Human Immunodeficiency Virus Type 1 Infection Of Cells Research Articles

The human immunodeficiency virus type-1 (HIV-1) Tat protein and Bcl-2 gene expression.

Tat protein of human immunodeficiency virus type-1 (HIV-1) plays a central role in viral replication and shows pleiotropic effects on the survival and growth of different cell types. Remarkably, Tat represents the first example of a viral protein, that can also be actively secreted by infected cells and shows a cytokine-like activity on both HIV-1 infected and uninfected cells. We previously reported that the stable expression of tat cDNA rescues Jurkat cell lines from apoptosis induced by a variety of stimuli, such as serum withdrawal, engagement of fas antigen or even a productive infection with HIV-1. These findings suggested that Tat was able to modulate the expression of one or more gene(s) relevant for the control of cell survival/death. Consistently, Jurkat cells stably transfected with tat show an upregulated expression of bcl-2. It is still unsettled whether Tat affects cell survival and bcl-2 expression directly or indirectly, modulating the expression of other cellular genes involved in the control of cell survival or encoding for cytokines. Blocking experiments performed with anti-Tat neutralizing antibodies revealed that TAt increases bcl-2 expression and prevent lymphoid T cells from apoptosis by acting, at least in part, through an autocrine/paracrine loop. While high (nM-microM) concentrations of extracellular Tat display a cytotoxic activity on the antigen-mediated induction of T cell proliferation, low (pM) concentrations of Tat were able to protect both Jurkat cells and primary peripheral blood mononuclear cells from apoptosis. Significantly, pM concentrations of Tat were detected in the sera of some HIV-1 infected individuals as well as in the culture supernatant of HIV-1 infected cells, raising the possibility that these levels of Tat protein may be present physiologically in vivo. The potential relevance of Tat-mediated upregulation of bcl-2 for the pathogenesis of HIV-1 disease is discussed.

Endoproteolytic cleavage of HIV-1 gp160 envelope precursor occurs after exit from the trans-Golgi network (TGN).

Endoproteolytic processing of human immunodeficiency virus type 1 (HIV-1) gp160 membrane glycoprotein precursor into gp 120 and gp41 is necessary for formation of infectious HIV particles [1]. We have studied the intracellular site of this processing using inhibition of transport at reduced temperature (20 degrees C). That reduced temperature (20 degrees C) inhibits the intracellular transport also in Jurkat-tat cells was demonstrated using the Semliki Forest virus p62 precursor processing as model. In HIV-1 infected Jurkat-tat cells the proteolytic processing of gp 160 precursor did not occur when the protein was accumulated in the TGN at 20 degrees C temperature. When the temperature was shifted to 37 degrees C the HIV-1 gp 160, which had accumulated in the TGN at the reduced temperature, was proteolytically processed. The processing of gp 160 was inhibited when the temperature reversion was carried out in the presence of brefeldin A (BFA) or aluminium fluoride (ALFn) indicating that the exit from the TGN is required for the proteolytic cleavage of HIV-1 gp160 precursor. The results suggest that the processing of gp 160 takes place at a yet unidentified transport step which is distal to the TGN/20 degrees C block site.

Expression of human immunodeficiency virus type 1 Nef antigen on the surface of acutely and persistently infected human T cells

The authors have previously shown the role of human immunodeficiency virus type 1 (HIV-1) Nef protein as a growth inhibitor to CD4+ T lymphocytes. We now report that the Nef antigen is partly expressed on both acutely and persistently infected human T cells. To investigate the cell surface expression of the Nef antigen, murine monoclonal antibodies (mAbs) were prepared by immunization with a recombinant Nef protein. The recombinant Nef was expressed by a baculovirus expression system as a truncated Nef protein with 22 kDa containing the middle to the C-terminus. Three clones were found to produce mAbs with IgM isotype against Nef protein by ELISA with the same truncated Nef. All these mAbs reacted on immunoblotting with two forms of Nef, 25 kDa and 27 kDa, in an HIV-1-infected human T-cell line and with the 27 kDa Nef in retroviral vector-infected mouse fibroblasts expressing a full-length Nef protein. Membrane immunofluorescence and flow cytometry with these mAbs revealed the Nef antigen to be expressed, at least in part, on the surface of these cells. Thus, the cell-surface form of Nef might play an important role in the selective depletion of CD4+ cells in HIV-1 infection.

