac were highly enriched in CD4 downstream signaling molecules. → HIV-1 latency can be maintained by the reduction of downstream signaling molecules. -- Abstract: HIV-1 can establish a latent infection in memory CD4 + T cells to evade the host immune response. CD4 molecules can act not only as the HIV-1 receptor for entry but also as the trigger in an intracellular signaling cascade for T-cell activation and proliferation via protein tyrosine kinases. Novel chronic HIV-1-infected A3.01-derived (NCHA) cells were used to examine the involvement of CD4 downstream signaling in HIV-1 latency. CD4 receptors in NCHA cells were dramatically downregulated on its surface but were slightly decreased in whole-cell lysates. The expression levels of CD4 downstream signaling molecules, including P56 Lck , ZAP-70, LAT, and c-Jun, were sharply decreased in NCHA cells. The lowered histone modifications of H3K4me3 and H3K9ac correlated with the downregulation of P56 Lck , ZAP-70, and LAT in NCHA cells. AP-1 binding activity was also reduced in NCHA cells. LAT and c-Jun suppressed in NCHA cells were highly induced after PMA treatment. In epigenetic analysis, other signal transduction molecules which are associated with active and/or latent HIV-1 infection showed normal states in HIV-1 latently infected cells compared to A3.01 cells. In conclusion, we demonstrated that the HIV-1 latent state is sustained by the reduction of downstream signaling molecules via the downregulation of CD4 and the attenuated activity of transcription factor as AP-1. The HIV-1 latency model via T-cell deactivation may provide some clues for the development of the new antireservoir therapy.

  20. The effect of CD4 receptor downregulation and its downstream signaling molecules on HIV-1 latency

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Kyung-Chang [National Institute of Health, Chungbuk (Korea, Republic of); School of Life Science and Biotechnology, Korea University, Seoul (Korea, Republic of); Kim, Hyeon Guk [National Institute of Health, Chungbuk (Korea, Republic of); Roh, Tae-Young [Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang, Gyeongbuk (Korea, Republic of); Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang, Gyeongbuk (Korea, Republic of); Park, Jihwan [Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang, Gyeongbuk (Korea, Republic of); Jung, Kyung-Min; Lee, Joo-Shil [National Institute of Health, Chungbuk (Korea, Republic of); Choi, Sang-Yun [School of Life Science and Biotechnology, Korea University, Seoul (Korea, Republic of); Kim, Sung Soon [National Institute of Health, Chungbuk (Korea, Republic of); Choi, Byeong-Sun, E-mail: byeongsun@korea.kr [National Institute of Health, Chungbuk (Korea, Republic of)

    2011-01-14

    Research highlights: {yields} CD4 receptors were downregulated on the surface of HIV-1 latently infected cells. {yields} CD4 downstream signaling molecules were suppressed in HIV-1 latently infected cells. {yields} HIV-1 progeny can be reactivated by induction of T-cell activation signal molecules. {yields} H3K4me3 and H3K9ac were highly enriched in CD4 downstream signaling molecules. {yields} HIV-1 latency can be maintained by the reduction of downstream signaling molecules. -- Abstract: HIV-1 can establish a latent infection in memory CD4 + T cells to evade the host immune response. CD4 molecules can act not only as the HIV-1 receptor for entry but also as the trigger in an intracellular signaling cascade for T-cell activation and proliferation via protein tyrosine kinases. Novel chronic HIV-1-infected A3.01-derived (NCHA) cells were used to examine the involvement of CD4 downstream signaling in HIV-1 latency. CD4 receptors in NCHA cells were dramatically downregulated on its surface but were slightly decreased in whole-cell lysates. The expression levels of CD4 downstream signaling molecules, including P56{sup Lck}, ZAP-70, LAT, and c-Jun, were sharply decreased in NCHA cells. The lowered histone modifications of H3K4me3 and H3K9ac correlated with the downregulation of P56{sup Lck}, ZAP-70, and LAT in NCHA cells. AP-1 binding activity was also reduced in NCHA cells. LAT and c-Jun suppressed in NCHA cells were highly induced after PMA treatment. In epigenetic analysis, other signal transduction molecules which are associated with active and/or latent HIV-1 infection showed normal states in HIV-1 latently infected cells compared to A3.01 cells. In conclusion, we demonstrated that the HIV-1 latent state is sustained by the reduction of downstream signaling molecules via the downregulation of CD4 and the attenuated activity of transcription factor as AP-1. The HIV-1 latency model via T-cell deactivation may provide some clues for the development of the new

  1. Intracellular route and biological activity of exogenously delivered Rep proteins from the adeno-associated virus type 2

    International Nuclear Information System (INIS)

    Awedikian, Rafi; Francois, Achille; Guilbaud, Mickael; Moullier, Philippe; Salvetti, Anna

    2005-01-01

    The two large Rep proteins, Rep78 and Rep68, from the adeno-associated virus type 2 (AAV-2) are required for AAV-2 DNA replication, site-specific integration, and for the regulation of viral gene expression. The study of their activities is dependent on the ability to deliver these proteins to the cells in a time and dose-dependent manner. We evaluated the ability of a protein transduction domain (PTD) derived from the human immunodeficiency virus 1 (HIV-1) TAT protein to drive the cellular internalization of exogenously delivered PTD-fused Rep68 proteins. This analysis unexpectedly revealed that recombinant Rep68 alone, in the absence of any PTD, could be endocytosed by the cells. Rep68 as the chimeric TAT-Rep68 proteins were internalized through endocytosis in clathrin-coated vesicles and retained in late endosomes/lysosomes with no detectable nuclear localization. In the presence of adenovirus, the Rep proteins could translocate into the nucleus where they displayed a biological activity. These findings support recent reports on the mechanism of entry of TAT-fused proteins and also revealed a new property of Rep68

  2. Viral linkage in HIV-1 seroconverters and their partners in an HIV-1 prevention clinical trial.

    Directory of Open Access Journals (Sweden)

    Mary S Campbell

    2011-03-01

    Full Text Available Characterization of viruses in HIV-1 transmission pairs will help identify biological determinants of infectiousness and evaluate candidate interventions to reduce transmission. Although HIV-1 sequencing is frequently used to substantiate linkage between newly HIV-1 infected individuals and their sexual partners in epidemiologic and forensic studies, viral sequencing is seldom applied in HIV-1 prevention trials. The Partners in Prevention HSV/HIV Transmission Study (ClinicalTrials.gov #NCT00194519 was a prospective randomized placebo-controlled trial that enrolled serodiscordant heterosexual couples to determine the efficacy of genital herpes suppression in reducing HIV-1 transmission; as part of the study analysis, HIV-1 sequences were examined for genetic linkage between seroconverters and their enrolled partners.We obtained partial consensus HIV-1 env and gag sequences from blood plasma for 151 transmission pairs and performed deep sequencing of env in some cases. We analyzed sequences with phylogenetic techniques and developed a Bayesian algorithm to evaluate the probability of linkage. For linkage, we required monophyletic clustering between enrolled partners' sequences and a Bayesian posterior probability of ≥ 50%. Adjudicators classified each seroconversion, finding 108 (71.5% linked, 40 (26.5% unlinked, and 3 (2.0% indeterminate transmissions, with linkage determined by consensus env sequencing in 91 (84%. Male seroconverters had a higher frequency of unlinked transmissions than female seroconverters. The likelihood of transmission from the enrolled partner was related to time on study, with increasing numbers of unlinked transmissions occurring after longer observation periods. Finally, baseline viral load was found to be significantly higher among linked transmitters.In this first use of HIV-1 sequencing to establish endpoints in a large clinical trial, more than one-fourth of transmissions were unlinked to the enrolled partner

  3. HIV-1 impairs human retinal pigment epithelial barrier function: possible association with the pathogenesis of HIV-associated retinopathy.

    Science.gov (United States)

    Tan, Suiyi; Duan, Heng; Xun, Tianrong; Ci, Wei; Qiu, Jiayin; Yu, Fei; Zhao, Xuyan; Wu, Linxuan; Li, Lin; Lu, Lu; Jiang, Shibo; Liu, Shuwen

    2014-07-01

    The breakdown of human retinal pigment epithelial (HRPE) barrier is considered as the etiology of retinopathy, which affects the quality of life of HIV/AIDS patients. Here we demonstrate that HIV-1 could directly impair HRPE barrier function, which leads to the translocation of HIV-1 and bacteria. HRPE cells (D407) were grown to form polarized, confluent monolayers and treated with different HIV-1 infectious clones. A significant increase of monolayer permeability, as measured by trans-epithelial electrical resistance (TEER) and apical-basolateral movements of sodium fluorescein, was observed. Disrupted tightness of HRPE barrier was associated with the downregulation of several tight junction proteins in D407 cells, including ZO-1, Occludin, Claudin-1, Claudin-2, Claudin-3, Claudin-4, and Claudin-5, after exposure to HIV-1, without affecting the viability of cells. HIV-1 gp120 was shown to participate in the alteration of barrier properties, as evidenced by decreased TEER and weakened expression of tight junction proteins in D407 monolayers after exposure to pseudotyped HIV-1, UV-inactivated HIV-1, and free gp120, but not to an envelope (Env)-defective mutant of HIV. Furthermore, exposure to HIV-1 particles could induce the release of pro-inflammatory cytokines in D407, including IL-6 and MCP-1, both of which downregulated the expression of ZO-1 in the HRPE barrier. Disrupted HRPE monolayer allowed translocation of HIV-1 and bacteria across the epithelium. Overall, these findings suggest that HIV-1 may exploit its Env glycoprotein to induce an inflammatory state in HRPE cells, which could result in impairment of HRPE monolayer integrity, allowing virus and bacteria existing in ocular fluids to cross the epithelium and penetrate the HRPE barrier. Our study highlights the role of HIV-1 in the pathogenesis of HIV/AIDS-related retinopathy and suggests potential therapeutic targets for this ocular complication.

  4. Human cyclin T1 expression ameliorates a T-cell-specific transcriptional limitation for HIV in transgenic rats, but is not sufficient for a spreading infection of prototypic R5 HIV-1 strains ex vivo

    Directory of Open Access Journals (Sweden)

    Littman Dan R

    2009-01-01

    Full Text Available Abstract Background Cells derived from native rodents have limits at distinct steps of HIV replication. Rat primary CD4 T-cells, but not macrophages, display a profound transcriptional deficit that is ameliorated by transient trans-complementation with the human Tat-interacting protein Cyclin T1 (hCycT1. Results Here, we generated transgenic rats that selectively express hCycT1 in CD4 T-cells and macrophages. hCycT1 expression in rat T-cells boosted early HIV gene expression to levels approaching those in infected primary human T-cells. hCycT1 expression was necessary, but not sufficient, to enhance HIV transcription in T-cells from individual transgenic animals, indicating that endogenous cellular factors are critical co-regulators of HIV gene expression in rats. T-cells from hCD4/hCCR5/hCycT1-transgenic rats did not support productive infection of prototypic wild-type R5 HIV-1 strains ex vivo, suggesting one or more significant limitation in the late phase of the replication cycle in this primary rodent cell type. Remarkably, we identify a replication-competent HIV-1 GFP reporter strain (R7/3 YU-2 Env that displays characteristics of a spreading, primarily cell-to-cell-mediated infection in primary T-cells from hCD4/hCCR5-transgenic rats. Moreover, the replication of this recombinant HIV-1 strain was significantly enhanced by hCycT1 transgenesis. The viral determinants of this so far unique replicative ability are currently unknown. Conclusion Thus, hCycT1 expression is beneficial to de novo HIV infection in a transgenic rat model, but additional genetic manipulations of the host or virus are required to achieve full permissivity.

  5. Molecular mechanisms of HIV-1 associated neurodegeneration

    Indian Academy of Sciences (India)

    Since identification of the human immunodeficiency virus-1 (HIV-1), numerous studies suggest a link between neurological impairments, in particular dementia, with acquired immunodeficiency syndrome (AIDS) with alarming occurrence worldwide. Approximately, 60% of HIV-infected people show some form of neurological ...

  6. Epidemiology of HIV-1 and emerging problems

    NARCIS (Netherlands)

    Lukashov, V. V.; de Ronde, A.; de Jong, J. J.; Goudsmit, J.

