Mono Mac 1: a new in vitro model system to study HIV-1 infection in human cells of the mononuclear phagocyte series.

Throughout the years, most researchers have used continuous cell lines as in vitro models to evaluate the immunopathogenesis of human immunodeficiency virus type-1 (HIV-1) infection. Unfortunately, the most commonly used monocytoid malignant cells have not been shown to adequately mimic primary human monocyte-derived macrophages, at least with respect to HIV-1 infection. The Mono Mac 1 cell line has been defined as a model system for studying biochemical, immunological, and genetic functions of human cells of the monocyte/macrophage lineage. In this study, we have investigated whether Mono Mac 1 represents an in vitro culture system for HIV-1 infection. Flow cytometric analyses revealed that Mono Mac 1 are positive for the HIV-1 primary receptor (CD4), as well as for the coreceptors (CXCR4, CCR5, and CCR3). Infectivity experiments conducted with recombinant luciferase-encoding and fully infectious viruses demonstrated that Mono Mac 1 can support a highly productive infection with both macrophage- and dual-tropic isolates of HIV-1. Furthermore, differentiation of such cells led to a marked increase in virus production. Data from semiquantitative polymerase chain reaction analysis and mobility shift assays indicated that enhanced virus production in differentiated Mono Mac 1 cells was most likely related to an increase in nuclear translocation of NF-kappaB. Mono Mac 1 can thus be considered as a human monocytoid cell line representing a proper in vitro system for studying the complex interactions between HIV-1 and cells of the monocyte/macrophage lineage.

HIV type 1 nef gene inhibits tumor necrosis factor alpha-induced apoptosis and promotes cell proliferation through the action of MAPK and JNK in human glial cells.

In neurodegenerative diseases associated with AIDS, reactive astrocytosis plays a central role in the neurotoxicity of the brain parenchyma. Whereas the HIV-1 nef gene is overexpressed during restricted HIV-1 infection of human astrocytes, our previous results have demonstrated that nef expressed in human U251MG glial cells activates the sphingomyelin pathway triggered by TNF-alpha, increasing ceramide production. Since ceramide is an important regulatory molecule of programmed cell death induced by TNF-alpha, we examined whether nef could alter TNF-alpha-induced apoptosis in the U251MG human astrocytoma cell line. Transfection studies indicated that nef could both prevent apoptosis and promote cell proliferation in response to TNF-alpha stimulation. MAPK and JNK activities were further analyzed in order to elucidate signaling cascades subsequent to the upregulation of ceramide production. After TNF-alpha treatment, both kinases were shown to be preferentially activated in the presence of nef. These experiments strongly suggest that the HIV-1 Nef protein might modulate the sensitivity of astrocytes to inflammatory molecules, thus contributing to the development of neurodegenerative diseases associated with AIDS.

Negative regulation of the NFAT1 factor by CD45: implication in HIV-1 long terminal repeat activation.

HIV-1 gene regulation is greatly dependent on the presence of the -104/-81 enhancer region which is regulated by both NF-kappaB and NFAT transcription factors. We have found that a greater induction in HIV-1 long terminal repeat-driven gene expression was observed upon PMA/ionomycin (Iono) stimulation of a CD45-deficient cell line (J45.01) in comparison to the parental Jurkat cells. Unlike NF-kappaB which was not affected by the absence of CD45, NFAT showed a much greater augmentation in nuclear translocation and transcriptional activity in J45.01 cells upon PMA/Iono stimulation. PMA/Iono-induced NFAT activation, NFAT translocation and calcium influx peaked at similar time points for both Jurkat and J45.01 cell lines. The NFAT-dependent promoters from the IL-2 and TNF-alpha genes were also more potently activated by PMA/Iono in J45.01 cells. Interestingly, higher levels of intracellular calcium were consistently demonstrated in PMA/Iono-induced CD45-deficient cell lines (J45.01 and HPB45.0). Furthermore, PMA/Iono induction of calcium mobilization in both Jurkat and J45.01 cell lines was observed to be EGTA-sensitive. Mechanistic studies revealed that CD3zeta and ZAP-70 were more heavily tyrosine phosphorylated in J45.01 cells than Jurkat cells. Analysis of the HIV-1 enhancer by EMSAs demonstrated that the bound NFAT complex was present at higher levels in J45.01 nuclear extracts and that the NFAT1 member was predominant. In conclusion, our results indicate that NFAT activation by stimuli acting in a more distal fashion from the TCR-mediated signaling pathway can be down-regulated by CD45 and that this CD45-dependent regulation in turn affects HIV-1 long terminal repeat activation.

Regulation of nuclear factor of activated T cells by phosphotyrosyl-specific phosphatase activity: a positive effect on HIV-1 long terminal repeat-driven transcription and a possible implication of SHP-1.

Although protein tyrosine phosphatase (PTP) inhibitors used in combination with other stimuli can induce interleukin 2 (IL-2) production in T cells, a direct implication of nuclear factor of activated T cells (NFAT) has not yet been demonstrated. This study reports that exposure of leukemic T cells and human peripheral blood mononuclear cells to bis-peroxovanadium (bpV) PTP inhibitors markedly induce activation and nuclear translocation of NFAT. NFAT activation by bpV was inhibited by the immunosuppressive drugs FK506 and cyclosporin A, as well as by a specific peptide inhibitor of NFAT activation. Mobility shift assays showed specific induction of the NFAT1 member by bpV molecules. The bpV-mediated NFAT activation was observed to be important for the up-regulation of the human immunodeficiency virus 1 (HIV-1) long terminal repeat (LTR) and the IL-2 promoter; NFAT1 was demonstrated to be particularly important in bpV-dependent positive action on HIV-1 LTR transcription. The active participation of p56(lck), ZAP-70, p21(ras), and calcium in the bpV-mediated signaling cascade leading to NFAT activation was confirmed, using deficient cell lines and dominant-negative mutants. Finally, overexpression of wild-type SHP-1 resulted in a greatly diminished activation of NFAT by bpV, suggesting an involvement of SHP-1 in the regulation of NFAT activation. These data were confirmed by constitutive NFAT translocation observed in Jurkat cells stably expressing a dominant-negative version of SHP-1. The study proposes that PTP activity attenuates constitutive kinase activities that otherwise would lead to constant NFAT activation and that this activation is participating in HIV-1 LTR stimulation by PTP inhibition.