Evidence for the role of human immunodeficiency virus type 1 Nef protein as a growth inhibitor to CD4+ T lymphocytes and for the blocking of the Nef function by anti-Nef antibodies

Human immunodeficiency virus type 1 (HIV-1) can lead to a profound CD4+ T-cell deficiency. To examine the functional role of HIV-1 Nef protein on the marked loss of CD4+ cells, Nef protein was expressed in and purified from Escherichia coli as a fusion protein with T7 phage gene10 product (Nef-gene10). When peripheral blood mononuclear cells (PBMC) from healthy donors were cultivated in the presence of Nef-gene10 or the gene10 product as well as interleukin-2 (IL-2), it was found that the Nef-gene10, but not the gene10 product, induced a remarkable decline in the CD4/CD8 ratio and in the response to phytohaemagglutinin of PBMC as well as of nylon wool-passed purified T cells. Nef-gene10 inhibited the proliferation of CD4+ cells, but did not kill the cells. This suppression of the IL-2-dependent proliferation of CD4+ cells by Nef-gene10 seemed to be due to enhanced production of several lymphokines, especially of interferon-γ. Thus, Nef protein might be partly responsible for the selective depletion of CD4+ cells in HIV-1 infection. Furthermore, the Nef-induced decline in the CD4 CD8 ratio was interrupted by anti-Nef antibodies, suggesting the possibility that a vaccine which resulted in the production of such functional Nef antibodies would be useful in the treatment of HIV-1-induced immunodysfunction.

Maleylated human serum albumin inhibits HIV-1 infection in vitro

Maleylated-human serum albumin (Mal-HSA) inhibited human immunodeficiency virus type-1 (HIV-1) infection of MT-4 cells in vitro. It was also found to inhibit the fusion between uninfected CD4 + (Molt-4 clone 8 cells) and HIV-1 infected cells (Molt-4/HIV-1) to form syncytia. To investigate the mechanism of the inhibition, a study was designed to determine whether Mal-HS could bind to Cd 4+ cells. Mal-HSA could bind to both MT-4 cells and Molt-4 clone 8 cells with high affinity, K d=2.0 nM and K d=5.8 nM, respectively. However, Mal-HSA could neither inhibit anti CD4 antibody Leu 3a binding to Molt-4 clone 8 cells nor modulate the expression of CD4 molecules on the surface of the cells. Mal-HSA binding to Molt-4 clone 8 cells was completely inhibited by sulfated polysaccharides bearing anti-HIV activity, such as dextran sulfate, fucoidan and carrageenan. Other HIV-1 susceptible human T-cell lines, such as Molt-4, CEM-5, H-9 and HuT-78 cells, also have Mal-HSA binding sites showing a high affinity, K d=0.9±0.4 nM. Mal-HSA binding proteins of Molt-4 clone 8 cells were identified by ligand blotting as 155 and 220 kDa proteins. Unlike dextran sulfate, Mal-HSA could not inhibit reverse transcriptase activity of HIV-1. These results indicate that Mal-HSA inhibits HIV-1 infection and syncytia formation, and suggest that 155 and/or kDa proteins of target cells are involvved in HIV-1 adsorption and/or the membrane fusion between HIV-1 and target cells.

