Persistent human immunodeficiency virus type 1 infection in human fetal glial cells reactivated by T-cell factor(s) or by the cytokines tumor necrosis factor alpha and interleukin-1 beta.

Human immunodeficiency virus type 1 (HIV-1) infection of the brain has been associated with a severe dementing illness in children and adults. However, HIV-1 antigens are most frequently found in macrophages and microglial cells. To determine the extent of susceptibility of neuroglial cells to infection, the HIV-1 genome was introduced into cells cultured from human fetal brain tissue. Astroglial cells rapidly transcribed the viral genome producing high levels of p24 protein and infectious virions which peaked two to three days posttransfection. Thereafter HIV-1 genome expression progressively diminished and a persistent phase of infection developed during which neither virus nor viral proteins could be demonstrated by immunodetection methods. Cocultivation with CD4+ T cells at any time during the persistent infection resulted in resumption of p24 synthesis and virus multiplication. The release of persistence did not require direct cell-cell contact between the glial and T cells, since separation of the two cell types across a permeable membrane resulted in a delayed but similar resumption of p24 synthesis and virus multiplication. The persistently infected glial cells could also be stimulated to produce viral p24 protein if either tumor necrosis factor alpha or interleukin-1 beta was added to the medium without T cells present. These results suggest that astrocytes may serve as an undetected reservoir for HIV-1 and disseminate the virus to other susceptible cells in the brain upon triggering by some cellular or biochemical signal.     

Human Mast cell progenitors can be infected by macrophagetropic human immunodeficiency virus type 1 and retain virus with maturation in vitro

Mast cells are critical components of innate and adaptive immunity that differentiate in tissues in situ from circulating committed progenitor cells. We now demonstrate that human cord blood-derived mast cell progenitors are susceptible to infection with macrophagetropic (M-tropic) and dualtropic human immunodeficiency virus type 1 (HIV-1) isolates but not with T-cell-tropic (T-tropic) strains. Mast cell progenitors (c-kit(+) CD13(+) cells with chloroacetate esterase activity) were purified from 4-week-old cultures of cord blood mononuclear cells maintained in stem cell factor, interleukin-6 (IL-6), and IL-10 using a CD14 depletion column. These progenitors expressed CCR3, CCR5, and CXCR4, as well as low levels of CD4. When infected in vitro with viruses pseudotyped with different HIV and simian immunodeficiency virus envelope glycoproteins, only M-tropic and dualtropic, but not T-tropic, viruses were able to enter mast cell progenitors. Both the CCR5-specific monoclonal antibody 2D7 and TAK-779, a nonpeptide inhibitor of CCR5-mediated viral entry, blocked HIV-1 strain ADA infection by >80%. Cultures infected with replication-competent virus produced progressively increasing amounts of virus for 21 days as indicated by p24 antigen detection. Mast cell progenitors that were exposed to an M-tropic, green fluorescent protein-expressing HIV-1 strain exhibited fluorescence indicative of viral entry and replication on a single-cell level and retained virus production during differentiation. The trafficking of mast cell progenitors to multiple tissues, combined with the long life span of mature mast cells, suggests that they could provide a widespread and persistent HIV reservoir in AIDS.     

Cell-specific differences in activation of NF-kappa B regulatory elements of human immunodeficiency virus and beta interferon promoters by tumor necrosis factor.

Three aspects of the involvement of tumor necrosis factor in human immunodeficiency virus (HIV) pathogenesis were examined. Tumor necrosis factor alpha (TNF-alpha) mRNA production was analyzed by polymerase chain reaction amplification in monocytic U937 cells and in a chronically HIV infected U937 cell line (U9-IIIB). TNF-alpha RNA was undetectable in U937 cells, whereas a low constitutive level was detected in U9-IIIB cells. Paramyxovirus infection induced a 5- to 10-fold increase in the steady-state level of TNF-alpha RNA in U9-IIIB cells compared with U937 cells, suggesting that HIV-infected monocytic cells produced higher levels of TNF-alpha than did normal cells after a secondary virus infection. The effects of TNF-alpha on gene expression were examined by transient expression assays using reporter chloramphenicol acetyltransferase plasmids linked to regulatory elements from the HIV long terminal repeat (LTR) and the beta interferon promoter. In U937 and Jurkat T lymphoid cells, the inducibility of the different hybrid promoters by TNF-alpha or phorbol ester varied in a cell type- and promoter context-specific manner; the levels of gene activity of NF-kappa B-containing plasmids correlated directly with induction of NF-kappa B DNA-binding activity. Although the intact beta interferon promoter was only weakly stimulated by phorbol ester or TNF-alpha, multimers of the PRDII NF-kappa B-binding domain were inducible by both agents. TNF-alpha was able to increase expression of the HIV LTR in T cells, but in monocytic cells, TNF-alpha did not induce the HIV LTR above a constitutive level of activity. This level of NF-kappa B-independent activity appears to be sufficient for virus multiplication, since TNF-alpha treatment had no effect on the kinetics of de novo HIV type 1 (HIV-1) infection and viral RNA production in U937 cells. However, in Jurkat cells, TNF-alpha dramatically enhanced the spread of HIV-1 through the cell population and increased viral RNA synthesis, indicating that in T cells HIV-1 multiplication was stimulated by TNF-alpha treatment.     

