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PubMed1991

HIV-1 indicator cell lines.

A simple quantitative bioassay for infectious HIV-1 has been developed. The assay is based on adherent CD4+ HeLa cell lines stably transfected with episomal vectors carrying the Escherichia coli beta-galactosidase gene under the control of the HIV long terminal repeat (LTR) promoter. HIV infection of these cell lines transactivates the LTR promoter inducing beta-galactosidase production. Infected cells and virus foci can be stained dark blue by the addition of the chromogenic substrate X-Gal. Alternatively, a readily automatable quantitative enzyme assay can be performed on the infected cultures. Because of its simplicity the bioassay may be useful for routine quantification of HIV-infected cultures, plaque purification, virus neutralization studies and for the screening of antiviral agents.

Biological Assay↗
PubMed1997

Induction of human immunodeficiency virus type 1 expression in monocytic cells by Cryptococcus neoformans and Candida albicans.

Because candidiasis and cryptococcosis are common in human immunodeficiency virus (HIV)-infected persons, the effect of Cryptococcus neoformans and Candida albicans on HIV expression in monocytic cells was examined. Stimulation of the latently HIV-infected myelomonocytic cell line OM-10.1 with C. neoformans and C. albicans in the presence of pooled human serum caused a ratio-dependent increase in HIV production. Induction of HIV by C. neoformans was enhanced by anti-capsular antibody, while induction by both organisms was inhibited by anti-TNF-alpha antibody. In THP-1 cells transfected with HIV plasmid constructs, both organisms induced transcription from the HIV long terminal repeat that was dependent on intact NF-kappaB binding sequences. Thus, C. neoformans and C. albicans enhance HIV expression in monocytic cells through a TNF-alpha- and NF-kappaB-dependent mechanism. In HIV-infected patients, such enhancement may further impair host immunity and could accelerate the course of HIV disease.

Binding Sites↗
PubMed1994

Phagocytosis of Mycobacterium tuberculosis modulates human immunodeficiency virus replication in human monocytic cells.

Macrophage activation resulting from phagocytosis has the potential to modulate human immunodeficiency virus (HIV) replication. We have determined the effects of phagocytosis of particulate stimuli on transcription and release of HIV. Using THP-1 and Mono Mac 6 human monocytic cell lines transfected with HIV long terminal repeat sequence chloramphenicol acetyltransferase (LTR CAT) constructs we have demonstrated that phagocytosis of Mycobacterium tuberculosis enhanced HIV-1 and -2 LTR CAT expression. However phagocytosis of zymosan or inert latex beads had little or no effect on CAT expression. Enhancement of HIV LTR CAT expression was dependent upon intact NF-kappa B binding sites and was independent of tumour necrosis factor alpha secretion. M. tuberculosis strains of different degrees of virulence induced similar levels of enhanced CAT expression. In contrast, phagocytosis of M. tuberculosis by HIV-1-infected THP-1 cells reduced supernatant reverse transcriptase (RT) activity without suppression of p24 antigen release. Phagocytosis of zymosan granules or latex particles did not alter released RT activity. However, phagocytosis of either M. tuberculosis, zymosan granules or latex particles by HIV-1-infected human peripheral blood monocyte-derived macrophages reduced supernatant RT activity. These data indicate that phagocytosis of M. tuberculosis may enhance HIV transcription in monocytic cells although it may reduce release of intact HIV.

Antibodies, Monoclonal↗
PubMed1995

Replication of HIV type 1 in rabbit cell lines is not limited by deficiencies in tat, rev, or long terminal repeat function.

HIV-1 infection has been documented in rabbits, but infection proceeds slowly in this species. Human and rabbit cell lines were compared in order to identify barriers to efficient HIV-1 infection of rabbit cells. A direct comparison of human and rabbit CD4 as receptor for HIV-1 indicated that the rabbit CD4 homolog did not function well even when expressed by human cells. Examination of viral RNA production indicated that the major HIV transcripts were produced in HIV-infected rabbit cells, but were present at levels significantly lower than those found for human cells. Ability of HIV-1 LTRs to direct protein expression in human and rabbit cells was compared using gene constructs with the chloramphenicol acetyltransferase (cat) gene flanked by HIV-1 LTRs. Chloramphenicol acetyltransferase protein expression was equivalent in rabbit and human cell lines transfected with the HIV-1/CAT constructs and cotransfections with the HIV-1 tat gene led to similar increases in CAT expression. Subsequent transfections with an infectious molecular HIV clone yielded approximately equal levels of HIV protein expression in rabbit and human cell lines, suggesting that major barriers to virus production in rabbit lines exist at steps prior to transcription of the viral genome. Because HTLV-I replicates with high efficiency in rabbit cells, a chimeric virus clone was constructed consisting of the 5' portion of HIV-1 through the nef coding sequence followed by the 3' HTLV-I LTR. Transfection of most rabbit cell lines with the chimera produced levels of p24gag protein higher than those transfected with the parent HIV-1 clone. By contrast, the unmodified HIV clone replicated more efficiently in all human cell lines tested.

