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.

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.

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.

Human immunodeficiency virus type 1 Nef epitopes recognized in HLA-A2 transgenic mice in response to DNA and peptide immunization.

We investigated the immune response against a human immunodeficiency virus type 1 (HIV-1) nef DNA sequence administered epidermally in mice transgenic for the human major histocompatibility complex (MHC) class I molecule HLA-A201. Ten potential HLA-A2 binding 9-mer Nef peptides were identified by a computer-based search algorithm. By a cell surface MHC class I stabilization assay, four peptides were scored as good binders, whereas two peptides bound weakly to HLA-A2. After DNA immunization, cytotoxic T lymphocyte (CTL) responses were predominantly directed against the Nef 44-52, 81-89, and 85-93 peptides. Interestingly, the 44-52 epitope resides outside the regions of Nef where previously described CTL epitopes are clustered. Dominance among Nef-derived peptides did not strictly correlate with HLA-A2 binding, in that only one of the high-affinity binding peptides was targeted in the CTL response. The 44-52, 85-93, and 139-147 peptides also generated specific CTLs in response to peptide immunization. T helper cell proliferation was detected after stimulation with 20-mer peptides in vitro. Three Nef regions (16-35, 106-125, and 166-185) dominated the T helper cell proliferation. The implications of these results for the development of DNA-based vaccines against HIV is discussed.

Human immunodeficiency virus type-1 Nef protein induces blood-brain barrier disruption in the rat: role of matrix metalloproteinase-9.

We recently showed that MMP-9 activity was detectable in the cerebrospinal fluid (CSF) of about half of neurologically symptomatic HIV-infected patients. Using an experimental animal model, we detected MMP-9 activity in CSF samples from rats that had been injected intracisternally with recombinant HIV-1 Nef protein, but not after injection of heat-treated Nef, gp120, gp160 or PBS. Nef also induced a breaching of the blood-brain barrier (BBB), which could be inhibited by pretreatment with the matrix metalloproteinase (MMP) inhibitor batimastat. In vitro Nef only slightly induced MMP-9 activity in freshly isolated human peripheral blood mononuclear cells and in the murine macrophage cell line RAW 264.7, but not in endothelial, neuronal or astroglial cell lines. Taken together, our findings indicate that HIV-1 Nef protein can induce BBB disruption in the rat - presumably via MMP induction.

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.

Prenatal transmission and pathogenicity of endogenous ecotropic murine leukemia virus Akv.

OBJECTIVE: Mouse strains carrying endogenous ecotropic murine leukemia viruses (MuLV) are capable of expressing infective virus throughout life. Risk of transplacental transmission of MuLV raises concerns of embryo infection and induction of pathogenic effects, and postnatal MuLV infection may lead to tumorigenesis. METHODS: Endogenous ecotropic MuLV-negative SWR/J embryos were implanted into Akv-infected viremic SWR/J mice, into spontaneously provirus-expressing AKR/J mice, and into noninfected SWR/J control mice; virus integration and virus expression were investigated at 14 days' gestation. Tumor development was monitored over 18 months. RESULTS: Of 111 embryos, 20 (18%) recovered from Akv-infected SWR/J mice, which had developed normally, were infected. New proviruses were detected in 10 of 111 (9%) embryos from Akv-infected SWR/J mice, and in 2 of 60 (3%) embryos from AKR/J mice; none expressed viral protein. Of 127 embryos recovered from Akv-infected SWR/J mice, 16 (13%) were dead; 4 of 5 (80%) were infected and expressed viral protein. Of 71 embryos from AKR/J mice, 11 (15%) were dead, and 2 of 2 had virus integration; virus expression was not detected. Numbers of dead embryos recovered from experimentally infected, viremic SWR/J mice and from spontaneously endogenous MuLV-expressing AKR/J mice were significantly higher, compared with numbers from nonviremic SWR/J control mice, and embryo lethality was significantly associated with prenatal provirus expression. Postnatal inoculation of Akv induced lymphoblastic lymphomas in 15 of 24 (61%) SWR/J mice within mean +/- SD latency of 14 +/- 2.4 months. Only 3 of 39 (8%) control mice developed lymphomas (P < 0.005). CONCLUSION: Embryos in MuLV-viremic dams are readily infected, and inappropriate prenatal expression of leukemogenic endogenous retroviruses may play a critical role in embryo lethality and decreased breeding performance in ecotropic provirus-positive mouse strains.