Signaling through Toll-like receptors triggers HIV-1 replication in latently infected mast cells

Evidence that human progenitor mast cells are susceptible to infection with CCR5-tropic strains of HIV-1 and that circulating HIV-1-infected FcepsilonRIalpha(+) cells with a similar progenitor phenotype have been isolated from AIDS patients has led to speculation that mast cells may serve as a potential reservoir for infectious HIV-1. In this study, progenitor mast cells, developed in vitro from CD34(+) cord blood stem cells, were experimentally infected with the CCR5-tropic strain HIV-1Bal after 28 days in culture as they reached their HIV-1-susceptible progenitor stage. HIV-1 p24 Ag levels were readily detectable by day 7 postinfection (PI), peaked at 2-3 wk PI as mature (tryptase/chymase-positive) HIV-1 infection-resistant mast cells emerged, and then steadily declined to below detectable limits by 10 wk PI, at which point integrated HIV-1 proviral DNA was confirmed by PCR quantitation in ( approximately 34% of) latently infected mast cells. Stimulation by ligands for Toll-like receptor (TLR) 2, TLR4, or TLR9 significantly enhanced viral replication in a dose- and time-dependent manner in both HIV-1-infected progenitor and latently infected mature mast cells, without promoting degranulation, apoptosis, cellular proliferation, or dysregulation of TLR agonist-induced cytokine production in infected mast cells. Limiting dilution analysis of TLR activated, latently infected mature mast cells indicated that one in four was capable of establishing productive infections in A301 sentinel cells. Taken together, these results indicate that mast cells may serve both as a viral reservoir and as a model for studying mechanisms of postintegration latency in HIV infection.     

Diminished Production of Human Immunodeficiency Virus Type 1 in Astrocytes Results from Inefficient Translation of gag, env, and nef mRNAs despite Efficient Expression of Tat and Rev

Astrocytes infected with human immunodeficiency virus type 1 (HIV-1) produce only minimal quantities of virus. The molecular events that limit acute-phase HIV-1 infection of astrocytes were examined after inducing acute-phase replication by transfection with the pNL4-3 proviral plasmid. The levels of HIV-1 mRNA were similarly high in both astrocytes and HeLa cells, but astrocytes produced approximately 50-fold less supernatant p24 than HeLa cells. We found that diminished HIV-1 production in astrocytes resulted from inefficient translation of gag, env, and nef mRNAs that were efficiently transported to the cytoplasm. Tat- or Rev-dependent reporter constructs showed no defect in Tat or Rev function in astrocytes compared with HeLa cells. HIV-1 mRNAs were correctly spliced, but only Rev and Tat proteins were efficiently translated from their native mRNAs. Pulse-chase labelling and immunoblot experiments revealed no defect in protein processing, but levels of Gag, Env, or Nef protein expressed were dramatically reduced in astrocytes compared to HeLa cells. These results demonstrate that inefficient translation of HIV-1 structural proteins underlies the restricted infection of astrocytes. The efficient expression of functional Tat and Rev by astrocytes may contribute to HIV-1 neuropathogenesis.     

TNF-alpha opens a paracellular route for HIV-1 invasion across the blood-brain barrier.

BACKGROUND: HIV-1 invades the central nervous system early after infection when macrophage infiltration of the brain is low but myelin pallor is suggestive of blood-brain-barrier damage. High-level plasma viremia is a likely source of brain infection. To understand the invasion route, we investigated virus penetration across in vitro models with contrasting paracellular permeability subjected to TNF-alpha. MATERIALS AND METHODS: Blood-brain-barrier models constructed with human brain microvascular endothelial cells, fetal astrocytes, and collagen I or fibronectin matrix responded in a dose-related fashion to cytokines and ligands modulating paracellular permeability and cell migration. Virus penetration was measured by infectious and quantitative HIV-1 RNA assays. Barrier permeability was determined using inulin or dextran. RESULTS: Cell-free HIV-1 was retained by the blood-brain barrier with close to 100% efficiency. TNF-alpha increased virus penetration by a paracellular route in a dose-dependent manner proportionately to basal permeability. Brain endothelial cells were the main barrier to HIV-1. HIV-1 with monocytes attracted monocyte migration into the brain chamber. CONCLUSIONS: Early after the infection, the blood-brain barrier protects the brain from HIV-1. Immune mediators, such as TNF-alpha, open a paracellular route for the virus into the brain. The virus and viral proteins stimulate brain microglia and macrophages to attract monocytes into the brain. Infiltrating macrophages cause progression of HIV-1 encephalitis.     