    2000-01-01

    Broad use of antiretroviral drugs is becoming a factor that is important to consider for understanding the HIV-1 epidemiology. Since 1993, we observe that a proportion of new infections within major risk groups in Amsterdam is caused by azidothymidine (AZT)-resistant viruses. After the introduction

  7. Detection of HIV-1 p24 Gag in plasma by a nanoparticle-based bio-barcode-amplification method.

    Science.gov (United States)

    Kim, Eun-Young; Stanton, Jennifer; Korber, Bette T M; Krebs, Kendall; Bogdan, Derek; Kunstman, Kevin; Wu, Samuel; Phair, John P; Mirkin, Chad A; Wolinsky, Steven M

    2008-06-01

    Detection of HIV-1 in patients is limited by the sensitivity and selectivity of available tests. The nanotechnology-based bio-barcode-amplification method offers an innovative approach to detect specific HIV-1 antigens from diverse HIV-1 subtypes. We evaluated the efficacy of this protein-detection method in detecting HIV-1 in men enrolled in the Chicago component of the Multicenter AIDS Cohort Study (MACS). The method relies on magnetic microparticles with antibodies that specifically bind the HIV-1 p24 Gag protein and nanoparticles that are encoded with DNA and antibodies that can sandwich the target protein captured by the microparticle-bound antibodies. The aggregate sandwich structures are magnetically separated from solution, and treated to remove the conjugated barcode DNA. The DNA barcodes (hundreds per target) were identified by a nanoparticle-based detection method that does not rely on PCR. Of 112 plasma samples from HIV-1-infected subjects, 111 were positive for HIV-1 p24 Gag protein (range: 0.11-71.5 ng/ml of plasma) by the bio-barcode-amplification method. HIV-1 p24 Gag protein was detected in only 23 out of 112 men by the conventional ELISA. A total of 34 uninfected subjects were negative by both tests. Thus, the specificity of the bio-barcode-amplification method was 100% and the sensitivity 99%. The bio-barcode-amplification method detected HIV-1 p24 Gag protein in plasma from all study subjects with less than 200 CD4(+) T cells/microl of plasma (100%) and 19 out of 20 (95%) HIV-1-infected men who had less than 50 copies/ml of plasma of HIV-1 RNA. In a separate group of 60 diverse international isolates, representative of clades A, B, C and D and circulating recombinant forms CRF01_AE and CRF02_AG, the bio-barcode-amplification method identified the presence of virus correctly. The bio-barcode-amplification method was superior to the conventional ELISA assay for the detection of HIV-1 p24 Gag protein in plasma with a breadth of coverage for diverse

  8. Interactions between the HIV-1 Unspliced mRNA and Host mRNA Decay Machineries

    Directory of Open Access Journals (Sweden)

    Daniela Toro-Ascuy

    2016-11-01

    Full Text Available The human immunodeficiency virus type-1 (HIV-1 unspliced transcript is used both as mRNA for the synthesis of structural proteins and as the packaged genome. Given the presence of retained introns and instability AU-rich sequences, this viral transcript is normally retained and degraded in the nucleus of host cells unless the viral protein REV is present. As such, the stability of the HIV-1 unspliced mRNA must be particularly controlled in the nucleus and the cytoplasm in order to ensure proper levels of this viral mRNA for translation and viral particle formation. During its journey, the HIV-1 unspliced mRNA assembles into highly specific messenger ribonucleoproteins (mRNPs containing many different host proteins, amongst which are well-known regulators of cytoplasmic mRNA decay pathways such as up-frameshift suppressor 1 homolog (UPF1, Staufen double-stranded RNA binding protein 1/2 (STAU1/2, or components of miRNA-induced silencing complex (miRISC and processing bodies (PBs. More recently, the HIV-1 unspliced mRNA was shown to contain N6-methyladenosine (m6A, allowing the recruitment of YTH N6-methyladenosine RNA binding protein 2 (YTHDF2, an m6A reader host protein involved in mRNA decay. Interestingly, these host proteins involved in mRNA decay were shown to play positive roles in viral gene expression and viral particle assembly, suggesting that HIV-1 interacts with mRNA decay components to successfully accomplish viral replication. This review summarizes the state of the art in terms of the interactions between HIV-1 unspliced mRNA and components of different host mRNA decay machineries.

  9. N-terminally truncated POM121C inhibits HIV-1 replication.

    Directory of Open Access Journals (Sweden)

    Hideki Saito

    Full Text Available Recent studies have identified host cell factors that regulate early stages of HIV-1 infection including viral cDNA synthesis and orientation of the HIV-1 capsid (CA core toward the nuclear envelope, but it remains unclear how viral DNA is imported through the nuclear pore and guided to the host chromosomal DNA. Here, we demonstrate that N-terminally truncated POM121C, a component of the nuclear pore complex, blocks HIV-1 infection. This truncated protein is predominantly localized in the cytoplasm, does not bind to CA, does not affect viral cDNA synthesis, reduces the formation of 2-LTR and diminished the amount of integrated proviral DNA. Studies with an HIV-1-murine leukemia virus (MLV chimeric virus carrying the MLV-derived Gag revealed that Gag is a determinant of this inhibition. Intriguingly, mutational studies have revealed that the blockade by N-terminally-truncated POM121C is closely linked to its binding to importin-β/karyopherin subunit beta 1 (KPNB1. These results indicate that N-terminally-truncated POM121C inhibits HIV-1 infection after completion of reverse transcription and before integration, and suggest an important role for KPNB1 in HIV-1 replication.

  10. In vitro protease cleavage and computer simulations reveal the HIV-1 capsid maturation pathway

    Science.gov (United States)

    Ning, Jiying; Erdemci-Tandogan, Gonca; Yufenyuy, Ernest L.; Wagner, Jef; Himes, Benjamin A.; Zhao, Gongpu; Aiken, Christopher; Zandi, Roya; Zhang, Peijun

    2016-12-01

    HIV-1 virions assemble as immature particles containing Gag polyproteins that are processed by the viral protease into individual components, resulting in the formation of mature infectious particles. There are two competing models for the process of forming the mature HIV-1 core: the disassembly and de novo reassembly model and the non-diffusional displacive model. To study the maturation pathway, we simulate HIV-1 maturation in vitro by digesting immature particles and assembled virus-like particles with recombinant HIV-1 protease and monitor the process with biochemical assays and cryoEM structural analysis in parallel. Processing of Gag in vitro is accurate and efficient and results in both soluble capsid protein and conical or tubular capsid assemblies, seemingly converted from immature Gag particles. Computer simulations further reveal probable assembly pathways of HIV-1 capsid formation. Combining the experimental data and computer simulations, our results suggest a sequential combination of both displacive and disassembly/reassembly processes for HIV-1 maturation.

  11. Reactivation of Latent HIV-1 Expression by Engineered TALE Transcription Factors.

    Science.gov (United States)

    Perdigão, Pedro; Gaj, Thomas; Santa-Marta, Mariana; Barbas, Carlos F; Goncalves, Joao

    2016-01-01

    The presence of replication-competent HIV-1 -which resides mainly in resting CD4+ T cells--is a major hurdle to its eradication. While pharmacological approaches have been useful for inducing the expression of this latent population of virus, they have been unable to purge HIV-1 from all its reservoirs. Additionally, many of these strategies have been associated with adverse effects, underscoring the need for alternative approaches capable of reactivating viral expression. Here we show that engineered transcriptional modulators based on customizable transcription activator-like effector (TALE) proteins can induce gene expression from the HIV-1 long terminal repeat promoter, and that combinations of TALE transcription factors can synergistically reactivate latent viral expression in cell line models of HIV-1 latency. We further show that complementing TALE transcription factors with Vorinostat, a histone deacetylase inhibitor, enhances HIV-1 expression in latency models. Collectively, these findings demonstrate that TALE transcription factors are a potentially effective alternative to current pharmacological routes for reactivating latent virus and that combining synthetic transcriptional activators with histone deacetylase inhibitors could lead to the development of improved therapies for latent HIV-1 infection.

  12. Digoxin reveals a functional connection between HIV-1 integration preference and T-cell activation.

    Science.gov (United States)

    Zhyvoloup, Alexander; Melamed, Anat; Anderson, Ian; Planas, Delphine; Lee, Chen-Hsuin; Kriston-Vizi, Janos; Ketteler, Robin; Merritt, Andy; Routy, Jean-Pierre; Ancuta, Petronela; Bangham, Charles R M; Fassati, Ariberto

    2017-07-01

    HIV-1 integrates more frequently into transcribed genes, however the biological significance of HIV-1 integration targeting has remained elusive. Using a selective high-throughput chemical screen, we discovered that the cardiac glycoside digoxin inhibits wild-type HIV-1 infection more potently than HIV-1 bearing a single point mutation (N74D) in the capsid protein. We confirmed that digoxin repressed viral gene expression by targeting the cellular Na+/K+ ATPase, but this did not explain its selectivity. Parallel RNAseq and integration mapping in infected cells demonstrated that digoxin inhibited expression of genes involved in T-cell activation and cell metabolism. Analysis of >400,000 unique integration sites showed that WT virus integrated more frequently than N74D mutant within or near genes susceptible to repression by digoxin and involved in T-cell activation and cell metabolism. Two main gene networks down-regulated by the drug were CD40L and CD38. Blocking CD40L by neutralizing antibodies selectively inhibited WT virus infection, phenocopying digoxin. Thus the selectivity of digoxin depends on a combination of integration targeting and repression of specific gene networks. The drug unmasked a functional connection between HIV-1 integration and T-cell activation. Our results suggest that HIV-1 evolved integration site selection to couple its early gene expression with the status of target CD4+ T-cells, which may affect latency and viral reactivation.

  13. Digoxin reveals a functional connection between HIV-1 integration preference and T-cell activation.

    Directory of Open Access Journals (Sweden)

    Alexander Zhyvoloup

    2017-07-01

    Full Text Available HIV-1 integrates more frequently into transcribed genes, however the biological significance of HIV-1 integration targeting has remained elusive. Using a selective high-throughput chemical screen, we discovered that the cardiac glycoside digoxin inhibits wild-type HIV-1 infection more potently than HIV-1 bearing a single point mutation (N74D in the capsid protein. We confirmed that digoxin repressed viral gene expression by targeting the cellular Na+/K+ ATPase, but this did not explain its selectivity. Parallel RNAseq and integration mapping in infected cells demonstrated that digoxin inhibited expression of genes involved in T-cell activation and cell metabolism. Analysis of >400,000 unique integration sites showed that WT virus integrated more frequently than N74D mutant within or near genes susceptible to repression by digoxin and involved in T-cell activation and cell metabolism. Two main gene networks down-regulated by the drug were CD40L and CD38. Blocking CD40L by neutralizing antibodies selectively inhibited WT virus infection, phenocopying digoxin. Thus the selectivity of digoxin depends on a combination of integration targeting and repression of specific gene networks. The drug unmasked a functional connection between HIV-1 integration and T-cell activation. Our results suggest that HIV-1 evolved integration site selection to couple its early gene expression with the status of target CD4+ T-cells, which may affect latency and viral reactivation.

  14. Synthesis of a Vpr-Binding Derivative for Use as a Novel HIV-1 Inhibitor.

    Science.gov (United States)

    Hagiwara, Kyoji; Ishii, Hideki; Murakami, Tomoyuki; Takeshima, Shin-nosuke; Chutiwitoonchai, Nopporn; Kodama, Eiichi N; Kawaji, Kumi; Kondoh, Yasumitsu; Honda, Kaori; Osada, Hiroyuki; Tsunetsugu-Yokota, Yasuko; Suzuki, Masaaki; Aida, Yoko

    2015-01-01

    The emergence of multidrug-resistant viruses compromises the efficacy of anti-human immunodeficiency virus type 1 (HIV-1) therapy and limits treatment options. Therefore, new targets that can be used to develop novel antiviral agents need to be identified. We previously identified a potential parent compound, hematoxylin, which suppresses the nuclear import of HIV-1 via the Vpr-importin α interaction and inhibits HIV-1 replication in a Vpr-dependent manner by blocking nuclear import of the pre-integration complex. However, it was unstable. Here, we synthesized a stable derivative of hematoxylin that bound specifically and stably to Vpr and inhibited HIV-1 replication in macrophages. Furthermore, like hematoxylin, the derivative inhibited nuclear import of Vpr in an in vitro nuclear import assay, but had no effect on Vpr-induced G2/M phase cell cycle arrest or caspase activity. Interestingly, this derivative bound strongly to amino acid residues 54-74 within the C-terminal α-helical domain (αH3) of Vpr. These residues are highly conserved among different HIV strains, indicating that this region is a potential target for drug-resistant HIV-1 infection. Thus, we succeeded in developing a stable hematoxylin derivative that bound directly to Vpr, suggesting that specific inhibitors of the interaction between cells and viral accessory proteins may provide a new strategy for the treatment of HIV-1 infection.