HIV-1 Nef alters the expression of betaII and epsilon 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 betaII and epsilon 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.

HIV type 1 Nef protein is a viral factor for leukocyte recruitment into the central nervous system.

Recombinant HIV-1 Nef protein, but not Tat, gp120, and gp160, provoked leukocyte recruitment into the CNS in a rat model. The strong reduction of bioactivity by heat treatment of Nef, and the blocking effect of the mAb 2H12, which recognizes the carboxy-terminal amino acid (aa) residues 171-190 (but not of mAb 3E6, an anti-Nef Ab of the same isotype, which maps the aa sequence 168-175, as well as a mixture of mAbs to CD4) provided evidence for the specificity of the observed Nef effects. Using a modified Boyden chamber technique, Nef exhibited chemotactic activity on mononuclear cells in vitro. Coadministration of the anti-Nef mAb 2H12, as well as treatment of Nef by heat inhibited Nef-induced chemotaxis. Besides soluble Nef, chemotaxis was also induced by a Nef-expressing human astrocytoma cell line, but not by control cells. These data suggest a direct chemotactic activity of soluble Nef. The detection of elevated levels of IL-6, TNF-alpha, and IFN-gamma in rat cerebrospinal fluid 6 h after intracisternal Nef injection hint at the additional involvement of indirect mechanisms in Nef-induced leukocyte migration into rat CNS. These data propose a mechanism by which HIV-1 Nef protein may be essential for AIDS neuropathogenesis, as a mediator of the recruitment of leukocytes that may serve as vehicles of the virus and perpetrators for disease through their production of neurotoxins.

Recognition of prominent viral epitopes induced by immunization with human immunodeficiency virus type 1 regulatory genes.

Mice immunized with the regulatory genes nef, rev, and tat from human immunodeficiency virus type 1 developed both humoral and cellular immune responses to the gene products Nef, Rev, and Tat. This study demonstrates that it is feasible to induce immune reactions to all of these regulatory gene products. Humoral responses were seen after DNA boosts, while potent T-cell proliferative responses were noted already after a single immunization. A Th1-directed immune response was demonstrated early after immunization. A 3- to 75-fold-stronger T-cell response was seen in animals receiving DNA epidermally compared to that in animals receiving intramuscular injections. Nef, Rev, and Tat putative B- and T-cell epitopes were clearly mapped by using peptides derived from the regulatory proteins and were similar to those which are detected in human immunodeficiency virus infection. Although immunization by the Nef, Rev, and Tat proteins raised high immunoglobulin G titers in serum, the epitope spreading appeared broader after DNA immunization. The combination of all of these regulatory genes together with two genes for structural proteins, the envelope and gag genes, demonstrated that a combined approach is feasible in that reactivities to all antigens persisted or were even augmented. No interference between plasmids was noted.

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.

Cleavage of recombinant and cell derived human immunodeficiency virus 1 (HIV-1) Nef protein by HIV-1 protease.

Recombinant purified Nef protein of HIV-1, as well as Nef protein derived from extracts of permanently HIV-1 infected glioblastoma cells and monocytes, are specifically cleaved by the HIV-1 protease. Nef cleavage products in cellular extracts treated with protease showed identical molecular weights as those obtained by digestion of purified Nef with recombinant HIV-1 protease. Since cellular extracts were prepared by detergent and mechanical lysis it cannot be excluded that physiological cytoplasmic conditions were altered. The lack of Nef cleavage by endogenous HIV-1 protease in infected cells might be due to low concentrations of viral protease and the presence of Gag precursor molecules as natural substrate. Using a panel of monoclonal antibodies two cleavage fragments of 19 kDa and 8 kDa were defined. The cleavage site was located by microsequencing between amino acid 57 and 58 (AW*LEAQEEEEVGF). The conserved cleavage motif within HIV-1 Nef suggests a potential biological function of Nef processing.