Nef enhances human immunodeficiency virus replication and responsiveness to interleukin-2 in human lymphoid tissue ex vivo

The nef gene is important for the pathogenicity associated with simian immunodeficiency virus infection in rhesus monkeys and with human immunodeficiency virus type 1 (HIV-1) infection in humans. The mechanisms by which nef contributes to pathogenesis in vivo remain unclear. We investigated the contribution of nef to HIV-1 replication in human lymphoid tissue ex vivo by studying infection with parental HIV-1 strain NL4-3 and with a nef mutant (DeltanefNL4-3). In human tonsillar histocultures, NL4-3 replicated to higher levels than DeltanefNL4-3 did. Increased virus production with NL4-3 infection was associated with increased numbers of productively infected cells and greater loss of CD4(+) T cells over time. While the numbers of productively infected T cells were increased in the presence of nef, the levels of viral expression and production per infected T cell were similar whether the nef gene was present or not. Exogenous interleukin-2 (IL-2) increased HIV-1 production in NL4-3-infected tissue in a dose-dependent manner. In contrast, DeltanefNL4-3 production was enhanced only marginally by IL-2. Thus, Nef can facilitate HIV-1 replication in human lymphoid tissue ex vivo by increasing the numbers of productively infected cells and by increasing the responsiveness to IL-2 stimulation.     

Enigma of HIV-1 latent infection in astrocytes: an in-vitro study using protein kinase C agonist as a latency reversing agent

Purging HIV-1 to cure the infection in patients undergoing suppressive antiretroviral therapy requires targeting all possible viral reservoirs. Other than the memory CD4⁺ T cells, several other HIV-1 reservoirs have been identified. HIV-1 infection in the brain as a reservoir is well documented, but not fully characterized. There, microglia, perivascular macrophages, and astrocytes can be infected by HIV-1. HIV-1 infection in astrocytes has been described as a nonproductive and primarily a latent infection. Using primary human astrocytes, we investigated latent HIV-1 infection and tested phorbol 12-myristate 13-acetate (PMA), a protein kinase C agonist, as an HIV-1-latency- reversing agent in infected astrocytes. Chloroquine (CQ) was used to facilitate initial HIV-1 escape from endosomes in astrocytes. CQ significantly increased HIV-1 infection. But treatment with PMA or viral Tat protein was similar to untreated HIV-1-infected astrocytes. Long-term follow-up of VSV-envelope-pseudotyped HIV-1 infected astrocytes showed persistent infection for 110 days, indicating the active state of the virus.     

TNF-alpha inhibits HIV-1 replication in peripheral blood monocytes and alveolar macrophages by inducing the production of RANTES and decreasing C-C chemokine receptor 5 (CCR5) expression.

The pathogenesis of HIV-1 infection is influenced by the immunoregulatory responses of the host. Macrophages present in the lymphoid tissue are susceptible to infection with HIV-1, but are relatively resistant to its cytopathic effects and serve as a reservoir for the virus during the course of disease. Previous investigators have demonstrated that increased serum levels of TNF-alpha contribute to the clinical symptoms of AIDS and that TNF-alpha stimulates the production of HIV-1 in chronically infected lymphocytic and monocytic cell lines by increasing HIV-1 gene expression. Although previous studies have suggested that TNF-alpha may increase HIV-1 infection of primary human mononuclear cells, some recent studies have indicated that TNF-alpha suppresses HIV-1 infection of macrophages. We now demonstrate that TNF-alpha suppresses HIV-1 replication in freshly infected peripheral blood monocytes (PBM) and alveolar macrophages (AM) in a dose-dependent manner. As TNF-alpha has been shown to increase the production of C-C chemokine receptor (CCR5)-binding chemokines under certain circumstances, we hypothesized that TNF-alpha inhibits HIV-1 replication by increasing the expression of these HIV-suppressive factors. We now show that TNF-alpha treatment of PBM and AM increases the production of the C-C chemokine, RANTES. Immunodepletion of RANTES alone or in combination with macrophage inflammatory protein-1alpha and -1beta block the ability of TNF-alpha to suppress viral replication in PBM and AM. In addition, we found that TNF-alpha treatment reduces CCR5 expression on PBM and AM. These findings suggest that TNF-alpha plays a significant role in inhibiting monocytotropic strains of HIV-1 by two distinct, but complementary, mechanisms.     

