Gene expression profiling reveals the profound upregulation of hypoxia-responsive genes in primary human astrocytes.

Oxygen is vital for the development and survival of mammals. In response to hypoxia, the brain initiates numerous adaptive responses at the organ level as well as at the molecular and cellular levels, including the alteration of gene expression. Astrocytes play critical roles in the proper functioning of the brain; thus the manner in which astrocytes respond to hypoxia is likely important in determining the outcome of brain hypoxia. Here, we used microarray gene expression profiling and data-analysis algorithms to identify and analyze hypoxia-responsive genes in primary human astrocytes. We also compared gene expression patterns in astrocytes with those in human HeLa cells and pulmonary artery endothelial cells (ECs). Remarkably, in astrocytes, five times as many genes were induced as suppressed, whereas in HeLa and pulmonary ECs, as many as or more genes were suppressed than induced. More genes encoding hypoxia-inducible functions, such as glycolytic enzymes and angiogenic growth factors, were strongly induced in astrocytes compared with HeLa cells. Furthermore, gene ontology and computational algorithms revealed that many target genes of the EGF and insulin signaling pathways and the transcriptional regulators Myc, Jun, and p53 were selectively altered by hypoxia in astrocytes. Indeed, Western blot analysis confirmed that two major signal transducers mediating insulin and EGF action, Akt and MEK1/2, were activated by hypoxia in astrocytes. These results provide a global view of the signaling and regulatory network mediating oxygen regulation in human astrocytes.

Detection of HIV-1 DNA in microglia/macrophages, astrocytes and neurons isolated from brain tissue with HIV-1 encephalitis by laser capture microdissection.

In HIV-1 encephalitis, HIV-1 replicates predominantly in macrophages and microglia. Astrocytes also carry HIV-1, but the infection of oligodendrocytes and neurons is debated. In this study we examined the presence of HIV-1 DNA in different brain cell types in 6 paraffin embedded, archival post-mortem pediatric and adult brain tissues with HIV-1 encephalitis by Laser Capture Microdissection (LCM). Sections from frontal cortex and basal ganglia were stained by immunohistochemistry for CD68 (microglia), GFAP (astrocytes), MAP2 (neurons), and p24 (HIV-1 positive cells) and different cell types were microdissected by LCM. Individual cells or pools of same type of cells were lysed, the cell lysates were subjected to PCR using HIV-1 gag SK38/SK39 primers, and presence of HIV-1 DNA was confirmed by Southern blotting. HIV-1 gag DNA was consistently detected by this procedure in the frontal cortex and basal ganglia in 1 to 20 p24 HIV-1 capsid positive cells, and in pools of 50 to 100 microglia/macrophage cells, 100 to 200 astrocytes, and 100 to 200 neurons in HIV-1 positive cases but not in HIV-1 negative controls. These findings suggest that in addition to microglia, the infection of astrocytes and neurons by HIV-1 may contribute to the development of HIV-1 disease in the brain.

Induction of rapid and extensive beta-chemokine synthesis in macrophages by human immunodeficiency virus type 1 and gp120, independently of their coreceptor phenotype.

Human immunodeficiency virus type 1 (HIV-1) interacts with its target cells through CD4 and a coreceptor, generally CCR5 or CXCR4. Macrophages display CD4, CCR5, and CXCR4 that are competent for binding and entry of virus. Virus binding also induces several responses by lymphocytes and macrophages that can be dissociated from productive infection. We investigated the responses of macrophages to exposure to a series of HIV-1 species, R5 species that productively infect and X4 species that do not infect macrophages. We chose to monitor production of several physiologically relevant factors within hours of treatment to resolve virally induced effects that may be unlinked to HIV-1 production. Our novel findings indicate that independently of their coreceptor phenotype and independently of virus replication, exposure to certain R5 and X4 HIV-1 species induced secretion of high levels of macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, RANTES, and tumor necrosis factor alpha. However two of the six R5 species tested, despite efficient infection, were unable to induce rapid chemokine production. The acute effects of virus on macrophages could be mimicked by exposure to purified R5 or the X4 HIV-1 envelope glycoprotein gp120. Depletion of intracellular Ca(2+) or inhibition of protein synthesis blocked the chemokine induction, implicating Ca(2+)-mediated signal transduction and new protein synthesis in the response. The group of viruses able to induce this chemokine response was not consistent with coreceptor usage. We conclude that human macrophages respond rapidly to R5 and X4 envelope binding by production of high levels of physiologically active proteins that are implicated in HIV-1 pathogenesis.