Fine mapping of HIV-1 Nef-epitopes by monoclonal antibodies.

A panel of newly isolated murine monoclonal antibodies is described which are specific for the Nef protein of the human immunodeficiency virus type 1 (HIV-1). Epitope mapping using recombinant Nef-related proteins, synthetic peptides and lipopeptides showed 3 independent antigenic determinants located within the regions of amino acids 83-93, 175-190 and 86-166 of the Nef protein. None of the monoclonal antibodies reacted with recombinant Nef proteins of HIV-2.

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

OBJECTIVES: The 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. DESIGN: Immunochemical methods are an appropriate tool to investigate expression and localization of cellular proteins. METHODS: Nef 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. RESULTS: Reactivities 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. CONCLUSIONS: Chronically 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.

Target cells for HIV in the central nervous system: macrophages or glial cells?

Infection of foetal or embryonic brain cells and cell lines from human astrocytomas and gliomas with HIV1 derived from T-lymphoma cultures leads to the expression of HIV in about 1 to 2% of the cells in culture. Single-cell cloning of astrocytoma cells shortly after infection resulted in the establishment of persistently HIV1-infected cell lines. These cultures were characterized by low production of virus and moderate intra- and extracellular expression of structural proteins. However, high expression of the nef regulatory protein was found. The virus could be rescued by cocultivation with T cells and primary macrophages giving rise to typical syncytia formation. In contrast to infection with HIV-infected T-lymphoma lines, cocultivation with HIV1-infected primary macrophages or monocytic cell lines induced a reduction in the growth of astrocytes and failed to induce productive infection. These in vitro observations support the hypothesis that astrocytes and glial cells may be a reservoir for HIV in the central nervous system and that macrophages may not carry the virus to the brain, but rather may be infected in the brain after having penetrated the blood-brain barrier.

Patient-specific heterogeneity of antinuclear antibodies as revealed by an isoelectric focusing immunoblot system.

Ninety-nine sera from patients with different rheumatic diseases (systemic lupus erythematosus, rheumatoid arthritis, progressive systemic sclerosis, and mixed and unidifferentiated connective tissue disease) were applied to a newly developed isoelectric focusing (IEF) immunoblot system for the demonstration of antinuclear antibodies. Nucleoproteins were separated according to their isoelectric points (pI) and immobilized onto nitrocellulose, and binding of serum antibodies was determined by an alkaline phosphatase labelled second antibody. 89.8% of all sera positive in indirect immunofluorescence assays with Hep 2 as substrate showed positive reactivity in IEF immunoblot. Furthermore, 88% of patients' sera negative on Hep 2 cells gave a positive reaction in IEF immunoblot. The predominant antibody banding pattern observed showed parallel bands in the acidic as well as the neutral pH ranges. Antibody specificities found in the IEF immunoblot system turned out to be patient-specific, but no marker antibody for a discrete disease entity was obtained. Even when monoclonal antibodies or WHO standard sera were applied to nuclear antigen they exhibited heterogeneity in their binding pattern. Bands with the same pI were observed using sera from patients with different rheumatic disease entities. Immunodeletion experiments suggest the recognition of identical antigenic proteins by the different patients' sera.

Myasthenia gravis: stimulation of antireceptor autoantibodies by autoreactive T cell lines.

We studied autoreactive acetylcholine receptor (AChR)-specific T cell lines from two patients with myasthenia gravis. Anti-AChR autoantibody production by peripheral blood mononuclear cells (PBM) from the donors of the T cell lines was measured with an enzyme-linked immunoadsorbent assay, using purified human AChR as antigen. Freshly isolated PBM produced barely detectable amounts of anti-AChR autoantibodies. If, however, autologous AChR-specific T cells were added to the cultures, the production of anti-AChR autoantibodies, and of total IgM and IgG, was markedly stimulated, depending on the number of T line cells and on the amount of AChR present in the cultures. AChR-specific functional helper T-lymphocytes may have a role in the immunoregulation of myasthenia gravis.