  15. Synthesis of a Vpr-Binding Derivative for Use as a Novel HIV-1 Inhibitor.

    Directory of Open Access Journals (Sweden)

    Kyoji Hagiwara

    Full Text Available The emergence of multidrug-resistant viruses compromises the efficacy of anti-human immunodeficiency virus type 1 (HIV-1 therapy and limits treatment options. Therefore, new targets that can be used to develop novel antiviral agents need to be identified. We previously identified a potential parent compound, hematoxylin, which suppresses the nuclear import of HIV-1 via the Vpr-importin α interaction and inhibits HIV-1 replication in a Vpr-dependent manner by blocking nuclear import of the pre-integration complex. However, it was unstable. Here, we synthesized a stable derivative of hematoxylin that bound specifically and stably to Vpr and inhibited HIV-1 replication in macrophages. Furthermore, like hematoxylin, the derivative inhibited nuclear import of Vpr in an in vitro nuclear import assay, but had no effect on Vpr-induced G2/M phase cell cycle arrest or caspase activity. Interestingly, this derivative bound strongly to amino acid residues 54-74 within the C-terminal α-helical domain (αH3 of Vpr. These residues are highly conserved among different HIV strains, indicating that this region is a potential target for drug-resistant HIV-1 infection. Thus, we succeeded in developing a stable hematoxylin derivative that bound directly to Vpr, suggesting that specific inhibitors of the interaction between cells and viral accessory proteins may provide a new strategy for the treatment of HIV-1 infection.

  16. Reactivation of Latent HIV-1 Expression by Engineered TALE Transcription Factors.

    Directory of Open Access Journals (Sweden)

    Pedro Perdigão

    Full Text Available The presence of replication-competent HIV-1 -which resides mainly in resting CD4+ T cells--is a major hurdle to its eradication. While pharmacological approaches have been useful for inducing the expression of this latent population of virus, they have been unable to purge HIV-1 from all its reservoirs. Additionally, many of these strategies have been associated with adverse effects, underscoring the need for alternative approaches capable of reactivating viral expression. Here we show that engineered transcriptional modulators based on customizable transcription activator-like effector (TALE proteins can induce gene expression from the HIV-1 long terminal repeat promoter, and that combinations of TALE transcription factors can synergistically reactivate latent viral expression in cell line models of HIV-1 latency. We further show that complementing TALE transcription factors with Vorinostat, a histone deacetylase inhibitor, enhances HIV-1 expression in latency models. Collectively, these findings demonstrate that TALE transcription factors are a potentially effective alternative to current pharmacological routes for reactivating latent virus and that combining synthetic transcriptional activators with histone deacetylase inhibitors could lead to the development of improved therapies for latent HIV-1 infection.

  17. Astrocytes sustain long-term productive HIV-1 infection without establishment of reactivable viral latency.

    Science.gov (United States)

    Barat, Corinne; Proust, Alizé; Deshiere, Alexandre; Leboeuf, Mathieu; Drouin, Jean; Tremblay, Michel J

    2018-02-21

    The "shock and kill" HIV-1 cure strategy proposes eradication of stable cellular reservoirs by clinical treatment with latency-reversing agents (LRAs). Although resting CD4 + T cells latently infected with HIV-1 constitute the main reservoir that is targeted by these approaches, their consequences on other reservoirs such as the central nervous system are still unknown and should be taken into consideration. We performed experiments aimed at defining the possible role of astrocytes in HIV-1 persistence in the brain and the effect of LRA treatments on this viral sanctuary. We first demonstrate that the diminished HIV-1 production in a proliferating astrocyte culture is due to a reduced proliferative capacity of virus-infected cells compared with uninfected astrocytes. In contrast, infection of non-proliferating astrocytes led to a robust HIV-1 infection that was sustained for over 60 days. To identify astrocytes latently infected with HIV-1, we designed a new dual-color reporter virus called NL4.3 eGFP-IRES-Crimson that is fully infectious and encodes for all viral proteins. Although we detected a small fraction of astrocytes carrying silent HIV-1 proviruses, we did not observe any reactivation using various LRAs and even strong inducers such as tumor necrosis factor, thus suggesting that these proviruses were either not transcriptionally competent or in a state of deep latency. Our findings imply that astrocytes might not constitute a latent reservoir per se but that relentless virus production by this brain cell population could contribute to the neurological disorders seen in HIV-1-infected persons subjected to combination antiretroviral therapy. © 2018 Wiley Periodicals, Inc.

  18. Involvement of histone methyltransferase GLP in HIV-1 latency through catalysis of H3K9 dimethylation

    International Nuclear Information System (INIS)

    Ding, Donglin; Qu, Xiying; Li, Lin; Zhou, Xin; Liu, Sijie; Lin, Shiguan; Wang, Pengfei; Liu, Shaohui; Kong, Chuijin; Wang, Xiaohui; Liu, Lin; Zhu, Huanzhang

    2013-01-01

    Understanding the mechanism of HIV-1 latency is crucial to eradication of the viral reservoir in HIV-1-infected individuals. However, the role of histone methyltransferase (HMT) G9a-like protein (GLP) in HIV-1 latency is still unclear. In the present work, we established four clonal cell lines containing HIV-1 vector. We found that the integration sites of most clonal cell lines favored active gene regions. However, we also observed hypomethylation of CpG of HIV 5′LTR in all four clonal cell lines. Additionally, 5′-deoxy-5′-methylthioadenosine (MTA), a broad-spectrum histone methyltransferase inhibitor, was used to examine the role of histone methylation in HIV-1 latency. MTA was found to decrease the level of H3K9 dimethylation, causing reactivation of latent HIV-1 in C11 cells. GLP knockdown by small interfering RNA clearly induced HIV-1 LTR expression. Results suggest that GLP may play a significant role in the maintenance of HIV-1 latency by catalyzing dimethylation of H3K9. - Highlights: ► We have established an in vitro model of HIV-1 latency. ► The integration sites of most clonal cell lines favor in active gene regions. ► Hypomethylation occurs in CpG islands of HIV 5′LTR in all four clonal cell lines. ► MTA can reactivate latent HIV-1 by decreasing the level of H3K9 me2 in C11 cells. ► HMT GLP may play a significant role in the maintenance of HIV-1 latency

  19. Binding of the mannose-specific lectin, Griffithsin, to HIV-1 gp120 exposes the CD4-binding site

    CSIR Research Space (South Africa)

    Alexandre, Kabamba B

    2011-09-01

    Full Text Available development. Anti- microb. Agents Chemother. 41:1521?1530. 7. Buchacher, A., et al. 1994. Generation of human monoclonal antibodies against HIV-1 proteins; electrofusion and Epstein-Barr virus transformation for peripheral blood lymphocyte immortalization...

  20. Tailored HIV-1 vectors for genetic modification of primary human dendritic cells and monocytes.

    Science.gov (United States)

    Durand, Stéphanie; Nguyen, Xuan-Nhi; Turpin, Jocelyn; Cordeil, Stephanie; Nazaret, Nicolas; Croze, Séverine; Mahieux, Renaud; Lachuer, Joël; Legras-Lachuer, Catherine; Cimarelli, Andrea

    2013-01-01

    Monocyte-derived dendritic cells (MDDCs) play a key role in the regulation of the immune system and are the target of numerous gene therapy applications. The genetic modification of MDDCs is possible with human immunodeficiency virus type 1 (HIV-1)-derived lentiviral vectors (LVs) but requires high viral doses to bypass their natural resistance to viral infection, and this in turn affects their physiological properties. To date, a single viral protein is able to counter this restrictive phenotype, Vpx, a protein derived from members of the HIV-2/simian immunodeficiency virus SM lineage that counters at least two restriction factors present in myeloid cells. By tagging Vpx with a short heterologous membrane-targeting domain, we have obtained HIV-1 LVs incorporating high levels of this protein (HIV-1-Src-Vpx). These vectors efficiently transduce differentiated MDDCs and monocytes either as previously purified populations or as populations within unsorted peripheral blood mononuclear cells (PBMCs). In addition, these vectors can be efficiently pseudotyped with receptor-specific envelopes, further restricting their cellular tropism almost uniquely to MDDCs. Compared to conventional HIV-1 LVs, these novel vectors allow for an efficient genetic modification of MDDCs and, more importantly, do not cause their maturation or affect their survival, which are unwanted side effects of the transduction process. This study describes HIV-1-Src-Vpx LVs as a novel potent tool for the genetic modification of differentiated MDDCs and of circulating monocyte precursors with strong potential for a wide range of gene therapy applications.

  1. Tat-PRAS40 prevent hippocampal HT-22 cell death and oxidative stress induced animal brain ischemic insults.

    Science.gov (United States)

    Shin, Min Jea; Kim, Dae Won; Jo, Hyo Sang; Cho, Su Bin; Park, Jung Hwan; Lee, Chi Hern; Yeo, Eun Ji; Choi, Yeon Joo; Kim, Ji An; Hwang, Jung Soon; Sohn, Eun Jeong; Jeong, Ji-Heon; Kim, Duk-Soo; Kwon, Hyeok Yil; Cho, Yong-Jun; Lee, Keunwook; Han, Kyu Hyung; Park, Jinseu; Eum, Won Sik; Choi, Soo Young

    2016-08-01

    Proline rich Akt substrate (PRAS40) is a component of mammalian target of rapamycin complex 1 (mTORC1) and is known to play an important role against reactive oxygen species-induced cell death. However, the precise function of PRAS40 in ischemia remains unclear. Thus, we investigated whether Tat-PRAS40, a cell-permeable fusion protein, has a protective function against oxidative stress-induced hippocampal neuronal (HT-22) cell death in an animal model of ischemia. We showed that Tat-PRAS40 transduced into HT-22 cells, and significantly protected against cell death by reducing the levels of H2O2 and derived reactive species, and DNA fragmentation as well as via the regulation of Bcl-2, Bax, and caspase 3 expression levels in H2O2 treated cells. Also, we showed that transduced Tat-PARS40 protein markedly increased phosphorylated RRAS40 expression levels and 14-3-3σ complex via the Akt signaling pathway. In an animal ischemia model, Tat-PRAS40 effectively transduced into the hippocampus in animal brain and significantly protected against neuronal cell death in the CA1 region. We showed that Tat-PRAS40 protein effectively transduced into hippocampal neuronal cells and markedly protected against neuronal cell damage. Therefore, we suggest that Tat-PRAS40 protein may be used as a therapeutic protein for ischemia and oxidative stress-induced brain disorders. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Interferon-Inducible CD169/Siglec1 Attenuates Anti-HIV-1 Effects of Alpha Interferon

    Science.gov (United States)

    Akiyama, Hisashi; Ramirez, Nora-Guadalupe Pina; Gibson, Gregory; Kline, Christopher; Watkins, Simon; Ambrose, Zandrea

    2017-01-01

    ABSTRACT A hallmark of human immunodeficiency virus type 1 (HIV-1) infection in vivo is chronic immune activation concomitant with type I interferon (IFN) production. Although type I IFN induces an antiviral state in many cell types, HIV-1 can replicate in vivo via mechanisms that have remained unclear. We have recently identified a type I IFN-inducible protein, CD169, as the HIV-1 attachment factor on dendritic cells (DCs) that can mediate robust infection of CD4+ T cells in trans. Since CD169 expression on macrophages is also induced by type I IFN, we hypothesized that type I IFN-inducible CD169 could facilitate productive HIV-1 infection in myeloid cells in cis and CD4+ T cells in trans and thus offset antiviral effects of type I IFN. In support of this hypothesis, infection of HIV-1 or murine leukemia virus Env (MLV-Env)-pseudotyped HIV-1 particles was enhanced in IFN-α-treated THP-1 monocytoid cells, and this enhancement was primarily dependent on CD169-mediated enhancement at the virus entry step, a phenomenon phenocopied in HIV-1 infections of IFN-α-treated primary monocyte-derived macrophages (MDMs). Furthermore, expression of CD169, a marker of type I IFN-induced immune activation in vivo, was enhanced in lymph nodes from pigtailed macaques infected with simian immunodeficiency virus (SIV) carrying HIV-1 reverse transcriptase (RT-SHIV), compared to uninfected macaques, and interestingly, there was extensive colocalization of p27gag and CD169, suggesting productive infection of CD169+ myeloid cells in vivo. While cell-free HIV-1 infection of IFN-α-treated CD4+ T cells was robustly decreased, initiation of infection in trans via coculture with CD169+ IFN-α-treated DCs restored infection, suggesting that HIV-1 exploits CD169 in cis and in trans to attenuate a type I IFN-induced antiviral state. IMPORTANCE HIV-1 infection in humans causes immune activation characterized by elevated levels of proinflammatory cytokines, including type I interferons (IFN

  3. Alterations in the nuclear proteome of HIV-1 infected T-cells

    Energy Technology Data Exchange (ETDEWEB)

    DeBoer, Jason [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); Jagadish, Teena; Haverland, Nicole A. [Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198 (United States); Madson, Christian J. [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); Ciborowski, Pawel [Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198 (United States); The Nebraska Center for Virology, University of Nebraska, Lincoln 68583 (United States); Belshan, Michael, E-mail: michaelbelshan@creighton.edu [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); The Nebraska Center for Virology, University of Nebraska, Lincoln 68583 (United States)

    2014-11-15

    Virus infection of a cell involves the appropriation of host factors and the innate defensive response of the cell. The identification of proteins critical for virus replication may lead to the development of novel, cell-based inhibitors. In this study we mapped the changes in T-cell nuclei during human immunodeficiency virus type 1 (HIV-1) at 20 hpi. Using a stringent data threshold, a total of 13 and 38 unique proteins were identified in infected and uninfected cells, respectively, across all biological replicates. An additional 15 proteins were found to be differentially regulated between infected and control nuclei. STRING analysis identified four clusters of protein–protein interactions in the data set related to nuclear architecture, RNA regulation, cell division, and cell homeostasis. Immunoblot analysis confirmed the differential expression of several proteins in both C8166-45 and Jurkat E6-1 T-cells. These data provide a map of the response in host cell nuclei upon HIV-1 infection. - Highlights: • We identify changes in the expression of nuclear proteins during HIV-1 infection. • 163 nuclear proteins were found differentially regulated during HIV-1 infection. • Bioinformatic analysis identified several nuclear pathways altered by HIV infection. • Candidate factors were validated in two independent cell lines.