Animals↗
PubMed1998

Antimicrobial peptides melittin and cecropin inhibit replication of human immunodeficiency virus 1 by suppressing viral gene expression.

Antimicrobial peptides are effectors of innate immunity, providing their hosts with rapid non-specific defence against parasitic invaders. In this report, the effects are assessed of two well-characterized antimicrobial amphipathic peptides (melittin and cecropin) on human immunodeficiency virus 1 (HIV-1) replication and gene expression in acutely infected cells at subtoxic concentrations. Production of infectious, cell-free virus was inhibited in a dose-dependent manner, with ID50 values in the range 0.9-1.5 microM for melittin and 2-3 microM for cecropin. Analysis of the effect of melittin on cell-associated virus production revealed decreased levels of Gag antigen and HIV-1 mRNAs. Transient transfection assays with HIV long terminal repeat (LTR)-driven reporter gene plasmids indicated that melittin has a direct suppressive effect on activity of the HIV LTR. HIV LTR activity was also reduced in human cells stably transfected with retroviral expression plasmids for the melittin or cecropin gene. It is concluded that antimicrobial peptides such as melittin and cecropin are capable of inhibiting cell-associated production of HIV-1 by suppressing HIV-1 gene expression.

Anti-Infective Agents↗
PubMed2002

Transient induction of cyclin T1 during human macrophage differentiation regulates human immunodeficiency virus type 1 Tat transactivation function.

The human immunodeficiency virus type 1 (HIV-1) Tat protein is essential for viral replication and stimulates transcription of the integrated provirus by recruiting the kinase complex TAK/P-TEFb, composed of cyclin T1 (CycT1) and Cdk9, to the viral TAR RNA element. TAK/P-TEFb phosphorylates the RNA polymerase II complex and stimulates transcriptional elongation. In this report, we investigated the regulation of TAK/P-TEFb in primary human macrophages, a major target cell of HIV infection. While Cdk9 levels remained constant, CycT1 protein expression in freshly isolated monocytes was very low, increased early during macrophage differentiation, and, unexpectedly, decreased to very low levels after about 1 week in culture. The kinase activity of TAK/P-TEFb paralleled the changes in CycT1 protein expression. RNA analysis indicated that the transient induction of CycT1 protein expression involves a posttranscriptional mechanism. In transient transfection assays, the ability of Tat to transactivate the HIV long terminal repeat (LTR) in the late differentiated macrophages was greatly diminished relative to its ability to transactivate the HIV LTR in early differentiated cells, strongly suggesting that CycT1 is limiting for Tat function in late differentiated macrophages. Interestingly, lipopolysaccharide, a component of the cell wall of gram-negative bacteria, reinduced CycT1 expression late in macrophage differentiation. These results raise the possibility that regulation of CycT1 expression may be involved in establishing latent infection in macrophages and that opportunistic infection may reactivate the virus by inducing CycT1 expression.

CD11c Antigen↗
PubMed2004

Mutation of the major 5' splice site renders a CMV-driven HIV-1 proviral clone Tat-dependent: connections between transcription and splicing.

Efficient transcription from the human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR) promoter is dependent on the viral transactivator Tat. To generate a Tat-independent proviral plasmid, we replaced the promoter in the HIV-1 LTR with the immediate early promoter of cytomegalovirus. Transfection of this plasmid yielded Tat-independent production of infectious HIV-1. Tat-independent expression was lost, however, when the major 5' splice site in the HIV-1 genome was mutated and no HIV-1-specific RNA or protein was detected. This defect was restored when a Tat expression plasmid was cotransfected. Our results support recent reports indicating an influence of the recognition of splice sites on efficient transcriptional elongation.

5' Untranslated Regions↗
PubMed1997

Contribution to the regulation of virus replication in cells latently infected with human immunodeficiency virus 1.

Monocytes/macrophages have been known to play an important role in the initiation and propagation of human immunodeficiency virus 1 (HIV-1) infection. To analyze the function of these cells during the clinical asymptomatic period of infection, we examined the effect of murine peritoneal macrophages and human peripheral blood macrophages on two cell lines latently infected with HIV-1, a promonocytic cell line, U1, and a T-cell line, ACH-2. Monokines of the murine peritoneal macrophages induced significant viral expression in U1, but not in ACH-2 cells. Experiments employing transient transfection of U937 and CEM cells with HIV long terminal repeat (LTR)-chloramphenicol acetyl transferase (CAT) plasmids indicated that the effect of these monokines was due to specific activation of the HIV LTR. In contrast, supernatants of human macrophages induced viral expression in both ACH-2 and U1 cells. These results suggest that several monokines are active in regulating the transition from the clinical asymptomatic period of HIV infection to progression to acquired immunodeficiency syndrome (AIDS).

Acquired Immunodeficiency Syndrome↗
PubMed1995

Infection and replication of Tat- human immunodeficiency viruses: genetic analyses of LTR and tat mutations in primary and long-term human lymphoid cells.

