Macrophage Bridging Conduit Trafficking of HIV-1 Through the Endoplasmic Reticulum and Golgi Network

Bridging conduits (BC) are tubular protrusions that facilitate cytoplasm and membrane exchanges between tethered cells. We now report that the human immunodeficiency virus type I (HIV-1) exploits these conduits to accelerate its spread and to shield it from immune surveillance. Endosome transport through BC drives HIV-1 intercellular transfers. How this occurs was studied in human monocyte-derived macrophages using proteomic, biochemical, and imaging techniques. Endosome, endoplasmic reticulum (ER), Golgi markers, and HIV-1 proteins were identified by proteomic assays in isolated conduits. Both the ER and Golgi showed elongated and tubular morphologies that extended into the conduits of polarized macrophages. Env and Gag antigen and fluorescent HIV-1 tracking demonstrated that these viral constituents were sequestered into endocytic and ER-Golgi organelles. Sequestered infectious viral components targeted the Golgi and ER by retrograde transport from early and Rab9 late endosomes. Disruption of the ER-Golgi network impaired HIV-1 trafficking in the conduit endosomes. This study provides, for the first time, mechanisms for how BC Golgi and ER direct cell–cell viral transfer

Functional Proteomic Analysis for Regulatory T Cell Surveillance of the HIV-1-Infected Macrophage

Regulatory T cells (Treg) induce robust neuroprotection in murine models of neuroAIDS, in part, through eliciting anti-inflammatory responses for HIV-1-infected brain mononuclear phagocytes (MP; macrophage and microglia). Herein, using both murine and human primary cell cultures in proteomic and cell biologic tests, we report that Treg promotes such neuroprotection by an even broader range of mechanisms than previously seen including inhibition of virus release, killing infected MP, and inducing phenotypic cell switches. Changes in individual Treg-induced macrophage proteins were quantified by iTRAQ labeling followed by mass spectrometry identifications. Reduction in virus release paralleled the upregulation of interferon-stimulated gene 15, an ubiquitin-like protein involved in interferon-mediated antiviral immunity. Treg killed virus-infected macrophages through caspase-3 and granzyme and perforin pathways. Independently, Treg transformed virus-infected macrophages from an M1 to an M2 phenotype by down- and up- regulation of inducible nitric oxide synthase and arginase 1, respectively. Taken together, Treg affects a range of virus-infected MP functions. The observations made serve to challenge the dogma of solitary Treg immune suppressor functions and provides novel insights into how Treg affects adaptive immunosurveillance for control of end organ diseases, notably neurocognitive disorders associated with advanced viral infection

Pulsed Stable Isotope Labeling of Amino Acids in Cell Culture Uncovers the Dynamic Interactions between HIV-1 and the Monocyte-Derived Macrophage

Dynamic interactions between human immunodeficiency virus-1 (HIV-1) and the macrophage govern the tempo of viral dissemination and replication in its human host. HIV-1 affects macrophage phenotype, and the macrophage, in turn, can modulate the viral life cycle. While these processes are linked to host–cell function and survival, the precise intracellular pathways involved are incompletely understood. To elucidate such dynamic virus–cell events, we employed pulsed stable isotope labeling of amino acids in cell culture. Alterations in <i>de novo</i> protein synthesis of HIV-1 infected human monocyte-derived macrophages (MDM) were examined after 3, 5, and 7 days of viral infection. Synthesis rates of cellular metabolic, regulatory, and DNA packaging activities were decreased, whereas, those affecting antigen presentation (major histocompatibility complex I and II) and interferon-induced antiviral activities were increased. Interestingly, enrichment of proteins linked to chromatin assembly or disassembly, DNA packaging, and nucleosome assembly were identified that paralleled virus-induced cytopathology and replication. We conclude that HIV-1 regulates a range of host MDM proteins that affect its survival and abilities to contain infection

Pulsed Stable Isotope Labeling of Amino Acids in Cell Culture Uncovers the Dynamic Interactions between HIV-1 and the Monocyte-Derived Macrophage

