L-Glutamate is the major excitatory neurotransmitter in the brain. Astrocytes maintain low levels of synaptic glutamate by high-affinity uptake and defects in this function may lead to neuronal cell death by excitotoxicity. We tested the effects of HIV-1 and its envelope glycoprotein gp120 upon glutamate uptake and expression of glutamate transporters EAAT1 and EAAT2 in fetal human astrocytes in vitro. Astrocytes isolated from fetal tissues between 16 and 19 weeks of gestation expressed EAAT1 and EAAT2 RNA and proteins as detected by Northern blot analysis and immunoblotting, respectively, and the cells were capable of specific glutamate uptake. Exposure of astrocytes to HIV-1 or gp120 significantly impaired glutamate uptake by the cells, with maximum inhibition within 6 h, followed by gradual decline during 3 days of observation. HIV-1-infected cells showed a 59% reduction in V(max) for glutamate transport, indicating a reduction in the number of active transporter sites on the cell surface. Impaired glutamate transport after HIV-1 infection or gp120 exposure correlated with a 40-70% decline in steady-state levels of EAAT2 RNA and protein. EAAT1 RNA and protein levels were less affected. Treatment of astrocytes with tumor necrosis factor-alpha (TNF-alpha) decreased the expression of both EAAT1 and EAAT2, but neither HIV-1 nor gp120 were found to induce TNF-alpha production by astrocytes. These findings demonstrate that HIV-1 and gp120 induce transcriptional downmodulation of the EAAT2 transporter gene in human astrocytes and coordinately attenuate glutamate transport by the cells. Reduction of the ability of HIV-1-infected astrocytes to take up glutamate may contribute to the development of neurological disease.
Neurodegeneration and human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) are the major disease manifestations of HIV-1 colonization of the central nervous system (CNS). In the brain, HIV-1 replicates in microglial cells and infiltrating macrophages and it persists in a low-productive, noncytolytic state in astrocytes. Astrocytes play critical roles in the maintenance of the brain microenvironment, responses to injury, and in neuronal signal transmission, and disruption of these functions by HIV-1 could contribute to HAD. To better understand the potential effects of HIV-1 on astrocyte biology, the authors investigated changes in gene expression using an efficient and sensitive rapid subtraction hybridization approach, RaSH. Primary human astrocytes were isolated from abortus brain tissue, low-passage cells were infected with HIV-1 or mock infected, and total cellular RNAs were isolated at multiple time points over a period of 1 week. This approach is designed to identify gene products modulated early and late after HIV-1 infection and limits the cloning of genes displaying normal cell-cycle fluctuations in astrocytes. By subtracting temporal cDNAs derived from HIV-1-infected astrocytes from temporal cDNAs made from uninfected cells, 10 genes displaying reduced expression in infected cells, termed astrocyte suppressed genes (ASGs), were identified and their suppression was confirmed by Northern blot hybridization. Both known and novel ASGs, not reported in current DNA databases, that are down-regulated by HIV-1 infection are described. Northern blotting confirms suppression of the same panel of ASGs by treatment of astrocytes with recombinant HIV-1 envelope glycoprotein, gp120. These results extend our previous analysis of astrocyte genes induced or enhanced by HIV-1 infection and together they suggest that HIV-1 and viral proteins have profound effects on astrocyte physiology, which may influence their function in the CNS.
The auxiliary protein Vif is essential for productive HIV-1 infection of primary lymphocytes and macrophages. Vif is required for the synthesis of infectious progeny virus and infection of peripheral blood lymphocytes (PBLs) by Vif-negative HIV-1 was thought to be confined to a single cycle. Here we define conditions for the maintenance of Vif-negative HIV-1 in PBLs during multiple rounds of viral infection. PBLs were infected with Vif-negative HIV-1 and then were serially cocultivated with uninfected PBLs. As determined by measurement of viral DNA, viral burdens declined but then rebounded and reached 1 copy per 30 cells after 7 weeks of culture. Viral core antigen p24 levels dropped and remained below detection limits after three cocultivations with no observed cytotoxicity. Viral RNA was also undetectable in cocultivated cells. The incapacitating deletion in vif was maintained during cocultivation as shown by the size of the vif amplicon. The presence of viral DNA in the absence of viral p24 RNA or protein suggested that the cells were capable of control of HIV-1 expression. This regulatory capacity was confirmed by the demonstration of resistance of PBLs or isolated CD4-positive cells to expression of exogenous wild-type R5 or X4 HIV-1. Resistant PBLs were susceptible to fusion with HIV-1 envelope-expressing cells and to reverse transcription of incoming viral DNA, indicating that the block to replication of exogenous virus was imposed after viral entry and DNA synthesis. Using a dual-chamber apparatus, we demonstrated that resistant Vif-negative HIV-1-infected PBLs secrete soluble factors that confer resistance on naive cells. These findings indicate that Vif-negative HIV-1 infection of primary CD4-positive lymphocytes results in maintenance of unexpressed virus and induces the production of soluble factors conferring resistance to wild-type HIV-1 replication on uninfected cells.