  4. Alterations in the nuclear proteome of HIV-1 infected T-cells

    International Nuclear Information System (INIS)

    DeBoer, Jason; Jagadish, Teena; Haverland, Nicole A.; Madson, Christian J.; Ciborowski, Pawel; Belshan, Michael

    2014-01-01

    Virus infection of a cell involves the appropriation of host factors and the innate defensive response of the cell. The identification of proteins critical for virus replication may lead to the development of novel, cell-based inhibitors. In this study we mapped the changes in T-cell nuclei during human immunodeficiency virus type 1 (HIV-1) at 20 hpi. Using a stringent data threshold, a total of 13 and 38 unique proteins were identified in infected and uninfected cells, respectively, across all biological replicates. An additional 15 proteins were found to be differentially regulated between infected and control nuclei. STRING analysis identified four clusters of protein–protein interactions in the data set related to nuclear architecture, RNA regulation, cell division, and cell homeostasis. Immunoblot analysis confirmed the differential expression of several proteins in both C8166-45 and Jurkat E6-1 T-cells. These data provide a map of the response in host cell nuclei upon HIV-1 infection. - Highlights: • We identify changes in the expression of nuclear proteins during HIV-1 infection. • 163 nuclear proteins were found differentially regulated during HIV-1 infection. • Bioinformatic analysis identified several nuclear pathways altered by HIV infection. • Candidate factors were validated in two independent cell lines

  5. Cellular Antiviral Factors that Target Particle Infectivity of HIV-1.

    Science.gov (United States)

    Goffinet, Christine

    2016-01-01

    In the past decade, the identification and characterization of antiviral genes with the ability to interfere with virus replication has established cell-intrinsic innate immunity as a third line of antiviral defense in addition to adaptive and classical innate immunity. Understanding how cellular factors have evolved to inhibit HIV-1 reveals particularly vulnerable points of the viral replication cycle. Many, but not all, antiviral proteins share type I interferon-upregulated expression and sensitivity to viral counteraction or evasion measures. Whereas well-established restriction factors interfere with early post-entry steps and release of HIV-1, recent research has revealed a diverse set of proteins that reduce the infectious quality of released particles using individual, to date poorly understood modes of action. These include induction of paucity of mature glycoproteins in nascent virions or self-incorporation into the virus particle, resulting in poor infectiousness of the virion and impaired spread of the infection. A better understanding of these newly discovered antiviral factors may open new avenues towards the design of drugs that repress the spread of viruses whose genomes have already integrated.

  6. The global transmission network of HIV-1.

    Science.gov (United States)

    Wertheim, Joel O; Leigh Brown, Andrew J; Hepler, N Lance; Mehta, Sanjay R; Richman, Douglas D; Smith, Davey M; Kosakovsky Pond, Sergei L

    2014-01-15

    Human immunodeficiency virus type 1 (HIV-1) is pandemic, but its contemporary global transmission network has not been characterized. A better understanding of the properties and dynamics of this network is essential for surveillance, prevention, and eventual eradication of HIV. Here, we apply a simple and computationally efficient network-based approach to all publicly available HIV polymerase sequences in the global database, revealing a contemporary picture of the spread of HIV-1 within and between countries. This approach automatically recovered well-characterized transmission clusters and extended other clusters thought to be contained within a single country across international borders. In addition, previously undescribed transmission clusters were discovered. Together, these clusters represent all known modes of HIV transmission. The extent of international linkage revealed by our comprehensive approach demonstrates the need to consider the global diversity of HIV, even when describing local epidemics. Finally, the speed of this method allows for near-real-time surveillance of the pandemic's progression.

  7. Anti-gp120 minibody gene transfer to female genital epithelial cells protects against HIV-1 virus challenge in vitro.

    Directory of Open Access Journals (Sweden)

    Ussama M Abdel-Motal

    Full Text Available Although cervico-vaginal epithelial cells of the female lower genital tract provide the initial defense system against HIV-1 infection, the protection is sometimes incomplete. Thus, enhancing anti-HIV-1 humoral immunity at the mucosal cell surface by local expression of anti-HIV-1 broadly neutralizing antibodies (BnAb that block HIV-1 entry would provide an important new intervention that could slow the spread of HIV/AIDS.This study tested the hypothesis that adeno-associated virus (AAV-BnAb gene transfer to cervico-vaginal epithelial cells will lead to protection against HIV-1. Accordingly, a recombinant AAV vector that encodes human b12 anti-HIV gp120 BnAb as a single-chain variable fragment Fc fusion (scFvFc, or "minibody" was constructed. The secreted b12 minibody was shown to be biologically functional in binding to virus envelope protein, neutralizing HIV-1 and importantly, blocking transfer and infectivity of HIV-1(bal in an organotypic human vaginal epithelial cell (VEC model. Furthermore, cervico-vaginal epithelial stem cells were found to be efficiently transduced by the optimal AAV serotype mediated expression of GFP.This study provides the foundation for a novel microbicide strategy to protect against sexual transmission of HIV-1 by AAV transfer of broadly neutralizing antibody genes to cervico-vaginal epithelial stem cells that could replenish b12 BnAb secreting cells through multiple menstrual cycles.

  8. Molecular cloning and anti-HIV-1 activities of APOBEC3s from northern pig-tailed macaques (Macaca leonina

    Directory of Open Access Journals (Sweden)

    Xiao-Liang ZHANG

    2016-07-01

    Full Text Available Northern pig-tailed macaques (NPMs, Macaca leonina are susceptible to HIV-1 infection largely due to the loss of HIV-1-restricting factor TRIM5α. However, great impediments still exist in the persistent replication of HIV-1 in vivo, suggesting some viral restriction factors are reserved in this host. The APOBEC3 proteins have demonstrated a capacity to restrict HIV-1 replication, but their inhibitory effects in NPMs remain elusive. In this study, we cloned the NPM A3A-A3H genes, and determined by BLAST searching that their coding sequences (CDSs showed 99% identity to the corresponding counterparts from rhesus and southern pig-tailed macaques. We further analyzed the anti-HIV-1 activities of the A3A-A3H genes, and found that A3G and A3F had the greatest anti-HIV-1 activity compared with that of other members. The results of this study indicate that A3G and A3F might play critical roles in limiting HIV-1 replication in NPMs in vivo. Furthermore, this research provides valuable information for the optimization of monkey models of HIV-1 infection.

  9. HIV-1 replication in cell lines harboring INI1/hSNF5 mutations.

    Science.gov (United States)

    Sorin, Masha; Yung, Eric; Wu, Xuhong; Kalpana, Ganjam V

    2006-08-31

    INI1/hSNF5 is a cellular protein that directly interacts with HIV-1 integrase (IN). It is specifically incorporated into HIV-1 virions. A dominant negative mutant derived from INI1 inhibits HIV-1 replication. Recent studies indicate that INI1 is associated with pre-integration and reverse transcription complexes that are formed upon viral entry into the target cells. INI1 also is a tumor suppressor, biallelically deleted/mutated in malignant rhabdoid tumors. We have utilized cell lines derived from the rhabdoid tumors, MON and STA-WT1, that harbor either null or truncating mutations of INI1 respectively, to assess the effect of INI1 on HIV-1 replication. We found that while HIV-1 virions produced in 293T cells efficiently transduced MON and STA-WT1 cells, HIV-1 particle production was severely reduced in both of these cells. Reintroduction of INI1 into MON and STA-WT1 significantly enhanced the particle production in both cell lines. HIV-1 particles produced in MON cells were reduced for infectivity, while those produced in STA-WT1 were not. Further analysis indicated the presence of INI1 in those virions produced from STA-WT1 but not from those produced from MON cells. HIV-1 produced in MON cells were defective for synthesis of early and late reverse transcription products in the target cells. Furthermore, virions produced in MON cells were defective for exogenous reverse transcriptase activity carried out using exogenous template, primer and substrate. Our results suggest that INI1-deficient cells exhibit reduced particle production that can be partly enhanced by re-introduction of INI1. Infectivity of HIV-1 produced in some but not all INI1 defective cells, is affected and this defect may correlate to the lack of INI1 and/or some other proteins in these virions. The block in early events of virion produced from MON cells appears to be at the stage of reverse transcription. These studies suggest that presence of INI1 or some other host factor in virions and

  10. HIV-1 replication in cell lines harboring INI1/hSNF5 mutations

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    Wu Xuhong

    2006-08-01

    Full Text Available Abstract Background INI1/hSNF5 is a cellular protein that directly interacts with HIV-1 integrase (IN. It is specifically incorporated into HIV-1 virions. A dominant negative mutant derived from INI1 inhibits HIV-1 replication. Recent studies indicate that INI1 is associated with pre-integration and reverse transcription complexes that are formed upon viral entry into the target cells. INI1 also is a tumor suppressor, biallelically deleted/mutated in malignant rhabdoid tumors. We have utilized cell lines derived from the rhabdoid tumors, MON and STA-WT1, that harbor either null or truncating mutations of INI1 respectively, to assess the effect of INI1 on HIV-1 replication. Results We found that while HIV-1 virions produced in 293T cells efficiently transduced MON and STA-WT1 cells, HIV-1 particle production was severely reduced in both of these cells. Reintroduction of INI1 into MON and STA-WT1 significantly enhanced the particle production in both cell lines. HIV-1 particles produced in MON cells were reduced for infectivity, while those produced in STA-WT1 were not. Further analysis indicated the presence of INI1 in those virions produced from STA-WT1 but not from those produced from MON cells. HIV-1 produced in MON cells were defective for synthesis of early and late reverse transcription products in the target cells. Furthermore, virions produced in MON cells were defective for exogenous reverse transcriptase activity carried out using exogenous template, primer and substrate. Conclusion Our results suggest that INI1-deficient cells exhibit reduced particle production that can be partly enhanced by re-introduction of INI1. Infectivity of HIV-1 produced in some but not all INI1 defective cells, is affected and this defect may correlate to the lack of INI1 and/or some other proteins in these virions. The block in early events of virion produced from MON cells appears to be at the stage of reverse transcription. These studies suggest that

  11. Curcumin protects microglia and primary rat cortical neurons against HIV-1 gp120-mediated inflammation and apoptosis.

