Lymphocyte migration through the blood-brain barrier (BBB) in feline immunodeficiency virus infection is significantly influenced by the pre-existence of virus and tumour necrosis factor (TNF)-alpha within the central nervous system (CNS): studies using an in vitro feline BBB model.

AIMS: In human immunodeficiency virus infection, macrophage-tropic and lymphotropic viruses exist in the host. Central nervous system (CNS) infection is an early and ongoing event, important to understand when developing strategies to treat infection. Some knowledge exists on macrophage-tropic virus interactions with the blood-brain barrier (BBB), and the aim of this study was to investigate lymphotropic lentivirus interactions with the BBB. METHODS: Interactions of the lymphotropic feline immunodeficiency virus (FIV) with an in vitro model of the feline BBB were evaluated in scenarios to mimic in vivo infections. RESULTS: Cell-free FIV crossed the BBB in very low quantities, and in the presence of tumour necrosis factor (TNF)-alpha, BBB integrity was unaffected. However, cell-associated FIV readily crossed the BBB, but BBB integrity was not significantly altered. Transmigration of uninfected and infected lymphocytes increased in response to TNF-alpha, accompanied by a moderate disruption of barrier integrity and an upregulation of vascular cell adhesion molecule-1 rather than intercellular adhesion molecule-1. Significant enhancement of migration and disruption of BBB tight junctions occurred when infected cells and TNF-alpha were added to the brain side of the BBB and this enhancement was not mediated through additional TNF-alpha production. CONCLUSIONS: Small quantities of virus in the brain together with TNF-alpha have the potential to stimulate greater cell and viral entry into the CNS and this is likely to involve important factors other than further TNF-alpha production. Lymphotropic lentivirus entry to the CNS is governed by many factors similar to macrophage-tropic strains.

Human immunodeficiency virus type 1 Nef mediates activation of STAT3 in immature dendritic cells.

Replication of immunodeficiency viruses (HIV-1 and SIV) in immature dendritic cell (DC)-T cell cocultures is dependent on Nef. In contrast, mature DCs promote the replication of wild-type and nef-defective SIV in concert with CD4(+) T cells. Transcription factor activation occurs on DC maturation and this study aimed to investigate whether Nef triggers similar events in immature DCs, rendering them more like mature DCs. Recombinant HIV nef-expressing adenovirus was used to selectively introduce nef into immature human or macaque DCs. These data provide the first evidence that the expression of HIV nef in immature DCs induced selective activation of STAT3 and, to a lesser extent, NF-kappaB. This highlights how Nef can signal primary immature DCs, suggesting one way in which Nef may modulate immature DCs to drive virus replication in the DC-T cell milieu.

HIV-1 Nef promotes survival of myeloid cells by a Stat3-dependent pathway.

Human immunodeficiency virus Nef is a small myristylated protein that plays a critical role in AIDS progression. Nef binds with high affinity to the SH3 domain of the myeloid-restricted tyrosine kinase Hck in vitro, identifying this Src-related kinase as a possible cellular target for Nef in macrophages. Here we show that Nef activates endogenous Hck in the granulocyte-macrophage colony-stimulating factor-dependent myeloid cell line, TF-1. Unexpectedly, Nef induced cytokine-independent TF-1 cell outgrowth and constitutive activation of the Stat3 transcription factor. Induction of survival required the Nef SH3 binding and membrane-targeting motifs and was blocked by dominant-negative Stat3 mutants. Nef also stimulated Stat3 activation in primary human macrophages, providing evidence for Stat3 as a Nef effector in a target cell for human immunodeficiency virus.

Modulation of HIV-1 infectivity by MAPK, a virion-associated kinase.

Infection of a cell by human immunodeficiency virus type 1 (HIV-1) results in the formation of a reverse transcription complex in which viral nucleic acids are synthesized. Efficient disengagement of the reverse transcription complex from the cell membrane and subsequent nuclear translocation require phosphorylation of reverse transcription complex components by a virion-associated kinase. In this study, we identify the virion-associated kinase as mitogen-activated protein kinase (ERK/MAPK). Upon density gradient fractionation, MAPK, but not its activating kinase MEK, co-sedimented with viral particles. Expression of a constitutively active, but not kinase-inactive, MEK1 in virus producer cells was able to activate virion-associated MAPK in trans. Stimulation of virion-associated MAPK activity in trans by the mitogen phorbol myristate acetate (PMA) increased viral infectivity. Conversely, suppression of virion-associated MAPK by specific inhibitors of the MAPK cascade markedly impaired viral infectivity. These studies demonstrate regulation of an early step in HIV-1 infection by the host cell MAPK signal transduction pathway.