We analyzed sequence variability and function of the long terminal repeat (LTR) from syncytium-inducing (SI) and non-syncytium-inducing (NSI) HIV-1. Twenty LTR DNA clones were obtained by polymerase chain reaction amplification and molecular cloning from short-term cultures of SI and NSI viruses from an AIDS patient and two asymptomatic individuals, respectively. All the LTR clones tested contained multiple nucleotide changes (mostly G-to-A transitions), compared to the subtype B consensus sequence, which were clustered within the negative regulatory element, including NF-AT, USF, and TCF-1 alpha binding sites. The core promoter/TAR region sequences were highly conserved. The basal and Tat-mediated transcriptional activities of selected LTR clones tested were 0.1 to 1 and 0.2 to 0.5 times that of the control, respectively, regardless of the SI or NSI origin of the clones. Phylogenetic analysis revealed interi-solate sequence divergence in the LTR that was similar but not identical to previously analyzed vif sequences from the same samples. In particular, the inter-isolate distances from reference sequences differed for the LTR and vif. This raises the possibility that recombination occurred between corresponding LTR and vif loci of the quasi-species present in the isolates described here.
CD4-positive membrane vesicles (MV) were isolated under isotonic conditions from human T lymphoblastoid cells MT-2 and CEM and tested for their ability to support reverse transcription of viral RNA upon exposure to human immunodeficiency virus, type 1 (HIV-1). MV contained cytoplasms as confirmed by the presence of mitochondrial DNA but were devoid of chromosomal DNA. Virus binding and vesicle lysis assays revealed that 4 -19% (depending upon virus dose) of MV-bound HIV-1 entered the vesicles. HIV-1 internalized in MV was able to initiate and complete viral DNA synthesis as determined by the detection of products of reverse transcription using polymerase chain reaction amplification of viral DNA using regions present in early (strong stop) transcripts and full-length double-stranded molecules. Viral DNA was undetectable in MV exposed to HIV-1 at 0°C, in MV exposed to UV-inactivated virus at 37°C, or after exposure to intact virus at 37°C in the presence of reverse transcriptase inhibitors 2,3-dideoxycytidine and a tetrahydroimidazo[4,5,1-jk](1,4)-benzodiazepin-2-(1H)-thione derivative, indicating that viral DNA detected in HIV-1-exposed MV was synthesized de novo. Kinetic studies revealed that HIV-1 DNA synthesis in MV was very rapid; full-length viral DNA was detected within 15 min of exposure at 37°C, and the DNA levels increased 90-fold after 1 h and declined thereafter. Strong stop viral DNA was 10-fold more abundant than full-length DNA after 1 h at 37°C, indicating that 10% of input viral genomes are fully transcribed in MV within this time frame. This system preserves the critical features of intact CD4-bearing cells to permit studies of HIV-1 entry, uncoating, and reverse transcription of viral RNA.Human immunodeficiency virus, type 1 (HIV-1) 1 enters T cells and macrophages by binding to surface CD4 receptors and fusion with plasma membranes (1-7). Following entry and uncoating, the viral RNA is used as a template by viral reverse transcriptase (RT) for synthesis of double-stranded DNA that then migrates into the nucleus and integrates into host chromosomal DNA (8). Reverse transcription of viral RNA is a critical process in the retroviral life cycle and a major target for anti-HIV-1 therapy (9). Several approaches have been employed to study the enzymatic activity and products of RT. The exogenous RT assay measures incorporation of deoxynucleoside 5Ј-triphosphates (dNTPs) into polymers using an exogenous homopolymeric template and has been extensively used for HIV-1 infectivity studies and for standardization of virus preparations (10 -13). The endogenous reverse transcription reaction utilizes detergent-permeabilized virions, exogenous dNTPs, and viral genomic RNA as a template (14,15). In contrast to the exogenous RT assay, the endogenous reaction products include genomic length minus strand and discontinuous plus strand viral DNA (16 -18), and thus the reaction closely models the reverse transcription reaction that occurs after viral entry into cells (19). In the past several years the process of re...
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