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    Luyan Guo

    Full Text Available Curcumin is a molecule found in turmeric root that has anti-inflammatory, antioxidant, and anti-tumor properties and has been widely used as both an herbal drug and a food additive to treat or prevent neurodegenerative diseases. To explore whether curcumin is able to ameliorate HIV-1-associated neurotoxicity, we treated a murine microglial cell line (N9 and primary rat cortical neurons with curcumin in the presence or absence of neurotoxic HIV-1 gp120 (V3 loop protein. We found that HIV-1 gp120 profoundly induced N9 cells to produce reactive oxygen species (ROS, tumor necrosis factor-α (TNF-α and monocyte chemoattractant protein-1 (MCP-1. HIV-1 gp120 also induced apoptosis of primary rat cortical neurons. Curcumin exerted a powerful inhibitory effect against HIV-1 gp120-induced neuronal damage, reducing the production of ROS, TNF-α and MCP-1 by N9 cells and inhibiting apoptosis of primary rat cortical neurons. Curcumin may exert its biological activities through inhibition of the delayed rectification and transient outward potassium (K(+ current, as curcumin effectively reduced HIV-1 gp120-mediated elevation of the delayed rectification and transient outward K(+ channel current in neurons. We conclude that HIV-1 gp120 increases ROS, TNF-α and MCP-1 production in microglia, and induces cortical neuron apoptosis by affecting the delayed rectification and transient outward K(+ channel current. Curcumin reduces production of ROS and inflammatory mediators in HIV-1-gp120-stimulated microglia, and protects cortical neurons against HIV-1-mediated apoptosis, most likely through inhibition of HIV-1 gp120-induced elevation of the delayed rectification and transient outward K(+ current.

  12. Tat-CBR1 inhibits inflammatory responses through the suppressions of NF-κB and MAPK activation in macrophages and TPA-induced ear edema in mice

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Young Nam [Department of Biomedical Science and Research Institute of Bioscience and Biotechnology, Hallym University, Chunchon 200-702 (Korea, Republic of); Kim, Dae Won [Department of Biochemistry and Molecular Biology, Research Institute of Oral Sciences, College of Dentistry, Kangnung-Wonju National University, Kangneung 210-702 (Korea, Republic of); Jo, Hyo Sang; Shin, Min Jea; Ahn, Eun Hee; Ryu, Eun Ji; Yong, Ji In; Cha, Hyun Ju; Kim, Sang Jin; Yeo, Hyeon Ji; Youn, Jong Kyu; Hwang, Jae Hyeok [Department of Biomedical Science and Research Institute of Bioscience and Biotechnology, Hallym University, Chunchon 200-702 (Korea, Republic of); Jeong, Ji-Heon; Kim, Duk-Soo [Department of Anatomy, College of Medicine, Soonchunhyang University, Cheonan-Si 330-090 (Korea, Republic of); Cho, Sung-Woo [Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, Seoul 138-736 (Korea, Republic of); Park, Jinseu [Department of Biomedical Science and Research Institute of Bioscience and Biotechnology, Hallym University, Chunchon 200-702 (Korea, Republic of); Eum, Won Sik, E-mail: wseum@hallym.ac.kr [Department of Biomedical Science and Research Institute of Bioscience and Biotechnology, Hallym University, Chunchon 200-702 (Korea, Republic of); Choi, Soo Young, E-mail: sychoi@hallym.ac.kr [Department of Biomedical Science and Research Institute of Bioscience and Biotechnology, Hallym University, Chunchon 200-702 (Korea, Republic of)

    2015-07-15

    Human carbonyl reductase 1 (CBR1) plays a crucial role in cell survival and protects against oxidative stress response. However, its anti-inflammatory effects are not yet clearly understood. In this study, we examined whether CBR1 protects against inflammatory responses in macrophages and mice using a Tat-CBR1 protein which is able to penetrate into cells. The results revealed that purified Tat-CBR1 protein efficiently transduced into Raw 264.7 cells and inhibited lipopolysaccharide (LPS)-induced cyclooxygenase-2 (COX-2), nitric oxide (NO) and prostaglandin E{sub 2} (PGE{sub 2}) expression levels. In addition, Tat-CBR1 protein leads to decreased pro-inflammatory cytokine expression through suppression of nuclear transcription factor-kappaB (NF-κB) and mitogen activated protein kinase (MAPK) activation. Furthermore, Tat-CBR1 protein inhibited inflammatory responses in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation when applied topically. These findings indicate that Tat-CBR1 protein has anti-inflammatory properties in vitro and in vivo through inhibition of NF-κB and MAPK activation, suggesting that Tat-CBR1 protein may have potential as a therapeutic agent against inflammatory diseases. - Highlights: • Transduced Tat-CBR1 reduces LPS-induced inflammatory mediators and cytokines. • Tat-CBR1 inhibits MAPK and NF-κB activation. • Tat-CBR1 ameliorates inflammation response in vitro and in vivo. • Tat-CBR1 may be useful as potential therapeutic agent for inflammation.

  13. SUN2 Modulates HIV-1 Infection and Latency through Association with Lamin A/C To Maintain the Repressive Chromatin.

    Science.gov (United States)

    Sun, Wei-Wei; Jiao, Shi; Sun, Li; Zhou, Zhaocai; Jin, Xia; Wang, Jian-Hua

    2018-05-01

    The postintegrational latency of HIV-1 is characterized by reversible silencing of long terminal repeat (LTR)-driven transcription of the HIV genome. It is known that the formation of repressive chromatin at the 5'-LTR of HIV-1 proviral DNA impedes viral transcription by blocking the recruitment of positive transcription factors. How the repressive chromatin is formed and modulated during HIV-1 infection remains elusive. Elucidation of which chromatin reassembly factor mediates the reorganization of chromatin is likely to facilitate the understanding of the host's modulation of HIV-1 transcription and latency. Here we revealed that "Sad1 and UNC84 domain containing 2" (SUN2), an inner nuclear membrane protein, maintained the repressive chromatin and inhibited HIV LTR-driven transcription of proviral DNA through an association with lamin A/C. Specifically, lamin A/C tethered SUN2 to the nucleosomes 1 and 2 of the HIV-1 5'-LTR to block the initiation and elongation of HIV-1 transcription. SUN2 knockdown converted chromatin to an active form and thus enhanced the phosphorylation of RNA polymerase II and its recruitment to the 5'-LTR HIV-1 proviral DNA, leading to reactivation of HIV-1 from latency. Conversely, the exogenous factors such as tumor necrosis factor alpha (TNF-α) induced reactivation, and the replication of HIV-1 led to the disassociation between SUN2 and lamin A/C, suggesting that disruption of the association between SUN2 and lamin A/C to convert the repressive chromatin to the active form might be a prerequisite for the initiation of HIV-1 transcription and replication. Together, our findings indicate that SUN2 is a novel chromatin reassembly factor that helps to maintain chromatin in a repressive state and consequently inhibits HIV-1 transcription. IMPORTANCE Despite the successful use of scores of antiretroviral drugs, HIV latency poses a major impediment to virus eradication. Elucidation of the mechanism of latency facilitates the discovery of new

  14. HIV-1 Eradication Strategies: Design and Assessment

    Science.gov (United States)

    Siliciano, Robert F.

    2014-01-01

    Purpose of review Recent developments have generated renewed interest in the possibility of curing HIV-1 infection. This review describes some of the practical challenges that will need to be overcome if curative strategies are to be successful. Recent findings The latent reservoir for HIV-1 in resting memory CD4+ T cells is the major barrier to curing the infection. The most widely discussed approach to curing the infection involves finding agents that reverse latency in resting CD4+ T cells, with the assumption that the cells will then die from viral cytopathic effects or be lysed by host cytolytic T lymphocytes (CTL). A major challenge is the development of in vitro models that can be used to explore mechanisms and identify latency reversing agents (LRA). Although several models have been developed, including primary cell models, none of them may fully capture the quiescent state of the cells that harbor latent HIV-1 in vivo. An additional problem is that LRA that do not cause T cell activation may not lead to the death of infected cells. Finally, measuring the effects of LRAs in vivo is complicated by the lack of correlation between different assays for the latent reservoir. Summary Progress on these practical issues is essential to finding a cure. PMID:23698561

  15. Alterations of HIV-1 envelope phenotype and antibody-mediated neutralization by signal peptide mutations.

    Directory of Open Access Journals (Sweden)

    Chitra Upadhyay

    2018-01-01

    Full Text Available HIV-1 envelope glycoprotein (Env mediates virus attachment and entry into the host cells. Like other membrane-bound and secreted proteins, HIV-1 Env contains at its N terminus a signal peptide (SP that directs the nascent Env to the endoplasmic reticulum (ER where Env synthesis and post-translational modifications take place. SP is cleaved during Env biosynthesis but potentially influences the phenotypic traits of the Env protein. The Env SP sequences of HIV-1 isolates display high sequence variability, and the significance of such variability is unclear. We postulate that changes in the Env SP influence Env transport through the ER-Golgi secretory pathway and Env folding and/or glycosylation that impact on Env incorporation into virions, receptor binding and antibody recognition. We first evaluated the consequences of mutating the charged residues in the Env SP in the context of infectious molecular clone HIV-1 REJO.c/2864. Results show that three different mutations affecting histidine at position 12 affected Env incorporation into virions that correlated with reduction of virus infectivity and DC-SIGN-mediated virus capture and transmission. Mutations at positions 8, 12, and 15 also rendered the virus more resistant to neutralization by monoclonal antibodies against the Env V1V2 region. These mutations affected the oligosaccharide composition of N-glycans as shown by changes in Env reactivity with specific lectins and by mass spectrometry. Increased neutralization resistance and N-glycan composition changes were also observed when analogous mutations were introduced to another HIV-1 strain, JRFL. To the best of our knowledge, this is the first study showing that certain residues in the HIV-1 Env SP can affect virus neutralization sensitivity by modulating oligosaccharide moieties on the Env N-glycans. The HIV-1 Env SP sequences thus may be under selective pressure to balance virus infectiousness with virus resistance to the host antibody

  16. A Tat-conjugated Peptide Nucleic Acid Tat-PNA-DR Inhibits Hepatitis B Virus Replication In Vitro and In Vivo by Targeting LTR Direct Repeats of HBV RNA

    Science.gov (United States)

    Zeng, Zhengyang; Han, Shisong; Hong, Wei; Lang, Yange; Li, Fangfang; Liu, Yongxiang; Li, Zeyong; Wu, Yingliang; Li, Wenxin; Zhang, Xianzheng; Cao, Zhijian

    2016-01-01

    Hepatitis B virus (HBV) infection is a major cause of chronic active hepatitis, cirrhosis, and primary hepatocellular carcinoma, all of which are severe threats to human health. However, current clinical therapies for HBV are limited by potential side effects, toxicity, and drug-resistance. In this study, a cell-penetrating peptide-conjugated peptide nucleic acid (PNA), Tat-PNA-DR, was designed to target the direct repeat (DR) sequences of HBV. Tat-PNA-DR effectively inhibited HBV replication in HepG2.2.15 cells. Its anti-HBV effect relied on the binding of Tat-PNA-DR to the DR, whereby it suppressed the translation of hepatitis B e antigen (HBeAg), HBsAg, HBV core, hepatitis B virus x protein, and HBV reverse transcriptase (RT) and the reverse transcription of the HBV genome. Furthermore, Tat-PNA-DR administered by intravenous injection efficiently cleared HBeAg and HBsAg in an acute hepatitis B mouse model. Importantly, it induced an 80% decline in HBV DNA in mouse serum, which was similar to the effect of the widely used clinical drug Lamivudine (3TC). Additionally, a long-term hydrodynamics HBV mouse model also demonstrated Tat-PNA-DR's antiviral effect. Interestingly, Tat-PNA-DR displayed low cytotoxicity, low mouse acute toxicity, low immunogenicity, and high serum stability. These data indicate that Tat-PNA-DR is a unique PNA and a promising drug candidate against HBV. PMID:26978579

  17. A Tat-conjugated Peptide Nucleic Acid Tat-PNA-DR Inhibits Hepatitis B Virus Replication In Vitro and In Vivo by Targeting LTR Direct Repeats of HBV RNA

    Directory of Open Access Journals (Sweden)

    Zhengyang Zeng

    2016-01-01

    Full Text Available Hepatitis B virus (HBV infection is a major cause of chronic active hepatitis, cirrhosis, and primary hepatocellular carcinoma, all of which are severe threats to human health. However, current clinical therapies for HBV are limited by potential side effects, toxicity, and drug-resistance. In this study, a cell-penetrating peptide-conjugated peptide nucleic acid (PNA, Tat-PNA-DR, was designed to target the direct repeat (DR sequences of HBV. Tat-PNA-DR effectively inhibited HBV replication in HepG2.2.15 cells. Its anti-HBV effect relied on the binding of Tat-PNA-DR to the DR, whereby it suppressed the translation of hepatitis B e antigen (HBeAg, HBsAg, HBV core, hepatitis B virus x protein, and HBV reverse transcriptase (RT and the reverse transcription of the HBV genome. Furthermore, Tat-PNA-DR administered by intravenous injection efficiently cleared HBeAg and HBsAg in an acute hepatitis B mouse model. Importantly, it induced an 80% decline in HBV DNA in mouse serum, which was similar to the effect of the widely used clinical drug Lamivudine (3TC. Additionally, a long-term hydrodynamics HBV mouse model also demonstrated Tat-PNA-DR's antiviral effect. Interestingly, Tat-PNA-DR displayed low cytotoxicity, low mouse acute toxicity, low immunogenicity, and high serum stability. These data indicate that Tat-PNA-DR is a unique PNA and a promising drug candidate against HBV.