Highly productive infection with pseudotyped human immunodeficiency virus type 1 (HIV-1) indicates no intracellular restrictions to HIV-1 replication in primary human astrocytes.

Human astrocytes can be infected with human immunodeficiency virus type 1 (HIV-1) in vitro and in vivo, but, in contrast to T lymphocytes and macrophages, virus expression is inefficient. To investigate the HIV-1 life cycle in human fetal astrocytes, we infected cells with HIV-1 pseudotyped with envelope glycoproteins of either amphotropic murine leukemia virus or vesicular stomatitis virus. Infection by both pseudotypes was productive and long lasting and reached a peak of 68% infected cells and 1.7 microg of viral p24 per ml of culture supernatant 7 days after virus inoculation and then continued with gradually declining levels of virus expression through 7 weeks of follow-up. This contrasted with less than 0.1% HIV-1 antigen-positive cells and 400 pg of extracellular p24 per ml at the peak of astrocyte infection with native HIV-1. Cell viability and growth kinetics were similar in infected and control cells. Northern blot analysis revealed the presence of major HIV-1 RNA species of 9, 4, and 2 kb in astrocytes exposed to pseudotyped (but not wild-type) HIV-1 at 2, 14, and 28 days after infection. Consistent with productive infection, the 9- and 4-kb viral transcripts in astrocytes infected by pseudotyped HIV-1 were as abundant as the 2-kb mRNA during 4 weeks of follow-up, and both structural and regulatory viral proteins were detected in infected cells by immunoblotting or cell staining. The progeny virus released by these cells was infectious. These results indicate that the major barrier to HIV-1 infection of primary astrocytes is at virus entry and that astrocytes have no intrinsic intracellular restriction to efficient HIV-1 replication.

Vif is largely absent from human immunodeficiency virus type 1 mature virions and associates mainly with viral particles containing unprocessed gag.

Vif is a human immunodeficiency virus type 1 (HIV-1) protein that is essential for the production of infectious virus. Most of Vif synthesized during HIV infection localizes within cells, and the extent of Vif packaging into virions and its function there remain controversial. Here we show that a small but detectable amount of Vif remains associated with purified virions even after their treatment with the protease subtilisin. However, treatment of these virions with 1% Triton X-100 revealed that most of the virion-associated Vif segregated with detergent-resistant virus particles consisting of unprocessed Gag, indicating that detergent-soluble, mature virions contain very little Vif. To investigate the control of Vif packaging in immature virus particles, we tested its association with Gag-containing virus-like particles (VLPs) in a Vif and Gag coexpression system in human cells. Only a small proportion of Vif molecules synthesized in this system became packaged into VLPs, and the VLP-associated Vif was protected from exogenous protease and detergent treatment, indicating that it is stably incorporated into immature virion-like cores. About 10-fold more Vpr than Vif was packaged into VLPs but most of the VLP-associated Vpr was removed by treatment with detergent. Mutagenesis of the C-terminal sequences in Gag previously shown to be responsible for interaction with Vif did not reduce the extent of Vif packaging into Gag VLPs. Surprisingly, short deletions in the capsid domain (CA) of Gag (amino acid residues 284 to 304 and 350 to 362) increased Vif packaging over 10-fold. The 350 to 363 deletion introduced into CA in HIV provirus also increased Vif incorporation into purified virions. Our results show that Vif can be packaged at low levels into aberrant virus particles or immature virions and that Vif is not present significantly in mature virions. Overall, these results indicate that the Vif content in virions is tightly regulated and also argue against a function of virion-associated Vif.

Cloning of differentially expressed genes in an HIV-1 resistant T cell clone by rapid subtraction hybridization, RaSH.