Derivation of a biologically contained replication system for human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) proviral mutants that lack viral regulatory genes are unable to replicate unless rescued by complementation in trans. Structurally intact virus can be produced by infecting recombinant cell lines expressing the deficient genes. A HIV-1 mutant functionally defective in tat and rev (vIIIB delta Tat/Rev), which replicates only in a recombinant T-cell line expressing tat and rev (CEMTART), is described in this report. Infection of the CEMTART cell line with vIIIB delta Tat/Rev permits the complete HIV-1 life cycle, including cytopathology, decreased expression of CD4, and production of viral structural proteins, to be biologically contained. Culture supernatants from infected CEMTART contain virus that is able to replicate only in uninfected CEMTART. No reversion of vIIIB delta Tat/Rev to wild-type HIV-1 was observed as measured either by sequencing proviral vIIIB delta Tat/Rev or by detecting the ability of vIIIB delta Tat/Rev to replicate in CEM or activated CD4-bearing T lymphocytes. Defective HIV-1 mutants produced by trans complementation of essential genes permit infection and analysis of defined genotypes on cellular function and phenotype. Authentic HIV-1 structural proteins and infected cells can be prepared in mass, and agents that interfere with the HIV-1 life cycle can be studied on a large scale with minimum risk of exposing workers to virulent HIV-1.

Inhibition of HIV-1 infection in vitro by murine monoclonal anti-p24 antibodies.

Murine monoclonal antibodies (Mabs) to the major core protein p24 of the human immunodeficiency virus type 1 (HIV-1) were tested for their ability to inhibit the replication and spread of the virus in permanent cell cultures (Molt4/8, K37, H9) and in the culture of II-2 stimulated T cells of healthy donors. After addition of ascitic fluid containing monoclonal anti-p24 antibodies or purified anti-p24 antibodies or the respective control to co-cultures of infected and non-infected cells, HIV-1 replication was evaluated by determining the percentage of infected cells and the activity of reverse transcriptase (RT) in cell-free supernatant. In addition, the supernatant's infectivity was determined. FACS analysis demonstrated p24 antigen in about 40% of unfixed HIV-1 infected cells at the cell membrane. Monoclonal anti-p24 antibodies of different epitope specificity added to the cells but not to the virus delayed the spread of HIV-1 infection in permanent cell culture. Furthermore, anti-p24 Mabs inhibited the release of RT-active virus particles by HIV-1 infected cell lines or II-2 stimulated T-lymphocytes, respectively, up to 60%. The mode of action of anti-p24 antibodies after HIV-1 infection is discussed on the basis of the data obtained.

An effective concentration method for human immunodeficiency virus type 1 (HIV-1)

We designed an effective virus concentration method to prevent the infection with human immunodeficiency virus type 1 (HIV-1) in laboratories. The absorbent of Minicon concentrators (Amicon Division, M.R. Grace & Co.-Conn.) was changed to chitin, a mucopolysaccharide extracted from the shells of Japanese pink crab. HIV-1 in the supernatant of HIV-1 infected Molt-4 cells was concentrated by Minicon and the new concentrators. The new concentrator showed good concentration rate and equality of concentration speed.

Bispecific single chain molecules (Janusins) target cytotoxic lymphocytes on HIV infected cells.

The human immunodeficiency virus type 1 (HIV-1) uses cell surface CD4 as a receptor to infect susceptible cells. Therefore, different forms of soluble CD4 (sCD4) molecules have been developed recently for potential therapeutic purposes. Here we describe a novel design of sCD4 molecules which exploit cytotoxic T cells as their effector function. The principle of bispecific antibodies was exploited and further developed to create new bispecific reagents which could retarget cytotoxic T cells of any specificity and thus, induce killing of HIV-1 infected cells. The most advanced molecules, Janusins, contain in one polypeptide chain the first two N-terminal CD4 domains and single chain combining site against the human CD3 complex (FvCD3).