  18. Quantitative live-cell imaging of human immunodeficiency virus (HIV-1) assembly.

    Science.gov (United States)

    Baumgärtel, Viola; Müller, Barbara; Lamb, Don C

    2012-05-01

    Advances in fluorescence methodologies make it possible to investigate biological systems in unprecedented detail. Over the last few years, quantitative live-cell imaging has increasingly been used to study the dynamic interactions of viruses with cells and is expected to become even more indispensable in the future. Here, we describe different fluorescence labeling strategies that have been used to label HIV-1 for live cell imaging and the fluorescence based methods used to visualize individual aspects of virus-cell interactions. This review presents an overview of experimental methods and recent experiments that have employed quantitative microscopy in order to elucidate the dynamics of late stages in the HIV-1 replication cycle. This includes cytosolic interactions of the main structural protein, Gag, with itself and the viral RNA genome, the recruitment of Gag and RNA to the plasma membrane, virion assembly at the membrane and the recruitment of cellular proteins involved in HIV-1 release to the nascent budding site.

  19. Synthetic, structural mimetics of the β-hairpin flap of HIV-1 protease inhibit enzyme function.

    Science.gov (United States)

    Chauhan, Jay; Chen, Shen-En; Fenstermacher, Katherine J; Naser-Tavakolian, Aurash; Reingewertz, Tali; Salmo, Rosene; Lee, Christian; Williams, Emori; Raje, Mithun; Sundberg, Eric; DeStefano, Jeffrey J; Freire, Ernesto; Fletcher, Steven

    2015-11-01

    Small-molecule mimetics of the β-hairpin flap of HIV-1 protease (HIV-1 PR) were designed based on a 1,4-benzodiazepine scaffold as a strategy to interfere with the flap-flap protein-protein interaction, which functions as a gated mechanism to control access to the active site. Michaelis-Menten kinetics suggested our small-molecules are competitive inhibitors, which indicates the mode of inhibition is through binding the active site or sterically blocking access to the active site and preventing flap closure, as designed. More generally, a new bioactive scaffold for HIV-1PR inhibition has been discovered, with the most potent compound inhibiting the protease with a modest K(i) of 11 μM. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Constitutively Active MAVS Inhibits HIV-1 Replication via Type I Interferon Secretion and Induction of HIV-1 Restriction Factors.

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    Sachin Gupta

    Full Text Available Type I interferon is known to inhibit HIV-1 replication through the induction of interferon stimulated genes (ISG, including a number of HIV-1 restriction factors. To better understand interferon-mediated HIV-1 restriction, we constructed a constitutively active form of the RIG-I adapter protein MAVS. Constitutive MAVS was generated by fusion of full length MAVS to a truncated form of the Epstein Barr virus protein LMP1 (ΔLMP1. Supernatant from ΔLMP1-MAVS-transfected 293T cells contained high levels of type I interferons and inhibited HIV replication in both TZM-bl and primary human CD4+ T cells. Supernatant from ΔLMP1-MAVS-transfected 293T cells also inhibited replication of VSV-G pseudotyped single cycle SIV in TZM-bl cells, suggesting restriction was post-entry and common to both HIV and SIV. Gene array analysis of ΔLMP1-MAVS-transfected 293T cells and trans-activated CD4+ T cells showed significant upregulation of ISG, including previously characterized HIV restriction factors Viperin, Tetherin, MxB, and ISG56. Interferon blockade studies implicated interferon-beta in this response. In addition to direct viral inhibition, ΔLMP1-MAVS markedly enhanced secretion of IFN-β and IL-12p70 by dendritic cells and the activation and maturation of dendritic cells. Based on this immunostimulatory activity, an adenoviral vector (Ad5 expressing ΔLMP1-MAVS was tested as a molecular adjuvant in an HIV vaccine mouse model. Ad5-Gag antigen combined with Ad5-ΔLMP1-MAVS enhanced control of vaccinia-gag replication in a mouse challenge model, with 4/5 animals showing undetectable virus following challenge. Overall, ΔLMP1-MAVS is a promising reagent to inhibit HIV-1 replication in infected tissues and enhance vaccine-mediated immune responses, while avoiding toxicity associated with systemic type I interferon administration.

  1. Recruitment of a SAP18-HDAC1 complex into HIV-1 virions and its requirement for viral replication.

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    Masha Sorin

    2009-06-01

    Full Text Available HIV-1 integrase (IN is a virally encoded protein required for integration of viral cDNA into host chromosomes. INI1/hSNF5 is a component of the SWI/SNF complex that interacts with HIV-1 IN, is selectively incorporated into HIV-1 (but not other retroviral virions, and modulates multiple steps, including particle production and infectivity. To gain further insight into the role of INI1 in HIV-1 replication, we screened for INI1-interacting proteins using the yeast two-hybrid system. We found that SAP18 (Sin3a associated protein 18 kD, a component of the Sin3a-HDAC1 complex, directly binds to INI1 in yeast, in vitro and in vivo. Interestingly, we found that IN also binds to SAP18 in vitro and in vivo. SAP18 and components of a Sin3A-HDAC1 complex were specifically incorporated into HIV-1 (but not SIV and HTLV-1 virions in an HIV-1 IN-dependent manner. Using a fluorescence-based assay, we found that HIV-1 (but not SIV virion preparations harbour significant deacetylase activity, indicating the specific recruitment of catalytically active HDAC into the virions. To determine the requirement of virion-associated HDAC1 to HIV-1 replication, an inactive, transdominant negative mutant of HDAC1 (HDAC1(H141A was utilized. Incorporation of HDAC1(H141A decreased the virion-associated histone deacetylase activity. Furthermore, incorporation of HDAC1(H141A decreased the infectivity of HIV-1 (but not SIV virions. The block in infectivity due to virion-associated HDAC1(H141A occurred specifically at the early reverse transcription stage, while entry of the virions was unaffected. RNA-interference mediated knock-down of HDAC1 in producer cells resulted in decreased virion-associated HDAC1 activity and a reduction in infectivity of these virions. These studies indicate that HIV-1 IN and INI1/hSNF5 bind SAP18 and selectively recruit components of Sin3a-HDAC1 complex into HIV-1 virions. Furthermore, HIV-1 virion-associated HDAC1 is required for efficient early post

  2. Where in the Cell Are You? Probing HIV-1 Host Interactions through Advanced Imaging Techniques

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    Brennan S. Dirk

    2016-10-01

    Full Text Available Viruses must continuously evolve to hijack the host cell machinery in order to successfully replicate and orchestrate key interactions that support their persistence. The type-1 human immunodeficiency virus (HIV-1 is a prime example of viral persistence within the host, having plagued the human population for decades. In recent years, advances in cellular imaging and molecular biology have aided the elucidation of key steps mediating the HIV-1 lifecycle and viral pathogenesis. Super-resolution imaging techniques such as stimulated emission depletion (STED and photoactivation and localization microscopy (PALM have been instrumental in studying viral assembly and release through both cell–cell transmission and cell–free viral transmission. Moreover, powerful methods such as Forster resonance energy transfer (FRET and bimolecular fluorescence complementation (BiFC have shed light on the protein-protein interactions HIV-1 engages within the host to hijack the cellular machinery. Specific advancements in live cell imaging in combination with the use of multicolor viral particles have become indispensable to unravelling the dynamic nature of these virus-host interactions. In the current review, we outline novel imaging methods that have been used to study the HIV-1 lifecycle and highlight advancements in the cell culture models developed to enhance our understanding of the HIV-1 lifecycle.

  3. Inhibition of HIV-1 infection by synthetic peptides derived CCR5 fragments

    International Nuclear Information System (INIS)

    Imai, Masaki; Baranyi, Lajos; Okada, Noriko; Okada, Hidechika

    2007-01-01

    HIV-1 infection requires interaction of viral envelope protein gp160 with CD4 and a chemokine receptor, CCR5 or CXCR4 as entry coreceptor. We designed HIV-inhibitory peptides targeted to CCR5 using a novel computer program (ANTIS), which searched all possible sense-antisense amino acid pairs between proteins. Seven AHBs were found in CCR5 receptor. All AHB peptides were synthesized and tested for their ability to prevent HIV-1 infection to human T cells. A peptide fragment (LC5) which is a part of the CCR5 receptor corresponding to the loop between the fifth and sixth transmembrane regions (amino acids 222-240) proved to inhibit HIV-1 IIIB infection of MT-4 cells. Interaction of these antisense peptides could be involved in sustaining HIV-1 infectivity. LC5 effectively indicated dose-dependent manner, and the suppression was enhanced additively by T20 peptide, which inhibits infection in vitro by disrupting the gp41 conformational changes necessary for membrane fusion. Thus, these results indicate that CCR5-derived AHB peptides could provide a useful tool to define the mechanism(s) of HIV infection, and may provide insight which will contribute to the development of an anti-HIV-1 reagent

  4. Nup153 and Nup98 bind the HIV-1 core and contribute to the early steps of HIV-1 replication

    International Nuclear Information System (INIS)

    Di Nunzio, Francesca; Fricke, Thomas; Miccio, Annarita; Valle-Casuso, Jose Carlos; Perez, Patricio; Souque, Philippe; Rizzi, Ermanno; Severgnini, Marco; Mavilio, Fulvio; Charneau, Pierre; Diaz-Griffero, Felipe

    2013-01-01

    The early steps of HIV-1 replication involve the entry of HIV-1 into the nucleus, which is characterized by viral interactions with nuclear pore components. HIV-1 developed an evolutionary strategy to usurp the nuclear pore machinery and chromatin in order to integrate and efficiently express viral genes. In the current work, we studied the role of nucleoporins 153 and 98 (Nup153 and Nup98) in infection of human Jurkat lymphocytes by HIV-1. We showed that Nup153-depleted cells exhibited a defect in nuclear import, while depletion of Nup 98 caused a slight defect in HIV integration. To explore the biochemical viral determinants for the requirement of Nup153 and Nup98 during HIV-1 infection, we tested the ability of these nucleoporins to interact with HIV-1 cores. Our findings showed that both nucleoporins bind HIV-1 cores suggesting that this interaction is important for HIV-1 nuclear import and/or integration. Distribution analysis of integration sites in Nup153-depleted cells revealed a reduced tendency of HIV-1 to integrate in intragenic sites, which in part could account for the large infectivity defect observed in Nup153-depleted cells. Our work strongly supports a role for Nup153 in HIV-1 nuclear import and integration. - Highlights: ► We studied the role of Nup98 and Nup153 in HIV-1 infection. ► Nup98 binds the HIV-1 core and is involved in HIV-1 integration. ► Nup153 binds the HIV-1 core and is involved in HIV-1 nuclear import. ► Depletion of Nup153 decreased the integration of HIV-1 in transcriptionally active sites

  5. Nup153 and Nup98 bind the HIV-1 core and contribute to the early steps of HIV-1 replication

    Energy Technology Data Exchange (ETDEWEB)

    Di Nunzio, Francesca, E-mail: francesca.di-nunzio@pasteur.fr [Molecular Virology and Vaccinology unit, CNRS URA 3015, Department of Virology, Institut Pasteur, 25-28 rue du Dr. Roux, 75015 Paris (France); Fricke, Thomas [Department of Microbiology and Immunology, Albert Einstein College of Medicine Bronx, NY 10461 (United States); Miccio, Annarita [University of Modena e Reggio Emilia, Centro di Medicina Rigenerativa, Modena (Italy); Valle-Casuso, Jose Carlos; Perez, Patricio [Department of Microbiology and Immunology, Albert Einstein College of Medicine Bronx, NY 10461 (United States); Souque, Philippe [Molecular Virology and Vaccinology unit, CNRS URA 3015, Department of Virology, Institut Pasteur, 25-28 rue du Dr. Roux, 75015 Paris (France); Rizzi, Ermanno; Severgnini, Marco [Institute of Biomedical Technologies, CNR, Milano (Italy); Mavilio, Fulvio [University of Modena e Reggio Emilia, Centro di Medicina Rigenerativa, Modena (Italy); Genethon, Evry (France); Charneau, Pierre [Molecular Virology and Vaccinology unit, CNRS URA 3015, Department of Virology, Institut Pasteur, 25-28 rue du Dr. Roux, 75015 Paris (France); Diaz-Griffero, Felipe, E-mail: felipe.diaz-griffero@einstein.yu.edu [Department of Microbiology and Immunology, Albert Einstein College of Medicine Bronx, NY 10461 (United States)

    2013-05-25

    The early steps of HIV-1 replication involve the entry of HIV-1 into the nucleus, which is characterized by viral interactions with nuclear pore components. HIV-1 developed an evolutionary strategy to usurp the nuclear pore machinery and chromatin in order to integrate and efficiently express viral genes. In the current work, we studied the role of nucleoporins 153 and 98 (Nup153 and Nup98) in infection of human Jurkat lymphocytes by HIV-1. We showed that Nup153-depleted cells exhibited a defect in nuclear import, while depletion of Nup 98 caused a slight defect in HIV integration. To explore the biochemical viral determinants for the requirement of Nup153 and Nup98 during HIV-1 infection, we tested the ability of these nucleoporins to interact with HIV-1 cores. Our findings showed that both nucleoporins bind HIV-1 cores suggesting that this interaction is important for HIV-1 nuclear import and/or integration. Distribution analysis of integration sites in Nup153-depleted cells revealed a reduced tendency of HIV-1 to integrate in intragenic sites, which in part could account for the large infectivity defect observed in Nup153-depleted cells. Our work strongly supports a role for Nup153 in HIV-1 nuclear import and integration. - Highlights: ► We studied the role of Nup98 and Nup153 in HIV-1 infection. ► Nup98 binds the HIV-1 core and is involved in HIV-1 integration. ► Nup153 binds the HIV-1 core and is involved in HIV-1 nuclear import. ► Depletion of Nup153 decreased the integration of HIV-1 in transcriptionally active sites.