An HIV-1 resistant T cell clone R1c2 has been generated that carries mutant, latent HIV-1 in a minority of the cell population. Resistant cells express HIV-1 receptors CD4 and CXCR4 and display resistance to infection by wild type (wt) HIV-1 at the level of virus transcription. To begin to define the repertoire of genes modulated in R1c2 cells that correlate with and potentially control expression of the HIV-1 resistance phenotype we have employed a rapid subtraction hybridization (RaSH) technique. For this approach, cDNA libraries were prepared from double-stranded cDNAs that were enzymatically digested into small fragments, ligated to adapters, PCR amplified followed by incubation of tester and driver PCR fragments. The RaSH scheme resulted in the cloning of genes displaying differential expression between HIV-1 resistant (R1c2) and susceptible (SupT1) cells, including known genes and those not described in current DNA databases. Analysis of the pattern of expression of the differentially expressed genes documented eleven genes with enhanced (HR clones) and six genes with reduced (HS clones) expression in HIV-1 resistant versus HIV-1 susceptible T-cell clones.

Prolonged infection of peripheral blood lymphocytes by Vif-negative HIV type 1 induces resistance to productive HIV type 1 infection through soluble factors.

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.

Human immunodeficiency virus type 1 can infect human retinal pigment epithelial cells in culture and alter the ability of the cells to phagocytose rod outer segment membranes.

Human immunodeficiency virus type 1 (HIV-1) has been found in the vitreous of persons with AIDS. Here we investigated the susceptibility of human retinal pigment epithelial (RPE) cells to HIV-1 infection in culture and the effects of HIV-1 on the phagocytic function of the RPE. We found that 10 of 11 populations of RPE cells isolated from different fetal or adult eyes were susceptible to low-level replication of HIV-1/NL4-3 as determined by the detection of viral DNA and spliced viral RNA encoding envelope. HIV-1 infection was not inhibited by recombinant soluble CD4, suggesting that CD4 is not required for virus entry into RPE cells. RPE cells fused with target cells constitutively expressing HIV-1 envelope glycoproteins, indicating that HIV-1 enters cells by receptor-mediated fusion. Exposure to HIV-1 or recombinant gp120 caused a two- to four-fold increase in the binding and uptake of isolated rod outer segments by RPE cells. These findings introduce a new cell target of HIV-1 replication in the eye and indicate that RPE cells function aberrantly when exposed to HIV-1 or its envelope glycoprotein.

Dependence of CD8+ T-cell-mediated suppression of HIV type 1 on viral phenotypes and mediation of phenotype-dependent suppression by viral envelope gene and not by beta-chemokines.

CD8+ T-cell-mediated HIV-1 suppressive activity has been shown against a number of strains of HIV-1 and HIV-2. In this study using a semiquantitative assay, we showed that CD8+ T cells from seropositive subjects and herpes virus saimiri transformed CD8+ T-cell clones from HIV-1-infected subjects exhibited 5 to 100-fold higher suppressive activity against slow replicating nonsyncytia-inducing strains (Slow/NSI) as compared to fast replicating syncytia-inducing strains (Fast/SI) of HIV-1. Such differential suppressive activity was not due to beta-chemokines as evidenced by the lack of blocking activity of antibodies to RANTES, MIP-1beta, and MIP-1alpha on the antiviral activities of CD8+ T cells. Moreover, there was no correlation between the level of CD8+ T-cell suppression and the level of these beta-chemokines in culture supernatant. Results from the CD8+ T-cell-mediated suppressive activity against two molecular cloned virus ME1 (Slow/NSI), ME46 (Fast/SI), and their interstrain recombinants indicate that the envelope gene carries a major genetic determinant responsible for this phenotypic-dependent differential suppressive activity.

Resistance against syncytium-inducing human immunodeficiency virus type 1 (HIV-1) in selected CD4(+) T cells from an HIV-1-infected nonprogressor: evidence of a novel pathway of resistance mediated by a soluble factor(s) that acts after virus entry.