Dynamic interactions between human immunodeficiency virus-1 (HIV-1) and the macrophage govern the tempo of viral dissemination and replication in its human host. HIV-1 affects macrophage phenotype, and the macrophage, in turn, can modulate the viral life cycle. While these processes are linked to host–cell function and survival, the precise intracellular pathways involved are incompletely understood. To elucidate such dynamic virus–cell events, we employed pulsed stable isotope labeling of amino acids in cell culture. Alterations in <i>de novo</i> protein synthesis of HIV-1 infected human monocyte-derived macrophages (MDM) were examined after 3, 5, and 7 days of viral infection. Synthesis rates of cellular metabolic, regulatory, and DNA packaging activities were decreased, whereas, those affecting antigen presentation (major histocompatibility complex I and II) and interferon-induced antiviral activities were increased. Interestingly, enrichment of proteins linked to chromatin assembly or disassembly, DNA packaging, and nucleosome assembly were identified that paralleled virus-induced cytopathology and replication. We conclude that HIV-1 regulates a range of host MDM proteins that affect its survival and abilities to contain infection

Pulsed Stable Isotope Labeling of Amino Acids in Cell Culture Uncovers the Dynamic Interactions between HIV-1 and the Monocyte-Derived Macrophage

Dynamic interactions between human immunodeficiency virus-1 (HIV-1) and the macrophage govern the tempo of viral dissemination and replication in its human host. HIV-1 affects macrophage phenotype, and the macrophage, in turn, can modulate the viral life cycle. While these processes are linked to host–cell function and survival, the precise intracellular pathways involved are incompletely understood. To elucidate such dynamic virus–cell events, we employed pulsed stable isotope labeling of amino acids in cell culture. Alterations in <i>de novo</i> protein synthesis of HIV-1 infected human monocyte-derived macrophages (MDM) were examined after 3, 5, and 7 days of viral infection. Synthesis rates of cellular metabolic, regulatory, and DNA packaging activities were decreased, whereas, those affecting antigen presentation (major histocompatibility complex I and II) and interferon-induced antiviral activities were increased. Interestingly, enrichment of proteins linked to chromatin assembly or disassembly, DNA packaging, and nucleosome assembly were identified that paralleled virus-induced cytopathology and replication. We conclude that HIV-1 regulates a range of host MDM proteins that affect its survival and abilities to contain infection

Pulsed Stable Isotope Labeling of Amino Acids in Cell Culture Uncovers the Dynamic Interactions between HIV-1 and the Monocyte-Derived Macrophage

Dynamic interactions between human immunodeficiency virus-1 (HIV-1) and the macrophage govern the tempo of viral dissemination and replication in its human host. HIV-1 affects macrophage phenotype, and the macrophage, in turn, can modulate the viral life cycle. While these processes are linked to host–cell function and survival, the precise intracellular pathways involved are incompletely understood. To elucidate such dynamic virus–cell events, we employed pulsed stable isotope labeling of amino acids in cell culture. Alterations in <i>de novo</i> protein synthesis of HIV-1 infected human monocyte-derived macrophages (MDM) were examined after 3, 5, and 7 days of viral infection. Synthesis rates of cellular metabolic, regulatory, and DNA packaging activities were decreased, whereas, those affecting antigen presentation (major histocompatibility complex I and II) and interferon-induced antiviral activities were increased. Interestingly, enrichment of proteins linked to chromatin assembly or disassembly, DNA packaging, and nucleosome assembly were identified that paralleled virus-induced cytopathology and replication. We conclude that HIV-1 regulates a range of host MDM proteins that affect its survival and abilities to contain infection

NaPB attenuates activation of NF-κB in mouse microglial cells.

<p>(A) BV-2 microglial cells preincubated with 0.5 mM NaPB for 6 h were stimulated with 1 µg/ml LPS. At different minute of stimulation, the level of phospho-IκBα was monitored by Western blot. B) Cells preincubated with different concentrations of NaPB for 6 h were stimulated with LPS for 1 h followed by monitoring the DNA-binding activity of NF-κB by EMSA. (C) Cells plated in 12-well plates were co-transfected with 0.25 µg of PBIIX-Luc (an NF-κB-dependent reporter construct) and 12.5 ng of pRL-TK. Twenty-four h after transfection, cells received different concentrations of NaPB. After 6 h of incubation, cells were stimulated with LPS for 4 h. Firefly (ff-Luc) and Renilla (r-Luc) luciferase activities were obtained by analyzing the total cell extract. Results are mean <u>+</u> S.D. of three different experiments. <i>^ap<0.001</i> vs control; <i>^bp<0.05</i> vs LPS; <i>^cp<0.001</i> vs LPS. D) Cells preincubated with 0.5 mM NaPB for 6 h were stimulated with LPS for 2 h followed by monitoring the recruitment of RelA p65 to the mouse iNOS promoter by ChIP assay. E) Cells were co-transfected with 0.25 µg of PBIIX-Luc and 12.5 ng of pRL-TK. Twenty-four hours after transfection, cells were incubated with NaPB in the presence or absence of HMG-CoA, mevalonate, FPP, GGPP, cholesterol, and coenzyme Q. After 6 h of incubation, cells were stimulated with LPS for 4 h followed by assay of firefly (ff-Luc) and Renilla (r-Luc) luciferase activities. Results are mean <u>+</u> S.D. of three different experiments. <i>^ap<0.001</i> vs control; <i>^bp<0.001</i> vs LPS; <i>^cp<0.001</i> vs LPS+NaPB.</p