  6. HIV-1 phylogenetic analysis shows HIV-1 transits through the meninges to brain and peripheral tissues.

    Science.gov (United States)

    Lamers, Susanna L; Gray, Rebecca R; Salemi, Marco; Huysentruyt, Leanne C; McGrath, Michael S

    2011-01-01

    Brain infection by the human immunodeficiency virus type 1 (HIV-1) has been investigated in many reports with a variety of conclusions concerning the time of entry and degree of viral compartmentalization. To address these diverse findings, we sequenced HIV-1 gp120 clones from a wide range of brain, peripheral and meningeal tissues from five patients who died from several HIV-1 associated disease pathologies. High-resolution phylogenetic analysis confirmed previous studies that showed a significant degree of compartmentalization in brain and peripheral tissue subpopulations. Some intermixing between the HIV-1 subpopulations was evident, especially in patients that died from pathologies other than HIV-associated dementia. Interestingly, the major tissue harboring virus from both the brain and peripheral tissues was the meninges. These results show that (1) HIV-1 is clearly capable of migrating out of the brain, (2) the meninges are the most likely primary transport tissues, and (3) infected brain macrophages comprise an important HIV reservoir during highly active antiretroviral therapy. Copyright © 2010 Elsevier B.V. All rights reserved.

  7. Uridine metabolism in HIV-1-infected patients: effect of infection, of antiretroviral therapy and of HIV-1/ART-associated lipodystrophy syndrome.

    Directory of Open Access Journals (Sweden)

    Pere Domingo

    Full Text Available BACKGROUND: Uridine has been advocated for the treatment of HIV-1/HAART-associated lipodystrophy (HALS, although its metabolism in HIV-1-infected patients is poorly understood. METHODS: Plasma uridine concentrations were measured in 35 controls and 221 HIV-1-infected patients and fat uridine in 15 controls and 19 patients. The diagnosis of HALS was performed following the criteria of the Lipodystrophy Severity Grading Scale. Uridine was measured by a binary gradient-elution HPLC method. Analysis of genes encoding uridine metabolizing enzymes in fat was performed with TaqMan RT-PCR. RESULTS: Median plasma uridine concentrations for HIV-1-infected patients were 3.80 µmol/l (interquartile range: 1.60, and for controls 4.60 µmol/l (IQR: 1.8 (P = 0.0009. In fat, they were of 6.0 (3.67, and 2.8 (4.65 nmol/mg of protein, respectively (P = 0.0118. Patients with a mixed HALS form had a median plasma uridine level of 4.0 (IC95%: 3.40-4.80 whereas in those with isolated lipoatrophy it was 3.25 (2.55-4.15 µmol/l/l (P = 0.0066. The expression of uridine cytidine kinase and uridine phosphorylase genes was significantly decreased in all groups of patients with respect to controls. A higher expression of the mRNAs for concentrative nucleoside transporters was found in HIV-1-infected patients with respect to healthy controls. CONCLUSIONS: HIV-1 infection is associated with a decrease in plasma uridine and a shift of uridine to the adipose tissue compartment. Antiretroviral therapy was not associated with plasma uridine concentrations, but pure lipoatrophic HALS was associated with significantly lower plasma uridine concentrations.

  8. APOBEC3G inhibits HIV-1 RNA elongation by inactivating the viral trans-activation response element.

    Science.gov (United States)

    Nowarski, Roni; Prabhu, Ponnandy; Kenig, Edan; Smith, Yoav; Britan-Rosich, Elena; Kotler, Moshe

    2014-07-29

    Deamination of cytidine residues in viral DNA is a major mechanism by which APOBEC3G (A3G) inhibits vif-deficient human immunodeficiency virus type 1 (HIV-1) replication. dC-to-dU transition following RNase-H activity leads to viral cDNA degradation, production of non-functional proteins, formation of undesired stop codons and decreased viral protein synthesis. Here, we demonstrate that A3G provides an additional layer of defense against HIV-1 infection dependent on inhibition of proviral transcription. HIV-1 transcription elongation is regulated by the trans-activation response (TAR) element, a short stem-loop RNA structure required for elongation factors binding. Vif-deficient HIV-1-infected cells accumulate short viral transcripts and produce lower amounts of full-length HIV-1 transcripts due to A3G deamination of the TAR apical loop cytidine, highlighting the requirement for TAR loop integrity in HIV-1 transcription. We further show that free single-stranded DNA (ssDNA) termini are not essential for A3G activity and a gap of CCC motif blocked with juxtaposed DNA or RNA on either or 3'+5' ends is sufficient for A3G deamination. These results identify A3G as an efficient mutator and that deamination of (-)SSDNA results in an early block of HIV-1 transcription. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. BET bromodomain inhibition as a novel strategy for reactivation of HIV-1.

    Science.gov (United States)

    Banerjee, Camellia; Archin, Nancie; Michaels, Daniel; Belkina, Anna C; Denis, Gerald V; Bradner, James; Sebastiani, Paola; Margolis, David M; Montano, Monty

    2012-12-01

    The persistence of latent HIV-1 remains a major challenge in therapeutic efforts to eradicate infection. We report the capacity for HIV reactivation by a selective small molecule inhibitor of BET family bromodomains, JQ1, a promising therapeutic agent with antioncogenic properties. JQ1 reactivated HIV transcription in models of latent T cell infection and latent monocyte infection. We also tested the effect of exposure to JQ1 to allow recovery of replication-competent HIV from pools of resting CD4(+) T cells isolated from HIV-infected, ART-treated patients. In one of three patients, JQ1 allowed recovery of virus at a frequency above unstimulated conditions. JQ1 potently suppressed T cell proliferation with minimal cytotoxic effect. Transcriptional profiling of T cells with JQ1 showed potent down-regulation of T cell activation genes, including CD3, CD28, and CXCR4, similar to HDAC inhibitors, but JQ1 also showed potent up-regulation of chromatin modification genes, including SIRT1, HDAC6, and multiple lysine demethylases (KDMs). Thus, JQ1 reactivates HIV-1 while suppressing T cell activation genes and up-regulating histone modification genes predicted to favor increased Tat activity. Thus, JQ1 may be useful in studies of potentially novel mechanisms for transcriptional control as well as in translational efforts to identify therapeutic molecules to achieve viral eradication.

  10. Tetherin restricts productive HIV-1 cell-to-cell transmission.

    Directory of Open Access Journals (Sweden)

    Nicoletta Casartelli

    2010-06-01

    Full Text Available The IFN-inducible antiviral protein tetherin (or BST-2/CD317/HM1.24 impairs release of mature HIV-1 particles from infected cells. HIV-1 Vpu antagonizes the effect of tetherin. The fate of virions trapped at the cell surface remains poorly understood. Here, we asked whether tetherin impairs HIV cell-to-cell transmission, a major means of viral spread. Tetherin-positive or -negative cells, infected with wild-type or DeltaVpu HIV, were used as donor cells and cocultivated with target lymphocytes. We show that tetherin inhibits productive cell-to-cell transmission of DeltaVpu to targets and impairs that of WT HIV. Tetherin accumulates with Gag at the contact zone between infected and target cells, but does not prevent the formation of virological synapses. In the presence of tetherin, viruses are then mostly transferred to targets as abnormally large patches. These viral aggregates do not efficiently promote infection after transfer, because they accumulate at the surface of target cells and are impaired in their fusion capacities. Tetherin, by imprinting virions in donor cells, is the first example of a surface restriction factor limiting viral cell-to-cell spread.

  11. Dynamics of Preferential Substrate Recognition in HIV-1 Protease: Redefining the Substrate Envelope

    Science.gov (United States)

    Özen, Ayşegül; Haliloğlu, Türkan; Schiffer, Celia A.

    2011-01-01

    HIV-1 protease (PR) permits viral maturation by processing the Gag and Gag-Pro-Pol polyproteins. Though HIV-1 PR inhibitors (PIs) are used in combination antiviral therapy, the emergence of drug resistance has limited their efficacy. The rapid evolution of HIV-1 necessitates the consideration of drug resistance in novel drug-design strategies. Drug-resistant HIV-1 PR variants, while no longer efficiently inhibited, continue to efficiently hydrolyze the natural viral substrates. Though highly diverse in sequence, the HIV-1 PR substrates bind in a conserved three-dimensional shape we defined as the “substrate envelope”. We previously showed that resistance mutations arise where PIs protrude beyond the substrate envelope, as these regions are crucial for drug binding but not for substrate recognition. Here, we extend this model by considering the role of protein dynamics in the interaction of HIV-1 PR with its substrates. Seven molecular dynamics simulations of PR-substrate complexes were performed to estimate the conformational flexibility of substrates in their complexes. Interdependency of the substrate-protease interactions may compensate for the variations in cleavage-site sequences, and explain how a diverse set of sequences can be recognized as substrates by the same enzyme. This diversity may be essential for regulating sequential processing of substrates. We also define a dynamic substrate envelope as a more accurate representation of PR-substrate interactions. This dynamic substrate envelope, described by a probability distribution function, is a powerful tool for drug design efforts targeting ensembles of resistant HIV-1 PR variants with the aim of developing drugs that are less susceptible to resistance. PMID:21762811

  12. Increased Risk of HIV-1 Transmission in Pregnancy: A Prospective Study among African HIV-1 Serodiscordant Couples

    Science.gov (United States)

    MUGO, Nelly R.; HEFFRON, Renee; DONNELL, Deborah; WALD, Anna; WERE, Edwin O.; REES, Helen; CELUM, Connie; KIARIE, James N.; COHEN, Craig R.; KAYINTEKORE, Kayitesi; BAETEN, Jared M.

    2011-01-01

    Background Physiologic and behavioral changes during pregnancy may alter HIV-1 susceptibility and infectiousness. Prospective studies exploring pregnancy and HIV-1 acquisition risk in women have found inconsistent results. No study has explored the effect of pregnancy on HIV-1 transmission risk from HIV-1 infected women to male partners. Methods In a prospective study of African HIV-1 serodiscordant couples, we evaluated the relationship between pregnancy and the risk of 1) HIV-1 acquisition among women and 2) HIV-1 transmission from women to men. Results 3321 HIV-1 serodiscordant couples were enrolled, 1085 (32.7%) with HIV-1 susceptible female partners and 2236 (67.3%) with susceptible male partners. HIV-1 incidence in women was 7.35 versus 3.01 per 100 person-years during pregnant and non-pregnant periods (hazard ratio [HR] 2.34, 95% confidence interval [CI] 1.33–4.09). This effect was attenuated and not statistically significant after adjusting for sexual behavior and other confounding factors (adjusted HR 1.71, 95% CI 0.93–3.12). HIV-1 incidence in male partners of infected women was 3.46 versus 1.58 per 100 person-years when their partners were pregnant versus not pregnant (HR 2.31, 95% CI 1.22–4.39). This effect was not attenuated in adjusted analysis (adjusted HR 2.47, 95% CI 1.26–4.85). Conclusions HIV-1 risk increased two-fold during pregnancy. Elevated risk of HIV-1 acquisition in pregnant women appeared in part to be explained by behavioral and other factors. This is the first study to show pregnancy increased the risk of female-to-male HIV-1 transmission, which may reflect biological changes of pregnancy that could increase HIV-1 infectiousness. PMID:21785321

  13. The RNA helicase DDX1 is involved in restricted HIV-1 Rev function in human astrocytes

    International Nuclear Information System (INIS)

    Fang Jianhua; Acheampong, Edward; Dave, Rajnish; Wang Fengxiang; Mukhtar, Muhammad; Pomerantz, Roger J.