Detection of human immunodeficiency virus type 1 in semen from seropositive men using culture and polymerase chain reaction deoxyribonucleic acid amplification techniques

We have demonstrated that the polymerase chain reaction is a valid and sensitive technique for the detection of human immunodeficiency virus type 1 (HIV-1) proviral deoxyribonucleic acid (DNA) in human semen. The combination of extraction, polymerase chain reaction, and liquid hybridization techniques used in this study was sensitive to a level of detection of one HIV-1 infected cell in 100,000 (or 3 infected cells/test sample). In a series of matched peripheral blood mononuclear cells (PBMC) and semen cells from 25 HIV-1 seropositive homosexual men, HIV-1 DNA was detected by polymerase chain reaction in 23 of 25 PBMC samples and 1 of 25 semen samples. By coculture on mitogen-activated peripheral blood leukocyte target cells, 19 of 24 PBMC and 4 of 24 semen samples were positive for infectious HIV-1. Of the four culture-positive semen samples, three were negative for the proviral form of the virus in the polymerase chain reaction assay. These data indicate that HIV-1 infected cells are not as prevalent in semen as in the peripheral blood. Furthermore, they indicate that the classical polymerase chain reaction approach, which only detects HIV-1 proviral DNA (infected cells), is not sufficient for clinical screening programs whose goal is the detection of HIV-1-infected semen samples. Accurate semen analysis by polymerase chain reaction may require enrichment of the infected cell population and/or a reverse transcriptase step to enable detection of the infectious ribonucleic acid form of the virus.

A defective proviral DNA with a 2.6-kb deletion of human immunodeficiency virus type 1 (HIV-1) in a persistently HIV-1 infected cell clone

A cell line (H2-5) producing defective doughnut-shaped particles of human immunodeficiency virus type I (HIV-1) was found to contain proviral DNA with a large deletion of 2558 bases, corresponding to the 3' half of pol gene, the vif and vpr genes, and the 5' terminal of the tat gene.

Human Immunodeficiency Virus (HIV) Leukoencephalopathy and the Microcirculation

We studied the brains of three patients with acquired immune deficiency syndrome (AIDS), all of whom developed subacutely progressive dementia unassociated with opportunistic infection or neoplasm in the central nervous system. Computed tomographic (CT) scans of the head revealed cortical atrophy, ventricular dilation, and diffuse hypodensity of the centrum semiovale. On microscopic examination, the cerebral and cerebellar white matter in all cases showed diffuse and focal, angiocentric regions of myelin pallor, focal vacuolization, and extensive gliosis. Variable axonal loss and axonal spheroids were evident. The microvasculature showed striking changes, including mural thickening, increased cellularity, and enlargement and pleomorphism of endothelial cells with variable numbers of macrophages and multinucleated giant cells (MNGC), which often contained hemosiderin pigment. Human immunodeficiency virus type 1 (HIV-1) antigens were identified immunocytochemically within perivascular macrophages and MNGC and in some microglial cells. We suggest that the morphologic abnormalities of the microcirculation may be associated with an alteration of the blood-brain barrier. The increased vascular permeability could contribute to damage and loss of the white matter including both myelin and axons, and result in subcortical cerebral atrophy. The HIV-1 infected cells present in relation to the microvasculature may play a role in mediating the vascular injury.

Transient expression of human immunodeficiency virus type 1 genome results in a nonproductive infection in human fetal dorsal root ganglia glial cells