A panel of CD4(+) T-cell clones were generated from peripheral blood lymphocytes from a patient with a nonprogressing infection of human immunodeficiency virus type 1 (HIV-1) by using herpesvirus saimiri as described recently. By and large, all of the clones expressed an activated T-cell phenotype (Th class 1) and grew without any further stimulation in interleukin-2-containing medium. None of these clones produced HIV-1, and all clones were negative for HIV-1 DNA. When these clones were infected with primary and laboratory (IIIB) strains of HIV-1 with syncytium-inducing (SI) phenotypes, dramatic variation of virus production was observed. While two clones were highly susceptible, other clones were relatively or completely resistant to infection with SI viruses. The HIV-resistant clones expressed CXCR4 coreceptors and were able to fuse efficiently with SI virus env-expressing cells, indicating that no block to virus entry was present in the resistant clones. Additionally, HIV-1 DNA was detectable after infection of the resistant clones, further suggesting that HIV resistance occurred in these clones after virus entry and probably after integration. We further demonstrate that the resistant clones secrete a factor(s) that can inhibit SI virus production from other infected cells and from a chronically infected producer cell line. Finally, we show that the resistant clones do not express an increased amount of ligands (stromal-derived factor SDF-1) of CXCR4 or other known HIV-inhibitory cytokines. Until now, the ligands of HIV coreceptors were the only natural substances that had been shown to play antiviral roles of any real significance in vivo. Our data from this study show that differential expression of another anti-HIV factor(s) by selected CD4(+) T cells may be responsible for the protection of these cells against SI viruses. Our results also suggest a novel mechanism of inhibition of SI viruses that acts at a stage after virus entry.

Replication of different clones of human immunodeficiency virus type 1 in primary fetal human astrocytes: enhancement of viral gene expression by Nef.

Dementia is a common complication of AIDS which is associated with human immunodeficiency virus type 1 (HIV-1) infection of brain macrophages and microglia. Recent studies have shown that astrocytes are also infected in the brain but HIV-1 replication in these cells is restricted. To determine virus specificity of this restriction we tested the expression of 15 HIV-1 molecular clones in primary human fetal astrocytes by infection and DNA transfection. Infection with cell-free viruses was poorly productive and revealed no clone-specific differences. In contrast, transfected cells produced transiently high levels of HIV-1 p24 core antigen, up to 50 nanograms per ml culture supernatant, and nanogram levels of p24 were detected 3-4 weeks after transfection of some viral clones. The average peak expression of HIV-1 in astrocytes varied as a function of viral clone used by a factor of 15 but the differences and the subsequent virus spread did not correlate with the tropism of the viral clones to T cells or macrophages. Functional vif, vpu, and vpr genes were dispensable for virus replication from transfected DNA, but intact nef provided a detectable enhancement of early viral gene expression and promoted maintenance of HIV-1 infection. We conclude that primary astrocytes present no fundamental barriers to moderate expression of different strains of HIV-1 and that the presence of functional Nef is advantageous to virus infection in these cells.

Peptide inhibitors of HIV-1 protease and viral infection of peripheral blood lymphocytes based on HIV-1 Vif.

We recently reported that HIV-1 Vif (virion infectivity factor) inhibits HIV-1 protease in vitro and in bacteria, suggesting that it may serve as the basis for the design of new protease inhibitors and treatment for HIV-1 infection. To evaluate this possibility, we synthesized peptide derivatives from the region of Vif, which inhibits protease, and tested their activity on protease. In an assay of cleavage of virion-like particles composed of HIV-1 Gag precursor polyprotein, full-length recombinant Vif, and a peptide consisting of residues 21-65 of Vif, but not a control peptide or BSA, inhibited protease activity. Vif21-65 blocked protease at a molar ratio of two to one. We then tested this peptide and a smaller peptide, Vif41-65, for their effects on HIV-1 infection of peripheral blood lymphocytes. Both Vif peptides inhibited virus expression below the limit of detection, but control peptides had no effect. To investigate its site of action, Vif21-65 was tested for its effect on Gag cleavage by protease during HIV-1 infection. We found that commensurate with its reduction of virus expression, Vif21-65 inhibited the cleavage of the polyprotein p55 to mature p24. These results are similar to those obtained by using Ro 31-8959, a protease inhibitor in clinical use. We conclude that Vif-derived peptides inhibit protease during HIV-1 infection and may be useful for the development of new protease inhibitors.

Endogenous production of beta-chemokines by CD4+, but not CD8+, T-cell clones correlates with the clinical state of human immunodeficiency virus type 1 (HIV-1)-infected individuals and may be responsible for blocking infection with non-syncytium-inducing HIV-1 in vitro.

