Ruth Brack-Werner

Astrocytes: HIV cellular reservoirs and important participants in neuropathogenesis.

IntroductionHIV can invade cells of the central nervous system (CNS) and cause progressive combined cognitive and motor impairment in infected individuals. The cellular basis and mechanisms underlying HIV-1 neuropathogenesis are complex and poorly understood. Astrocytes are the most abundant cell ty

Macrophages and their relevance in Human Immunodeficiency Virus Type I infection.

Macrophages are important target cells for the Human Immunodeficiency Virus Type I (HIV-1) in vivo. Several studies have assessed the molecular biology of the virus in this cell type, and a number of differences towards HIV-1 infection of CD4+ T cells have been described. There is a broad consensus that macrophages resist HIV-1 infection much better than CD4+ T cells. Among other reasons, this is due to the presence of the recently identified host cell restriction factor SamHD1, which is strongly expressed in cells of the myeloid lineage. Furthermore, macrophages produce and release relatively low amounts of infectious HIV-1 and are less sensitive to viral cytotoxicity in comparison to CD4+ T cells. Nevertheless, macrophages play a crucial role in the different phases of HIV-1 infection. In this review, we summarize and discuss the significance of macrophages for HIV-1 transmission, the acute and chronic phases of HIV-1 infection, the development of acquired immunodeficiency syndrome (AIDS) and HIV-associated diseases, including neurocognitive disorders. We propose that interaction of HIV-1 with macrophages is crucial during all stages of HIV-1 infection. Thus, long-term successful treatment of HIV-1 infected individuals requires potent strategies to prevent HIV-1 from entering and persisting in these cells.

Infection of human brain cells by HIV-1: Restricted virus production in chronically infected human glial cell lines

ObjectiveTo study expression of HIV-1 in human glial cell lines. DesignChronically HIV-1-infected glial cell lines were established to evade potential artefacts resulting from unphysiological viral entry (i.e., transfection). These cell lines were used to study viral expression and regulation. MethodsChronically infected glial cell lines were established by terminal dilution cloning of human glioma cells exposed to HIV-1. Virus production and expression were assayed by measuring reverse transcriptase activity, p24-antigen levels and syncytia-inducing capacity in C8166 target cells (extracellular), or by indirect immunoperoxidase staining, immunoblot analysis, and p24− and Nef-antigen-capture enzyme-linked immunosorbent assays (intracellular). HIV-long terminal repeat (LTR)-dependent expression of the chloramphenicol acetyltransferase reporter gene was determined in transient transfection assays. ResultsCulture supernatant from chronically HIV-1-infected glial cells contained only low levels of virus compared with chronically HIV-infected fibroblasts and T-lymphoma cells. Detailed study of HIV-antigen expression in representative glial cell line TH4–7-5 indicated the presence of all major structural proteins, albeit at low levels, and of Vif, Tat, Rev and Nef. Intracellular levels of Nef exceeded p24-antigen levels by approximately 10-fold. Virus was recovered from TH4–7-5 cells by cocultivation with blood-derived target cells, indicating that low-level virus production is not due to defective provirus. Prominent negative regulatory element (NRE)-mediated suppression of exogenous HIV-LTR activity was observed in TH4–7-5 cells and was unequalled by chronically HIV-producing fibroblast cells or by uninfected fibroblast and glial cells. ConclusionsOur results suggest that restricted virus production by chronically infected glial cells involves LTR-mediated regulation of virus expression.

Long-term HIV-1 infection of neural progenitor populations.

Background:HIV can reside in the brain for many years. While astrocytes are known to tolerate long-term HIV infection, the potential of other neural cell types to harbour HIV is unclear. Objective:To investigate whether HIV can persist in neural progenitor cell populations. Design:A multipotent human neural stem cell line (HNSC.100) was used to compare HIV infection in neural progenitor and astrocyte cell populations. Methods:Expression of cellular genes/proteins was analysed by real-time reverse transcriptase PCR, Western blot, immunocytochemistry and flow cytometry. Morphological properties of cells were measured by quantitative fluorescent image analysis. Virus release by cells exposed to HIV-1IIIB was monitored by enzyme-linked immunosorbent assay for Gag. Proviral copy numbers were determined by real-time PCR and early HIV transcripts by reverse transcriptase PCR. Rev activity was determined with a fluorescent-based reporter assay. Results:Progenitor populations differed from astrocyte populations by showing much lower glial fibrillary acidic protein (GFAP) production, higher cell-surface expression of the CXCR4 chemokine receptor, higher Rev activity and distinct cell morphologies. HIV-exposed progenitor cultures released moderate amounts of virus for over 2 months and continued to display cell-associated HIV markers (proviral DNA, early HIV transcripts) during the entire observation period (115 days). Differentiation of HIV-infected progenitor cells to astrocytes was associated with transient activation of virus production. Long-term HIV infection of progenitor populations led to upregulation of GFAP and changes in cell morphology. Conclusion:These studies suggest that neural progenitor populations can contribute to the reservoir for HIV in the brain and undergo changes as a consequence of HIV persistence.