Activation of small G proteins (p21^ras and p21^rac) and NF-κB in ventral midbrain of MPTP-intoxicated mice is NaPB-sensitive.

<p>A) Mice were treated with NaPB (200 mg/kg body wt/d) via gavage from 1 d prior to MPTP injection. Six h after the last injection of MPTP, activation of p21^ras and p21^rac was monitored in ventral midbrain tissues. Experiment was repeated three times each time using two animal in each group. B) Bands from three different mice were quantified and activation of p21^ras and p21^rac is shown as percent of control. C) Mice were treated with NaPB (200 mg/kg body wt/d) from 3 h after the last injection of MPTP. Twenty-four h after the last injection of MPTP, ventral midbrain sections were immunostained for p65 (low magnification). Midbrain sections of MPTP-intoxicated mice were also double-labeled for p65 and glial cell markers (GFAP for astrocytes and CD11b for microglia). Results represent three independent experiments. D) NF-κB p65 positive cells counted in four nigral sections (two images per slide) from each of four mice in an Olympus IX81 fluorescence microscope using the MicroSuite™ imaging software are mentioned as cells/mm². <i>^ap<0.0001</i> vs saline-control; <i>^bp<0.0001</i> vs MPTP.</p

NaPB protects dopaminergic neurons in MPTP-intoxicated mice.

<p>Mice receiving NaPB (200 mg/kg body wt/day) from 3 h after the last injection of MPTP were sacrificed 7 d after the last injection of MPTP followed by TH immunostaining of SNpc (A) and striatum (B), counting of TH-positive neurons in SNpc (C), quantification of TH-positive fibers in striatum (D), assay of neurotransmitters in striatum (E), and quantification of GSH in nigra (F). Data are means <u>+</u> SEM of eight mice per group. <i>^ap<0.0001</i> vs saline group; <i>^bp<0.0001</i> vs the MPTP group.</p

Dose-dependent inhibition of NO production by NaPB in mouse and human glial cells.

<p>Primary mouse microglia were treated with different concentrations of NaPB for 6 h followed by stimulation with LPS under serum-free condition. After 24 h of stimulation, concentrations of nitrite were measured in supernatants (A) and the level of iNOS protein was monitored in cells by Western blot (B). Results are mean <u>+</u> SD of three different experiments. <i>^ap<0.001</i> vs control; <i>^bp<0.05</i> vs LPS; <i>^cp<0.001</i> vs LPS. After 5 h of stimulation, the expression of iNOS mRNA was monitored by semi-quantitative RT-PCR (C). Cells preincubated with different concentration of trichostatin A (TSA) and sodium butyrate (NaBu) for 6 h were stimulated with LPS for 24 h under serum-free condition followed by monitoring the level of nitrite in supernatants (D). Results are mean <u>+</u> S.D. of three different experiments. <i>^ap<0.001</i> vs control; <i>^bp<0.05</i> vs LPS; <i>^c,dp<0.001</i> vs LPS. Primary human astroglia isolated from fetal brain tissues were treated with different concentrations of NaPB for 6 h followed by stimulation with IL-1β under serum-free condition. After 48 h of stimulation, concentrations of nitrite (E) were measured in supernatants. Human astroglia plated at 70–80% confluence in 12-well plates were cotransfected with 0.25 µg of phiNOS(7.2)Luc and 12.5 ng of pRL-TK using the Lipofectamine-Plus (Invitrogen). Twenty-four h after transfection, cells received NaPB. After 6 h of incubation, cells were stimulated with IL-1β (20 ng/ml) for 12 h. Firefly (ff-Luc) and Renilla (r-Luc) luciferase activities were obtained by analyzing the total cell extract (F). Data are mean <u>+</u> S.D. of three different experiments. <i>^ap</i><0.001 versus control; <i>^bp</i><0.05 versus IL-1β; <i>^cp</i><0.001 versus IL-1β.</p