Recent studies have demonstrated that the beta-chemokines RANTES, MIP-1alpha, and MIP-1beta suppress human immunodeficiency virus type 1 (HIV-1) replication in vitro and may play an important role in protecting exposed but uninfected individuals from HIV-1 infection. However, levels of beta-chemokines in AIDS patients are comparable to and can exceed levels in nonprogressing individuals, indicating that global beta-chemokine production may have little effect on HIV-1 disease progression. We sought to clarify the role of beta-chemokines in nonprogressors and AIDS patients by examination of beta-chemokine production and HIV-1 infection in patient T-lymphocyte clones established by herpesvirus saimiri immortalization. Both CD4+ and CD8+ clones were established, and they resembled primary T cells in their phenotypes and expression of activated T-cell markers. CD4+ T-cell clones from all patients had normal levels of mRNA-encoding CCR5, a coreceptor for non-syncytium-inducing (NSI) HIV-1. CD4+ clones from nonprogressors and CD8+ clones from AIDS patients secreted high levels of RANTES, MIP1alpha, and MIP-1beta. In contrast, CD4+ clones from AIDS patients produced no RANTES and little or no MIP-1alpha or MIP-1beta. The infection of CD4+ clones with the NSI HIV-1 strain ADA revealed an inverse correlation to beta-chemokine production; clones from nonprogressors were poorly susceptible to ADA replication, but clones from AIDS patients were highly infectable. The resistance to ADA infection in CD4+ clones from nonprogressors could be partially reversed by treatment with anti-beta-chemokine antibodies. These results indicate that CD4+ cells can be protected against NSI-HIV-1 infection in culture through endogenously produced factors, including beta-chemokines, and that beta-chemokine production by CD4+, but not CD8+, T cells may constitute one mechanism of disease-free survival for HIV-1-infected individuals.

The redox state of cysteines in human immunodeficiency virus type 1 Vif in infected cells and in virions.

HIV-1 Vif has two conserved cysteine residues in positions 114 and 133, both of which were found to be essential for HIV-1 infection and for Vif function in transcomplementation assays (X-Y. Ma, P. Sova, W. Chao, and D. J. Volsky, 1994, J. Virol. 68: 1714-1720). We evaluated here the redox status and disulfide bond formation of Vif cysteines inside cells or in virions and tested the role of Vif cysteines in Vif distribution in cells and in virions. Immunoblot analysis of Vif in wild type virus-infected cells and virions under different redox conditions revealed that the cysteine residues are readily accessible to chemical interaction but they do not form intramolecular disulfide bonds either inside cells or in virions, nor do they form covalent bonds with other proteins in either compartment. Cysteine mutants of Vif resembled wildtype Vif in their intracellular and virion distribution, indicating that Vif cysteines do not affect intracellular Vif transport and packaging into virions. We conclude that the cysteines in Vif do not form sulfhydryl bonds either intracellularly or in virions and may contribute to Vif activity rather than structure.

Human immunodeficiency virus type 1 (HIV-1) protein Vif inhibits the activity of HIV-1 protease in bacteria and in vitro.

Human immunodeficiency virus type 1 (HIV-1) Vif is required for productive infection of T lymphocytes and macrophages. Virions produced in the absence of Vif have abnormal core morphology and those produced in primary T cells carry immature core proteins and low levels of mature capsid (M. Simm, M. Shahabuddin, W. Chao, J. S. Allan, and D. J. Volsky, J. Virol. 69:4582-4586, 1995). To investigate whether Vif influences the activity of HIV-1 protease (PR), the viral enzyme which is responsible for processing Gag and Gag-Pol precursor polyproteins into mature virion components, we transformed bacteria to inducibly express truncated Gag-Pol fusion proteins and Vif. We examined the cleavage of polyproteins consisting of matrix to PR (Gag-PR), capsid to PR (CA-PR), and p6Pol to PR (p6Pol-PR) and evaluated HIV-1 protein processing at specific sites by Western blotting using antibodies against matrix, capsid, and PR proteins. We found that Vif modulates HIV-1 PR activity in bacteria mainly by preventing the release of mature MA and CA from Gag-PR, CA from CA-PR, and p6Pol from p6Pol-PR, with other cleavages being less affected. Using subconstructs of Vif, we mapped this activity to the N-terminal half of the molecule, thus identifying a new functional domain of Vif. Kinetic study of p6Pol-PR autocatalysis in the presence or absence of Vif revealed that Vif and N'Vif reduce the rate of PR-mediated proteolysis of this substrate. In an assay of in vitro proteolysis of a synthetic peptide substrate by purified recombinant PR we found that recombinant Vif and the N-terminal half of the molecule specifically inhibit PR activity at a molar ratio of the N-terminal half of Vif to PR of about 1. These results suggest a mechanism and site of action of Vif in HIV-1 replication and demonstrate novel regulation of a lentivirus PR by an autologous viral protein acting in trans.

