Birgit Kohleisen

Cellular localization of Nef expressed in persistently HIV-1-infected low-producer astrocytes

ObjectivesThe characterization and localization of HIV-1 Nef highly expressed in permanently infected astrocytes (TH4–7-5) as a model for latent infection of human brain cells. DesignImmunochemical methods are an appropriate tool to investigate expression and localization of cellular proteins. MethodsNef expression was analysed by Western blot and immunoperoxidase staining using a panel of monoclonal and polyclonal antibodies. Cellular localization studies were performed by indirect immunofluorescence and subcellular fractionation of TH4–7-5 cells. Myristoylation of Nef was investigated by immunoprecipitation of [3H]myristic acid-labelled cell extract. TH4–7-5 nef gene was cloned and amplified by polymerase chain reaction and the nef nucleotide sequence analysed. ResultsReactivities of various Nef-specific antibodies with Nef antigen in TH4–7-5 cells were demonstrated by Western blot analysis. Immunofluorescence revealed cytoplasmic perinuclear staining of Nef with most antibodies. However, one monoclonal antibody against amino acids 168–175 of Nef showed intense homogeneous nuclear staining in TH4–7-5 cells. Reactivity of this Nef antibody was blocked with recombinant Nef derived from TH4–7-5 cells. After subcellular fractionation, Nef was detected in nuclear, membrane and cytosolic fractions of TH4–7-5 cells. No myristoylated Nef antigen was detectable, perhaps because of a serine residue at position 2 of the TH4–7-5 nef gene instead of the glycine residue required for myristoylation. ConclusionsChronically HIV-1-infected astrocytoma cells with restricted virus production express different antigenic forms of Nef, which can be distinguished by their subcellular localization. Variant subcellular targeting of Nef suggests the existence of multiple activities of Nef within HIV-infected cells.

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.

DNA vaccination in mice using HIV-1 nef, rev and tat genes in self-replicating pBN-vector.

The immunogenicity of a self-replicating DNA-vector containing HIV-1 nef gene (pBN-Nef) was characterized using various DNA delivery methods. In addition, gene gun immunisation was used for assessing immunogenicity of two other HIV-1 genes (rev and tat) given in the same vector. The pBN-Nef was the most immunogenic raising both humoral and cell-mediated immune responses in mice; these responses lasted for up to six months. The pBN-Nef vector was immunogenic also when given intramuscularly or intradermally. The pBN-Rev construct did not elicit humoral responses but did elicit proliferative as well as CTL-response against the corresponding protein. The pBN-Tat was a poor immunogen in all respects. The antibodies elicited with various DNA delivery methods belonged to different antibody subclasses; however, two main epitopes in Nef were frequently recognized by all of them.

HIV Type 1 Nef Promotes Neoplastic Transformation of Immortalized Neural Cells

To study the effects of HIV-1 Nef on CNS-derived cells in vivo, an expression system based on the murine neural stem cell line C17.2 was established. Stable expression of LAV-1(Bru)-nef in these cells induced a transformed phenotype and enhanced cell growth in soft agar. Further experiments using previously established nef-expressing human astrocytoma cell lines as well as nef-expressing murine fibroblasts suggested a brain cell-specific transforming activity of Nef. After implantation into syngeneic or nude mice both murine and human nef-expressing CNS-derived cells induced tumor development. Interestingly, human astrocytoma cells expressing a Nef mutant carrying a disrupted SH3-binding motif involved in protein-protein interactions failed to induce tumor formation. These in vivo data suggest that Nef promotes neoplastic transformation of immortalized murine neural stem cells and enhances malignancy of low-tumorigenic human astrocytoma cells. Nef may therefore be involved in the development of AIDS-associated brain tumors.

Heparin-binding capacity of the HIV-1 NEF-protein allows one-step purification and biochemical characterization.

Recombinant Nef-protein of HIV-1 Bru derived from Escherichia coli revealed heparin-binding activity. This property was used to purify the Nef-protein by a one-step procedure, yielding about 90% homogenous Nef-protein as evaluated by silver staining. The Nef-protein was soluble without denaturing agents. Native folding of Nef was demonstrated with antibodies against conformational epitopes of Nef by a slot blot assay under native conditions. Despite its affinity to heparin and its nuclear localization in persistently HIV-1 infected glioblastoma cells (Kohleisen et al., 1992), Nef did not show DNA-binding properties by slot blot/hybridization assay and South/Western blot. In nucleotide-binding assays a strong autophosphorylation activity with [gamma-32P]ATP was observed. Nef-protein was not a substrate for ADP-ribosylation by bacterial toxins arguing against G-protein-like activities of Nef. Recombinant Nef did not interact with membranes as shown by the lack of increased fluorescence emission of Nef in the presence of liposomes. The recombinant Nef-protein obtained by one-step heparin-based purification shares immunological properties with native Nef and should prove useful for further studies of Nef function and immunogenicity.