Human immunodeficiency virus type 1 (HIV-1), the etiologic agent of acquired immunodeficiency syndrome (AIDS), has been implicated in the generation of AIDS-associated neurologic dysfunction. We are currently examining the replicative processes involved in HIV-1 infection of selected human fetal neural cell populations in vitro. To determine whether infection of the human fetal dorsal root ganglia (DRG) glial cell population culminates in the production and release of infectious HIV-1, cocultivation and reverse transcriptase (RT) assays were performed. Direct assay of HIV-1 infected neural cell supernatants as well as exposure of permissive SupTi cells to these HIV-1-infected neural cell supernatants detected no RT activity in eitherthe HIV-1-infected DRG glial cell supernatants orthe SupTi cell supernatants. When SupTl cells were cocultivated with the HIV-1-infected neural cells for 24-hr intervals, RT activity was detected in the SupTi supernatants from cocultures initiated less than 2 days after infection (most likely resulting from infectious input virus) but not from cocultures initiated on 3, 5, 10, and 30 days after infection. Hybridization analysis demonstrated transient expression of HIV-1 cytoplasmic mRNA with accumulation reaching a maximum level by 2 to 3 days postinfection, declining thereafter with low, but detectable, levels at 16 days postinfection. In addition, polymerase chain reaction amplification in conjunction with DNA blot hybridization detected HIV-1-specific proviral DNA at 3 days postinfection. Cumulatively, these data suggest that HIV-1 infection of human fetal DRG glial cells culminates in a nonproductive infection with expression of at least a fraction of the virus genome but no detectable infectious virus production.

Syncytia formation in HIV-1 infected cells is associated with an increase in cellular oleic acid

Infection of cells with the human immunodeficiency virus type-1 (HIV-1) usually results in the formation of giant multinuclear cells (syncytia) [(1986) Nature 322, 470-474; (1986) Nature 322, 725-728; (1985) Hum. Pathol. 18, 760-765; (1987) Arm. Neurol. 21, 490-496]. The appearance of syncytia is associated with an increase in the monounsaturated oleic acid content. This report describes experiments which compare the activity of known antiviral agents with that of saturated fatty acid derivatives in inhibiting oleic acid and syncytia formation. A concept is introduced which proposes that infection of cells with the human immunodeficiency virus causes a rise in cellular oleic acid which leads to increased membrane fluidity.

Expression of human immunodeficiency virus type 1 (HIV-1) gag antigens on the surface of a cell line persistently infected with HIV-1 that highly expresses HIV-1 antigens

MT-4 cells persistently infected with human immunodeficiency virus type 1 (HIV-1) (MT-4/HIV-1) were recently isolated (K. Ikuta, C. Morita, M. Nakai, N. Yamamoto, and S. Kato, Japan. J. Cancer Res. (Gann), 79, 418–423, 1988). Mouse hybridoma cell clones producing monoclonal antibodies (MoAbs) to HIV-1 gag p24 and pl8, and pol reverse transcriptase (RT) were isolated by using this MT-4/HIV-1 cell line for the screening of MoAb production by the immunofluorescence (IF) test. By indirect IF tests of acetone-fixed cells with these MoAbs, the IF intensities in MT-4/HIV-1 cells were found to be higher than those in the other HIV-1 infected cells, such as MOLT-4/HIV-1, HL-60/HIV-1, and U937/HIV-1 cells. Cell surface expression of the HIV-1 gag p24 and p18 antigens examined by IF and radioimmune techniques with these MoAbs revealed the p24 and pl8 antigens to be expressed strongly on the cell surface of MT4/HIV-1 cells and faintly on the cell surface of MOLT-4/HIV-1 cells, respectively. However, monoclonal antibody isolated in the present study failed to detect pol RT antigen on the surface of MT-4/HIV-1 cells. These results indicate that the gag p24 and p18 antigens are expressed, at least in part, on the surface of HIV-1-infected cells.

Augmentation and stable expression of a novel transcription factor SII in CD4-positive cells on infection with human immunodeficiency virus type-1(HIV-1)

We have examined the expression of a novel transcription factor SII in HIV-infected cells. Concomitant with the increasing expression of HIV-1 mRNA, the viability of HIV infected cells dropped and the expression of beta-actin gene also diminished. Concurrently, the expression of SII mRNA was stabilized or augmented. The data presented in this communication suggests that in HIV-1 infected cells, selective transcriptional controls were taking place and that a novel transcriptional factor, SII, plays an essential role in HIV-1 expression leading to cytocidal effects.