Permanent occupancy of the human immunodeficiency virus type 1 enhancer by NF-kappa B is needed for persistent viral replication in monocytes.

This work aimed to ascertain the role of kappaB-responsive elements of the human immunodeficiency virus type 1 (HIV-1) enhancer not only in early initiation but also in long-term maintenance of proviral transcription in cells of the monocytic lineage. For this purpose, we used three main approaches. The first was to abruptly terminate tumor necrosis factor-induced NF-kappaB binding to the enhancer sequences in U1 monocytic cells, using a short pulse of exogenous tumor necrosis factor. This resulted in concomitant decrease in nuclear NF-kappaB DNA-binding activity and endogenous long terminal repeat transcriptional activity. The second was to suppress the permanent NF-kappaB translocation induced by HIV-1 replication itself in chronically infected U937 cells, using a specific proteasome inhibitor (Z-LLL-H). As early as 2 h after addition of the inhibitor to the culture medium, there was an inhibition of both constitutive activation of NF-kappaB and HIV-1 genome expression. The third approach was to monitor the replication competence in U937 cells of an infectious HIV-1 provirus carrying point mutations in the kappaB-responsive elements of both long terminal repeats. Compared with its wild-type counterpart, this mutated provirus showed a profoundly decreased, Z-LLL-H-insensitive transcriptional and replicative activity in U937 monocytes. Together, our results indicate that occupancy of the viral enhancer by NF-kappaB (p50/p65) heterodimers is required for ongoing transcription of integrated HIV provirus in monocytes, even in cells chronically infected and permanently producing functional HIV Tat protein. Thus, the ability of HIV-1 replication to activate NF-kappaB is crucial to the intense self-perpetuated viral transcription observed in cells of the monocytic lineage.

Role of IkappaBalpha ubiquitination in signal-induced activation of NFkappaB in vivo.

In unstimulated cells, the transcription factor NF-kappaB is held in the cytoplasm in an inactive state by the inhibitor protein IkappaBalpha. Stimulation of cells results in rapid phosphorylation and degradation of IkappaBalpha, thus releasing NF-kappaB, which translocates to the nucleus and activates transcription of responsive genes. Here we demonstrate that in cells where proteasomal degradation is inhibited, signal induction by tumor necrosis factor alpha results in the rapid accumulation of higher molecular weight forms of IkappaBalpha that dissociate from NF-kappaB and are consistent with ubiquitin conjugation. Removal of the high molecular weight forms of IkappaBalpha by a recombinant ubiquitin carboxyl-terminal hydrolase and reactivity of the immunopurified material with a monoclonal antibody specific for ubiquitin indicated that IkappaBalpha was conjugated to multiple copies of ubiquitin. Western blot analysis of immunopurified IkappaBalpha from cells expressing epitope-tagged versions of IkappaBalpha and ubiquitin revealed the presence of multiple copies of covalently bound tagged ubiquitin. An S32A/S36A mutant of IkappaBalpha that is neither phosphorylated nor degraded in response to signal induction fails to undergo inducible ubiquitination in vivo. Thus signal-induced activation of NF-kappaB involves phosphorylation-dependent ubiquitination of IkappaBalpha, which targets the protein for rapid degradation by the proteasome and releases NF-kappaB for translocation to the nucleus.

HIV genome transcription induced by polyoma virus middle T antigen through both enhancer- and promoter-dependent LTR activation.

In order to understand the regulation of HIV genome transcription induced by cell stimulation through transmembrane receptors, we have transfected cells with polyoma middle T antigen (PyMT) expression vectors, thus mimicking activated receptor-dependent cell stimulation. PyMT-expressing Cos7 cells provided an environment where transcription of an HIV provirus was activated. PyMT expression induced the activity of both enhancer- and promoter-dependent HIV-LTR luciferase vectors. Induction of the HIV promoter domain depended on Sp1-binding sites and could be blocked by Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K). This indicates that PyMT-induced HIV transcription and replication are controlled by both the enhancer and promoter domains of the HIV-LTR. The latter, but not the former, was induced in a PI3K-dependent way. Thus at least 2 different transduction pathways appear to collaborate for induction of full HIV genome transcription in activated cells.

Absolute dependence on kappa B responsive elements for initiation and Tat-mediated amplification of HIV transcription in blood CD4 T lymphocytes.