A Conformationally Frozen Peptoid Boosts CXCR4 Affinity and Anti‐HIV Activity

The chemokine receptor subtype CXCR4 belongs to the G-protein coupled receptors (GPCRs) and is, together with itsnatural ligand CXCL12 (or SDF-1), a central part of thesignaling system in the human body. Its functions range fromstem-cell trafficking during embryogenesis, through cardio-vascular, hematopoietic, and brain development, to signalingin the nervous and immune system.

Relationship of Nef‐positive and GFAP‐reactive astrocytes to drug use in early and late HIV infection

Reactive astrocytosis is a well‐documented feature of HIV encephalitis (HIVE), but it is unclear whether restricted infection of astrocytes contributes to this phenomenon. In addition, the part played by reactive and/or infected astrocytes in AIDS‐related dementia is not fully understood. In this study of patients at different stages of the human immunodeficiency virus (HIV) infection, who had been treated at most with one antiretroviral drug, reactive astrocytes were identified by immunopositivity for glial fibrillary acidic protein (GFAP) and infected astrocytes by positivity for HIV Nef protein. Results were compared for drug‐using AIDS patients with (n = 9) and without (n = 7) HIVE, for presymptomatic HIV‐positive drug users (n = 12) and for control HIV‐negative subjects (n = 20), including a group who used drugs (n = 10). GFAP‐reactive astrocytes in both grey and white matter were significantly more numerous in HIVE subjects than in each of the other groups but did not correlate with viral load. Nef‐positive astrocytes were confined to HIVE cases and to white matter, but were numerous in only one subject who was treatment‐naive. Nef‐positive microglia were identified in all HIVE cases and in occasional AIDS and presymptomatic subjects who did not have HIVE. The results suggest that astrocytes may form an additional viral reservoir in late HIV infection and may contribute to HIVE. However, the number of GFAP‐positive astrocytes was neither increased in pre‐AIDS nor in drug abuse, in contrast with microglia which we have shown previously to be up‐regulated in both states.

Stable expression of HIV-1 Nef induces changes in growth properties and activation state of human astrocytes.

OBJECTIVE Nef was shown to be the predominant viral protein expressed in HIV-1-infected astrocytes in vivo and in vitro suggesting a distinct role of Nef in this cell type. Nef-induced activation of T cells is well described, whereas the functional activities of Nef in astrocytes are unknown. Our aim was to examine the effect of Nef on growth properties and activation of astrocytes. DESIGN Human Nef-expressing astrocytic cell lines were established by stable transfection with different wild-type and mutant nef genes derived from laboratory isolates and brain tissue. METHODS Nef-expressing astrocytes were characterized in terms of growth properties (proliferation, growth in soft agar, focus formation) and morphology. Apoptotic cell death and expression of activation markers were determined by fluorescent antibody cell sorting. RESULTS Astrocytic cell lines revealed persistent Nef expression--detectable at the levels of mRNA and protein--and showed altered growth properties and morphology. Elevated expression of activation markers such as glial fibrillary acidic protein and CD88 (complement receptor C5a) was observed; these are regarded as markers for inflammatory processes in the brain. This effect was independent of the nef type or the expression level of the Nef protein. In contrast with previous reports no evidence for increased apoptotic cell death was found in astrocytes expressing Nef stably. CONCLUSIONS Our findings suggest that Nef changes the cellular properties of astrocytes, thus contributing to astrocyte activation and induction of astrogliosis in the central nervous system of individuals with AIDS.

The root extract of the medicinal plant Pelargonium sidoides is a potent HIV-1 attachment inhibitor.