Improvement of Herpesvirus saimiri T cell immortalization procedure to generate multiple CD4+ T-cell clones from peripheral blood lymphocytes of AIDS patients.

Herpesvirus saimiri (HVS) can infect and immortalize human T lymphocytes of both CD4- and CD8-positive phenotypes. We have previously shown that infection of peripheral blood lymphocytes (PBL) from AIDS patients with HVS predominantly yielded immortalized CD8-positive T cell clones. Here we show that CD4-positive T cells from AIDS patients can be efficiently immortalized by HVS if patient PBL are enriched for CD4-positive T cell subpopulation prior to HVS infection. Such cells can be cloned and maintained in culture for prolonged times, and they exhibit activated T cell phenotype of Th1 class and are susceptible to HIV-1 infection. Several immortalized T cell clones obtained from one out of three AIDS patients tested here were HIV-1 positive and produced infectious virus. This approach permits efficient generation of multiple CD4-positive T cell clones from AIDS patients for functional and virological studies.

Human immunodeficiency virus type 1 (HIV-1) infection of herpesvirus saimiri-immortalized human CD4-positive T lymphoblastoid cells: evidence of enhanced HIV-1 replication and cytopathic effects caused by endogenous interferon-gamma.

Herpesvirus saimiri (HVS) is a nonhuman primate gamma herpesvirus which can immortalize human T lymphocytes similar to Epstein-Barr virus immortalization of B cells. The HVS-immortalized T cell lines can be cloned and they remain functional, including susceptibility of CD4 expressing T cells to infection with human immunodeficiency virus type 1 (HIV-1). In this report, we have used five such HVS-transformed CD4-positive T cell clones to reevaluate the role of endogenous interferon gamma (IFN gamma) in HIV-1 replication in T cells. All five clones had similar phenotypes; and four clones constitutively produced IFN gamma and one clone did not. All five clones could be efficiently infected with HIV-1. HIV-1 infection of the IFN gamma-positive cells also upregulated IFN gamma mRNA production and IFN gamma secretion but not production of IL-2 or IL-4. In contrast, infection of IFN gamma-negative cells did not induce IFN gamma, IL-2, or IL-4. Exposure to anti-IFN gamma antibodies after HIV-1 infection significantly reduced virus production and inhibited virus-induced death of IFN gamma-positive cells but had no effect on IFN gamma-negative cells. We conclude that in CD4-positive T lymphocytes immortalized by HVS endogenous IFN gamma does not inhibit HIV-1 but enhances HIV-1 replication and cytolysis. The potential augmenting effects of IFN gamma on HIV-1 replication in CD4-positive T cells recommend caution in a therapeutic use of this cytokine in AIDS.

Isolation and long-term culture of primary ocular human immunodeficiency virus type 1 isolates in primary astrocytes.

Vitreous specimens from 14 HIV-1 infected persons undergoing medically indicated vitrectomy were assayed for the presence of infectious HIV-1 and viral tropism. Human primary fetal astrocytes, adult lymphocytes, or macrophages were exposed to vitreous in culture and and cells were then assayed for HIV-1 DNA by polymerase chain reaction amplification. We found that 11 of 14 patients tested carried ocular HIV-1 which replicated in one or more primary cell types; of the 13 vitreous samples tested in astrocytes, eight contained transmissible HIV-1. The three patients with no culturable ocular virus were in antiviral therapy at the time of vitrectomy. Comparison of envelope V3 sequences from astrocytes infected in culture to that in uncultured blood cells revealed 21% sequence divergence indicating that ocular HIV-1 transmitted to astrocytes was not recently derived from virus present in the blood. Two ocular samples transmissible to astrocytes were tested further and found capable of sustained replication by serial passage to uninfected astrocytes. However, the viral structural proteins produced by infected astrocytes were abnormal, p24 was absent and higher molecular weight Gag proteins were present. We conclude that the eye is a central nervous system compartment which frequently contains HIV-1 capable of replication in human astrocytes.