HIV-1 Nef alters the expression of ?II and ? isoforms of protein kinase c and the activation of the long terminal repeat promoter in human astrocytoma cells

In the human immunodeficiency virus type 1 (HIV‐1)‐infected brain, the virus does not replicate in astrocytes, but a synthesis of viral regulatory proteins occurs in these cells, leading to accumulation of Nef. As an approach to understand the effects of Nef on astrocyte functional activity, we analyzed whether intracellular Nef interferes with the expression and activation of the enzyme protein kinase C (PKC), which is an important regulator of astroglial functions and HIV‐1 replication. Astrocytoma clones (U251 MG) not expressing Nef (Neo), or expressing wild‐type Nef (Bru) or nonmyristoylated Nef (TH) were used to monitor the expression and activation of 10 PKC isoforms. The same clones were used to evaluate the effect of Nef on the viral long terminal repeat (LTR) promoter after activation of PKC with the phorbol ester 12‐myristate 13‐acetate (PMA). PKC intracellular distribution and activation were evaluated by Western blot analysis of cytosolic and membrane fractions of control and Nef‐expressing clones. PMA‐induced LTR activation was analyzed in clones transfected with a plasmid encoding for the CAT reporter gene controlled by the LTR promoter, by using an enzyme‐linked immunosorbent assay to measure CAT expression. Nef selectively downregulated the expression and activation of βII and ϵ PKC isoforms in astrocytoma cells. Such downregulation correlated with an inhibition of LTR activation after PMA stimulation. The myristoylation of Nef and its membrane localization were essential for these effects. These results suggest that Nef may alter astrocytic functions by interfering with PKC expression and activation and contribute to the restriction of HIV‐1 replication in astrocytes. GLIA 27:143–151, 1999.

HIV-1 Nef co-localizes with the astrocyte-specific cytoskeleton protein GFAP in persistently nef-expressing human astrocytes.

In T-cells HIV-1 Nef exerts various functions and interacts with actin. In astrocytes interaction of Nef with cellular proteins is poorly understood. Therefore, human astrocytic cell clones stably transfected with nef-genes derived from HIV-1 Bru and its myristoylation-defective TH-variant were investigated by confocal laser scanning microscopy for expression of Nef and cytoskeleton proteins actin and GFAP, a marker for activated astrocytes. Myristoylated Nef was detected in cytoplasm, Golgi and plasmamembrane, while non-myristoylated Nef was exclusively cytoplasmic. Nef co-localised with GFAP in the perinuclear region of astrocytes. In contrast, Nef did not interact with actin filaments in human astrocytes. Nef/GFAP interaction could contribute to changes in morphology and activation state of astrocytes shown previously which are both critical for development of astrogliosis in HIV-1 infected brain.

Molecular Interaction of HIV-1 in Glioma Cells

Analysis of brain specimens of HIV-1-infected individuals by immuno-chemistry as well as in situ hybridization indicate that the major cell types expressing HIV-1 antigens and nucleic acids in the central nervous system (CNS) are of macrophage origin and include microglia, macrophages and derivative multinucleated cells.1,2 However, HIV-1 expression has been described in other cell types, including capillary endothelial cells, astrocytes, and oligodendrocytes.3–7 Productive HIV-1 infection in brains of patients with AIDS-encephalitis is in many cases not abundant and often not in keeping with the severity of the disease.8 On the other hand, high levels of HIV-1 DNA have been detected in brain tissue by Southern blot analysis9,10 as well as the polymerase chain reaction (PCR) method.11–13 Quantitative estimates of viral DNA in HIV-encephalitis autopsy samples indicate DNA levels comparable to or exceeding those in lymphoid tissues.10,12 This suggests the presence of a latently infected virus reservoir in the brain. An indication that HIV-1 can directly infect cells of the nervous system came from in vitro infection studies of cultured human astrocytoma and neuroblastoma cells with HIV-1.14–19 In addition, primary human brain cells expressing glial fibrillary acidic protein (GFAP) have also been found to be infected with HIV-1 in vitro. 20–22 In vitro infection of human glioma cells with HIV 1 differs markedly from infection of susceptible T-cell lines or peripheral blood mononuclear cells (PBMCs) by: (a) absence of virusinduced cytopathic effects in target cells; (b) lack of requirement for the presence of the CD4 cell surface receptor; and (c) the prevalence of a nonproductive infection phenotype.