Global HIV-1 treatment would benefit greatly from safe herbal medicines with scientifically validated novel anti-HIV-1 activities. The root extract from the medicinal plant Pelargonium sidoides (PS) is licensed in Germany as the herbal medicine EPs®7630, with numerous clinical trials supporting its safety in humans. Here we provide evidence from multiple cell culture experiments that PS extract displays potent anti-HIV-1 activity. We show that PS extract protects peripheral blood mononuclear cells and macrophages from infection with various X4 and R5 tropic HIV-1 strains, including clinical isolates. Functional studies revealed that the extract from PS has a novel mode-of-action. It interferes directly with viral infectivity and blocks the attachment of HIV-1 particles to target cells, protecting them from virus entry. Analysis of the chemical footprint of anti-HIV activity indicates that HIV-1 inhibition is mediated by multiple polyphenolic compounds with low cytotoxicity and can be separated from other extract components with higher cytotoxicity. Based on our data and its excellent safety profile, we propose that PS extract represents a lead candidate for the development of a scientifically validated herbal medicine for anti-HIV-1 therapy with a mode-of-action different from and complementary to current single-molecule drugs.

Peptides derived from HIV-1 integrase that bind Rev stimulate viral genome integration.

Background The human immunodeficiency virus type 1 (HIV-1) integrase protein (IN), catalyzes the integration of viral DNA into the host cell genome. IN catalyzes the first step of the integration process, namely the 3′-end processing in which IN removes a pGT dinucleotide from the 3′ end of each viral long terminal repeat (LTR). Following nuclear import of the viral preintegration complex, the host chromosomal DNA becomes accessible to the viral cDNA and the second step of the integration process, namely the strand-transfer step takes place. This ordered sequence of events, centered on integration, is mandatory for HIV replication. Methodology/Principal Findings Using an integrase peptide library, we selected two peptides, designated INr-1 and INr-2, which interact with the Rev protein and probably mediate the Rev-integrase interaction. Using an in-vitro assay system, we show that INr-1 and INr-2 are able to abrogate the inhibitory effects exerted by Rev and Rev-derived peptides on integrase activity. Both INr-1 and INr-2 were found to be cell-permeable and nontoxic, allowing a study of their effect in HIV-1-infected cultured cells. Interestingly, both INr peptides stimulated virus infectivity as estimated by production of the viral P24 protein, as well as by determination of the appearance of newly formed virus particles. Furthermore, kinetics studies revealed that the cell-permeable INr peptides enhance the integration process, as was indeed confirmed by direct determination of viral DNA integration by real-time PCR. Conclusions/Significance The results of the present study raise the possibility that in HIV-infected cells, the Rev protein may be involved in the integration of proviral DNA by controlling/regulating the activity of the integrase. Release from such inhibition leads to stimulation of IN activity and multiple viral DNA integration events.

EASY-HIT: HIV Full-Replication Technology for Broad Discovery of Multiple Classes of HIV Inhibitors

ABSTRACT HIV replication assays are important tools for HIV drug discovery efforts. Here, we present a full HIV replication system (EASY-HIT) for the identification and analysis of HIV inhibitors. This technology is based on adherently growing HIV-susceptible cells, with a stable fluorescent reporter gene activated by HIV Tat and Rev. A fluorescence-based assay was designed that measures HIV infection by two parameters relating to the early and the late phases of HIV replication, respectively. Validation of the assay with a panel of nine reference inhibitors yielded effective inhibitory concentrations consistent with published data and allowed discrimination between inhibitors of early and late phases of HIV replication. Finer resolution of the effects of reference drugs on different steps of HIV replication was achieved in secondary time-of-addition assays. The EASY-HIT assay yielded high Z′ scores (>0.9) and signal stabilities, confirming its robustness. Screening of the LOPAC1280 library identified 10 compounds (0.8%), of which eight were known to inhibit HIV, validating the suitability of this assay for screening applications. Studies evaluating anti-HIV activities of natural products with the EASY-HIT technology led to the identification of three novel inhibitory compounds that apparently act at different steps of HIV-1 replication. Furthermore, we demonstrate successful evaluation of plant extracts for HIV-inhibitory activities, suggesting application of this technology for the surveillance of biological extracts with anti-HIV activities. We conclude that the EASY-HIT technology is a versatile tool for the discovery and characterization of HIV inhibitors.