The role of NF-kappa B-dependent signals in activating the transcriptional activity of the HIV regulatory region (LTR) was analyzed by systematic comparison of HIV LTR activity in human CD4 T cells purified from peripheral blood and a transformed lymphoblastoid T cell line. In normal CD4 T cells we also analyzed the role played by the viral kappa B responsive elements in HIV replication. Analysis of nuclear extracts of resting, normal T lymphocytes revealed the presence of the p50, but not the p65, NF-kappa B subunit and the induction by phorbol esters of bona fide (p50-p65) NF-kappa B complexes. In parallel, we observed clear enhancer-dependent HIV LTR transactivation comparable in intensity with that observed in lymphoblastoid cells. We show that unstimulated CD4 T lymphocytes offer a cellular environment of very low permissivity to HIV LTR functioning. This was in sharp contrast to the high spontaneous LTR activity observed in lymphoblastoid T cells, where LTR activity was essentially independent of kappa B-responsive elements. Due to the low basal LTR activity in resting T lymphocytes, NF-kappa B-dependent transactivation was a sine qua non event for induction of the HIV LTR. Surprisingly, even the function of HIV Tat in resting CD4 T lymphocytes was found to be absolutely dependent on LTR kappa B responsive elements. The relevance of these observations obtained in transient transfections was confirmed by the incapacity of blood CD4 T lymphocytes infected with an HIV infectious provirus carrying critical point mutations in the kappa B responsive elements to show any detectable transcriptional activity upon cell activation and prolonged culture in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of Sp1 phosphorylation and NF-kappa B-independent HIV promoter domain activity in T lymphocytes stimulated by okadaic acid.

In contrast to the purely enhancer-dependent effect of cytokines such as TNF on the activity of the HIV regulatory region (LTR), we observed that okadaic acid (OKA) activates HIV transcription through both the enhancer, responding to the factor NF-kappa B, and the promoter domain of the LTR. The inducibility of HIV LTR-driven luciferase expression constructs in lymphoblastoid cells stimulated by OKA depended on both functional Sp1 binding elements and the ability of the TATA box to bind the protein TBP. In both transformed and normal lymphocytes, OKA stimulation induced intense phosphorylation of the constitutively expressed Sp1 protein in the nucleus, a property of OKA not shared by TNF, phorbol ester, or PHA and interleukin 2. Responsiveness of LTR constructs deleted of kappa B elements to HIV Tat expression was increased upon OKA but not TNF stimulation. Our results suggest that SP1 phosphorylation induced by OKA, a selective inhibitor of the serine-threonine phosphatase PP2A, facilitates the formation of a transcription complex involving general transcription factors, HIV Tat, and Sp1 proteins. The formation of this complex would increase, independently of an in synergy with NF-kappa B, the low basal activity of the HIV LTR observed in normal T lymphocytes.

Phosphatidylcholine hydrolysis activates NF-kappa B and increases human immunodeficiency virus replication in human monocytes and T lymphocytes.

We have tested whether breakdown of phosphatidylcholine (PC) initiated by exogenous addition of a PC-specific phospholipase C (PC-PLC) from Bacillus cereus or by endogenous overexpression of PC-PLC induces functional activation of NF-kappa B and increases human immunodeficiency virus (HIV) enhancer activity. PC-PLC-activated hydrolysis of PC was found to induce bona fide p50/p65 NF-kappa B binding activity in three different cell lines of human or murine origin. No significant changes in the turnover of other cellular phospholipids were detected in PC-PLC-treated cells. Induction of NF-kappa B by PC-PLC did not depend on de novo synthesis of proteins or autocrine secretion of either tumor necrosis factor or interleukin 1. In human monocytic and lymphoblastoid T-cell lines, induction of NF-kappa B by PC-PLC resulted in clear induction of luciferase expression vectors placed under the control of synthetic kappa B enhancers or wild type, but not kappa B-mutated, HIV long terminal repeat constructs. HIV replication was increased by PC-PLC in chronically infected monocytes and T lymphocytes. NF-kappa B activation promoted by addition of exogenous PC-PLC correlated with an intense production of diacylglycerol. However, addition of a phosphatidylinositol-specific PLC from B. cereus also induced diacylglycerol but did not activate kappa B enhancer-directed vectors. PC-PLC-induced NF-kappa B activation could not be blocked by a specific inhibitor of phorbol ester-inducible protein kinases C. These results indicate that a cellular transduction pathway, dependent on specific PC breakdown, is functional in T lymphocytes and monocytes and may be used by various transmembrane receptors to activate HIV transcription through NF-kappa B-dependent induction of the HIV enhancer.