    2005-01-01

    Productive infection by human immunodeficiency virus type I (HIV-1) in the central nervous system (CNS) involves mainly macrophages and microglial cells. A frequency of less than 10% of human astrocytes is estimated to be infectable with HIV-1. Nonetheless, this relatively low percentage of infected astrocytes, but associated with a large total number of astrocytic cells in the CNS, makes human astrocytes a critical part in the analyses of potential HIV-1 reservoirs in vivo. Investigations in astrocytic cell lines and primary human fetal astrocytes revealed that limited HIV-1 replication in these cells resulted from low-level viral entry, transcription, viral protein processing, and virion maturation. Of note, a low ratio of unspliced versus spliced HIV-1-specific RNA was also investigated, as Rev appeared to act aberrantly in astrocytes, via loss of nuclear and/or nucleolar localization and diminished Rev-mediated function. Host cellular machinery enabling Rev function has become critical for elucidation of diminished Rev activity, especially for those factors leading to RNA metabolism. We have recently identified a DEAD-box protein, DDX1, as a Rev cellular co-factor and now have explored its potential importance in astrocytes. Cells were infected with HIV-1 pseudotyped with envelope glycoproteins of amphotropic murine leukemia viruses (MLV). Semi-quantitative reverse transcriptase-polymerase chain reactions (RT-PCR) for unspliced, singly-spliced, and multiply-spliced RNA clearly showed a lower ratio of unspliced/singly-spliced over multiply-spliced HIV-1-specific RNA in human astrocytes as compared to Rev-permissive, non-glial control cells. As well, the cellular localization of Rev in astrocytes was cytoplasmically dominant as compared to that of Rev-permissive, non-glial controls. This endogenous level of DDX1 expression in astrocytes was demonstrated directly to lead to a shift of Rev sub-cellular distribution dominance from nuclear and/or nucleolar to

  14. États financiers 2015–2016

    International Development Research Centre (IDRC) Digital Library (Canada)

    une assurance raisonnable qu'il pourra réaliser ses objectifs. .... les employés et leur procure des services de santé avant ...... indépendance, à un audit des états financiers annuels, conformément aux normes d'audit généralement reconnues.

  15. Requirements for capsid-binding and an effector function in TRIMCyp-mediated restriction of HIV-1

    International Nuclear Information System (INIS)

    Diaz-Griffero, Felipe; Vandegraaff, Nick; Li Yuan; McGee-Estrada, Kathleen; Stremlau, Matthew; Welikala, Sohanya; Si Zhihai; Engelman, Alan; Sodroski, Joseph

    2006-01-01

    In owl monkeys, a retrotransposition event replaced the gene encoding the retroviral restriction factor TRIM5α with one encoding TRIMCyp, a fusion between the RING, B-box 2 and coiled-coil domains of TRIM5 and cyclophilin A. TRIMCyp restricts human immunodeficiency virus (HIV-1) infection by a mechanism dependent on the interaction of the cyclophilin A moiety and the HIV-1 capsid protein. Here, we show that infection by retroviruses other than HIV-1 can be restricted by TRIMCyp, providing an explanation for the evolutionary retention of the TRIMCyp gene in owl monkey lineages. The TRIMCyp-mediated block to HIV-1 infection occurs before the earliest step of reverse transcription. TRIMCyp-mediated restriction involves at least two functions: (1) capsid binding, which occurs most efficiently for trimeric TRIMCyp proteins that retain the coiled-coil and cyclophilin A domains, and (2) an effector function that depends upon the B-box 2 domain

  16. Selected HIV-1 Env trimeric formulations act as potent immunogens in a rabbit vaccination model

    DEFF Research Database (Denmark)

    Heyndrickx, Leo; Stewart-Jones, Guillaume; Jansson, Marianne Bendixen

    2013-01-01

    Ten to 30% of HIV-1 infected subjects develop broadly neutralizing antibodies (bNAbs) during chronic infection. We hypothesized that immunizing rabbits with viral envelope glycoproteins (Envs) from these patients may induce bNAbs, when formulated as a trimeric protein and in the presence of an ad...

  17. Selected HIV-1 Env Trimeric Formulations Act as Potent Immunogens in a Rabbit Vaccination Model

    NARCIS (Netherlands)

    Heyndrickx, Leo; Stewart-Jones, Guillaume; Jansson, Marianne; Schuitemaker, Hanneke; Bowles, Emma; Buonaguro, Luigi; Grevstad, Berit; Vinner, Lasse; Vereecken, Katleen; Parker, Joe; Ramaswamy, Meghna; Biswas, Priscilla; Vanham, Guido; Scarlatti, Gabriella; Fomsgaard, Anders

    2013-01-01

    Ten to 30% of HIV-1 infected subjects develop broadly neutralizing antibodies (bNAbs) during chronic infection. We hypothesized that immunizing rabbits with viral envelope glycoproteins (Envs) from these patients may induce bNAbs, when formulated as a trimeric protein and in the presence of an

  18. Presenting native-like HIV-1 envelope trimers on ferritin nanoparticles improves their immunogenicity

    NARCIS (Netherlands)

    Sliepen, Kwinten; Ozorowski, Gabriel; Burger, Judith A.; van Montfort, Thijs; Stunnenberg, Melissa; Labranche, Celia; Montefiori, David C.; Moore, John P.; Ward, Andrew B.; Sanders, Rogier W.

    2015-01-01

    Background: Presenting vaccine antigens in particulate form can improve their immunogenicity by enhancing B cell activation. Findings: We describe ferritin-based protein nanoparticles that display multiple copies of native-like HIV-1 envelope glycoprotein trimers (BG505 SOSIP.664). Trimer-bearing

  19. Inhibition of HIV-1 Integrase gene expression by 10-23 DNAzyme

    Indian Academy of Sciences (India)

    We have designed three novel DNAzymes, DIN54, DIN116, and DIN152, against HIV-1 Integrase gene using Mfold software and evaluated them for target site cleavage activity on the in vitro transcribed mRNA. All DNAzymes were tested for its inhibition of expression of HIV Integrase protein in the transiently transfected cell ...

  20. HIV-1 Nef control of cell signalling molecules: multiple strategies to ...

    Indian Academy of Sciences (India)

    Unknown

    The Nef protein of HIV-1 plays a fundamental role in the virus life cycle. ... to modulate the expression of key cellular receptors important for cell ... of the Src family kinases, leading to an effect on host cell function is likely to ..... Bad, Nef serves to balance the apoptosis inducing effects ..... ties of vpu, env, and nef; J. Virol.

  1. A Conserved Target Site in HIV-1 Gag RNA is Accessible to Inhibition by Both an HDV Ribozyme and a Short Hairpin RNA

    Directory of Open Access Journals (Sweden)

    Robert J Scarborough

    2014-01-01

    Full Text Available Antisense-based molecules targeting HIV-1 RNA have the potential to be used as part of gene or drug therapy to treat HIV-1 infection. In this study, HIV-1 RNA was screened to identify more conserved and accessible target sites for ribozymes based on the hepatitis delta virus motif. Using a quantitative screen for effects on HIV-1 production, we identified a ribozyme targeting a highly conserved site in the Gag coding sequence with improved inhibitory potential compared to our previously described candidates targeting the overlapping Tat/Rev coding sequence. We also demonstrate that this target site is highly accessible to short hairpin directed RNA interference, suggesting that it may be available for the binding of antisense RNAs with different modes of action. We provide evidence that this target site is structurally conserved in diverse viral strains and that it is sufficiently different from the human transcriptome to limit off-target effects from antisense therapies. We also show that the modified hepatitis delta virus ribozyme is more sensitive to a mismatch in its target site compared to the short hairpin RNA. Overall, our results validate the potential of a new target site in HIV-1 RNA to be used for the development of antisense therapies.

  2. Membrane topology analysis of HIV-1 envelope glycoprotein gp41

    Directory of Open Access Journals (Sweden)

    Xiao Dan

    2010-11-01

    Full Text Available Abstract Background The gp41 subunit of the HIV-1 envelope glycoprotein (Env has been widely regarded as a type I transmembrane protein with a single membrane-spanning domain (MSD. An alternative topology model suggested multiple MSDs. The major discrepancy between the two models is that the cytoplasmic Kennedy sequence in the single MSD model is assigned as the extracellular loop accessible to neutralizing antibodies in the other model. We examined the membrane topology of the gp41 subunit in both prokaryotic and mammalian systems. We attached topological markers to the C-termini of serially truncated gp41. In the prokaryotic system, we utilized a green fluorescent protein (GFP that is only active in the cytoplasm. The tag protein (HaloTag and a membrane-impermeable ligand specific to HaloTag was used in the mammalian system. Results In the absence of membrane fusion, both the prokaryotic and mammalian systems (293FT cells supported the single MSD model. In the presence of membrane fusion in mammalian cells (293CD4 cells, the data obtained seem to support the multiple MSD model. However, the region predicted to be a potential MSD is the highly hydrophilic Kennedy sequence and is least likely to become a MSD based on several algorithms. Further analysis revealed the induction of membrane permeability during membrane fusion, allowing the membrane-impermeable ligand and antibodies to cross the membrane. Therefore, we cannot completely rule out the possible artifacts. Addition of membrane fusion inhibitors or alterations of the MSD sequence decreased the induction of membrane permeability. Conclusions It is likely that a single MSD model for HIV-1 gp41 holds true even in the presence of membrane fusion. The degree of the augmentation of membrane permeability we observed was dependent on the membrane fusion and sequence of the MSD.

  3. Fragment Based Strategies for Discovery of Novel HIV-1 Reverse Transcriptase and Integrase Inhibitors.

    Science.gov (United States)

    Latham, Catherine F; La, Jennifer; Tinetti, Ricky N; Chalmers, David K; Tachedjian, Gilda

    2016-01-01

    Human immunodeficiency virus (HIV) remains a global health problem. While combined antiretroviral therapy has been successful in controlling the virus in patients, HIV can develop resistance to drugs used for treatment, rendering available drugs less effective and limiting treatment options. Initiatives to find novel drugs for HIV treatment are ongoing, although traditional drug design approaches often focus on known binding sites for inhibition of established drug targets like reverse transcriptase and integrase. These approaches tend towards generating more inhibitors in the same drug classes already used in the clinic. Lack of diversity in antiretroviral drug classes can result in limited treatment options, as cross-resistance can emerge to a whole drug class in patients treated with only one drug from that class. A fresh approach in the search for new HIV-1 drugs is fragment-based drug discovery (FBDD), a validated strategy for drug discovery based on using smaller libraries of low molecular weight molecules (FBDD is aimed at not only finding novel drug scaffolds, but also probing the target protein to find new, often allosteric, inhibitory binding sites. Several fragment-based strategies have been successful in identifying novel inhibitory sites or scaffolds for two proven drug targets for HIV-1, reverse transcriptase and integrase. While any FBDD-generated HIV-1 drugs have yet to enter the clinic, recent FBDD initiatives against these two well-characterised HIV-1 targets have reinvigorated antiretroviral drug discovery and the search for novel classes of HIV-1 drugs.

  4. Ex vivo analysis identifies effective HIV-1 latency–reversing drug combinations

    Science.gov (United States)

    Laird, Gregory M.; Bullen, C. Korin; Rosenbloom, Daniel I.S.; Martin, Alyssa R.; Hill, Alison L.; Durand, Christine M.; Siliciano, Janet D.; Siliciano, Robert F.

    2015-01-01

    Reversal of HIV-1 latency by small molecules is a potential cure strategy. This approach will likely require effective drug combinations to achieve high levels of latency reversal. Using resting CD4+ T cells (rCD4s) from infected individuals, we developed an experimental and theoretical framework to identify effective latency-reversing agent (LRA) combinations. Utilizing ex vivo assays for intracellular HIV-1 mRNA and virion production, we compared 2-drug combinations of leading candidate LRAs and identified multiple combinations that effectively reverse latency. We showed that protein kinase