Low TRBP levels support an innate human immunodeficiency virus type 1 resistance in astrocytes by enhancing the PKR antiviral response

Acute human immunodeficiency virus type 1 (HIV-1) replication in astrocytes produces minimal new virus particles due, in part, to inefficient translation of viral structural proteins despite high levels of cytoplasmic viral mRNA. We found that a highly reactive double-stranded (ds) RNA-binding protein kinase (PKR) response in astrocytes underlies this inefficient translation of HIV-1 mRNA. The dsRNA elements made during acute replication of HIV-1 in astrocytes triggers PKR activation and the specific inhibition of HIV-1 protein translation. The heightened PKR response results from relatively low levels of the cellular antagonist of PKR, the TAR RNA binding protein (TRBP). Efficient HIV-1 production was restored in astrocytes by inhibiting the innate PKR response to HIV-1 dsRNA with dominant negative PKR mutants, or PKR knockdown by siRNA gene silencing. Increasing the expression of TRBP in astrocytes restored acute virus production to levels comparable to those observed in permissive cells. Therefore, the robust innate PKR antiviral response in astrocytes results from relatively low levels of TRBP expression and contributes to their restricted infection. Our findings highlight TRBP as a novel cellular target for therapeutic interventions to block productive HIV-1 replication in cells that are fully permissive for HIV-1 infection.     

Replication of parvovirus B19 in hematopoietic progenitor cells generated in vitro from normal human peripheral blood.

Erythroid progenitor cells generated in vitro from peripheral human blood in the presence of interleukin-3 and erythropoietin were infected with human parvovirus B19. B19 virus DNA replication was highest 48 to 72 h after infection, and maximum levels of B19 virus proteins were detected in culture supernatants at 72 to 96 h after infection. B19 virus propagated in vitro was infectious. This cell culture system with peripheral blood cells facilitates studies in vitro of B19 virus replication.     

Quantitation of human immunodeficiency virus type 1 infection kinetics.

Tissue culture infections of CD4-positive human T cells by human immunodeficiency virus type 1 (HIV-1) proceed in three stages: (i) a period following the initiation of an infection during which no detectable virus is produced; (ii) a phase in which a sharp increase followed by a peak of released progeny virions can be measured; and (iii) a final period when virus production declines. In this study, we have derived equations describing the kinetics of HIV-1 accumulation in cell culture supernatants during multiple rounds of infection. Our analyses indicated that the critical parameter affecting the kinetics of HIV-1 infection is the infection rate constant k = Inn/ti, where n is the number of infectious virions produced by one cell (about 10(2)) and ti is the time required for one complete cycle of virus infection (typically 3 to 4 days). Of particular note was our finding that the infectivity of HIV-1 during cell-to-cell transmission is 10(2) to 10(3) times greater than the infectivity of cell-free virus stocks, the inocula commonly used to initiate tissue culture infections. We also demonstrated that the slow infection kinetics of an HIV-1 tat mutant is not due to a longer replication time but reflects the small number of infectious particles produced per cycle.     

Purified astrocytes promote the in vitro division of a bipotential glial progenitor cell.

Optic nerves of neonatal rats contain a bipotential glial progenitor cell which can be induced by tissue culture conditions to differentiate into either an oligodendrocyte (the myelin-forming cell of the CNS) or a type 2 astrocyte (an astrocyte population found only in the myelinated tracts of the CNS). In our previous studies most oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells differentiated within 3 days in vitro with relatively little division of the progenitors or their differentiated progeny. We have now found that the O-2A progenitors are stimulated to divide in culture by purified populations of type 1 astrocytes, another glial cell-type found in the rat optic nerve. This cell-cell interaction appears to be mediated by a soluble factor(s) and results in the production of large numbers of both progenitor cells and oligodendrocytes. As type 1 astrocytes are the major glial cell-type in the optic nerve when oligodendrocytes first begin to be produced in large numbers in vivo, our results suggest that this astrocyte subpopulation may play an important role in expanding the oligodendrocyte population during normal development.     

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside⁺ (GalC) and O4⁺ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.     

Replication of B19 parvovirus in highly enriched hematopoietic progenitor cells from normal human bone marrow.

The target cell specificity of the B19 parvovirus infection was examined by isolating highly enriched hematopoietic progenitor and stem cells from normal human bone marrow. The efficiency of the B19 parvovirus replication in enriched erythroid progenitor cells was approximately 100-fold greater than that in unseparated bone marrow cells. The more-primitive progenitor cells identical to or closely related to the human pluripotent hematopoietic stem cells, on the other hand, did not support viral replication. The B19 progeny virus produced by the enriched erythroid progenitor cells was infectious and strongly suppressed erythropoiesis in vitro. The susceptibility of both the more-primitive erythroid progenitors (burst-forming units-erythroid) and the more-mature erythroid progenitors (CFU-erythroid) to the cytolytic response of the virus and the lack of effect on the myeloid progenitors (CFU-granulocyte-macrophage) further give evidence to the remarkable tropism of the B19 parvovirus for human hematopoietic cells of erythroid lineage.