Tat is an essential regulatory protein for the replication of human immunodeficiency virus (HIV). Mutations in the tat gene have been shown to block HIV replication in human T cells. Several studies have established that Tat releases an elongation block to the transcription of HIV long terminal repeat (LTR); however, it is not known whether this mechanism alone is sufficient to explain the block to HIV replication in human T cells when Tat is absent. It is possible that Tat is also needed for other functions during HIV replication. To test these hypotheses, we studied several tat mutants, including two stop codon mutants and one deletion mutant using replication-competent HIV-1 constructs carrying wild-type or mutant LTRs with modifications in the NF-kappa B and/or Sp1 binding sites. In this study, we show that Tat- HIV-1 with wild-type LTRs can replicate in HeLa cells, and the virus produced from HeLa cells can infect primary peripheral blood lymphocytes and macrophages. It was found that the propagation of the Tat mutants containing wild-type LTRs was less efficient than that of the LTR-modified Tat mutants. Large amounts of viral RNA and particles were synthesized in infections established using the tat mutants that contain modified LTRs. However, this efficient propagation of the LTR-modified tat mutants was restricted to some lymphoid cell lines that have been transformed with other viruses. Thus, despite its essential role for releasing an elongation block, Tat is not otherwise absolutely required for synthesis of full-length HIV transcripts and assembly of virus particles. Direct sequencing of the viral genomes and reinfection kinetics showed no evidence of wild-type reversion even after prolonged infection with the Tat- virus. The implications for in vivo HIV-1 replication and potential application of this system to the study of alternative Tat function are discussed.

Animals↗
PubMed1989

Inhibition of human immunodeficiency virus (HIV) replication by HIV-trans-activated alpha 2-interferon.

We have prepared stable cell lines, derived from Vero cells and A3.01 cells, that express a hybrid human alpha 2-interferon gene under control of the human immunodeficiency virus (HIV) long terminal repeat. These cells constitutively produced low levels (50-150 units/ml) of alpha 2-interferon. However, high levels of interferon (10(3) units/ml) could be induced upon trans-activation by the product of the tat gene (pIIIextatIII), and de novo infection by HIV resulted in a moderate increase (400 units/ml) in alpha 2-interferon synthesis. In contrast to the fully permissive HIV replication, in transfected Vero cells or infected A3.01 cells, the transcription and replication of HIV in Vero or A3.01 cells containing the HIV long terminal repeat--alpha 2-interferon hybrid gene (VN89 and A3N89 cells, respectively) was completely inhibited. These data suggest that virus-trans-activated alpha 2-interferon synthesis can be used as a selective inhibitor of HIV replication.

Animals↗
PubMed1993

HIV-1 Nef protein inhibits the recruitment of AP-1 DNA-binding activity in human T-cells.

The human immunodeficiency virus type 1 long terminal repeat, HIV-1-LTR, contains binding sites for several cellular transcription factors which contribute to HIV-1 gene expression. Our previous studies on the function of the HIV-1-encoded Nef protein suggested that Nef may be an inhibitor HIV-1 transcription. To determine whether Nef affects the binding of cellular factors implicated in HIV-1 regulation, 32P-labeled oligonucleotides corresponding to the binding sites were incubated with nuclear extracts prepared from Nef-expressing T-cell lines that were not stimulated or were stimulated with T-cell mitogens. We found that Nef inhibited the recruitment of AP-1 DNA-binding activity in mitogen-stimulated human T-cells. Additionally, Nef expressing cells were transiently transfected with a plasmid in which HIV-1 AP-1 DNA recognition sequences were cloned downstream of the chloramphenicol acetyltransferase (CAT) gene. Mitogen-mediated transcriptional activation of the CAT gene in this construct was inhibited in Nef-expressing cells but not in control cells. These studies suggest that, by inhibiting AP-1 activation, Nef may play a role in regulating HIV-1 gene expression in infected T-cells.

Base Sequence↗
PubMed1992

Infection of human brain cells by HIV-1: restricted virus production in chronically infected human glial cell lines.

OBJECTIVE: To study expression of HIV-1 in human glial cell lines. DESIGN: Chronically HIV-1-infected glial cell lines were established to evade potential artefacts resulting from unphysiological viral entry (i.e., transfection). These cell lines were used to study viral expression and regulation. METHODS: Chronically infected glial cell lines were established by terminal dilution cloning of human glioma cells exposed to HIV-1. Virus production and expression were assayed by measuring reverse transcriptase activity, p24-antigen levels and syncytia-inducing capacity in C8166 target cells (extracellular), or by indirect immunoperoxidase staining, immunoblot analysis, and p24- and Nef-antigen-capture enzyme-linked immunosorbent assays (intracellular). HIV-long terminal repeat (LTR)-dependent expression of the chloramphenicol acetyltransferase reporter gene was determined in transient transfection assays. RESULTS: Culture supernatant from chronically HIV-1-infected glial cells contained only low levels of virus compared with chronically HIV-infected fibroblasts and T-lymphoma cells. Detailed study of HIV-antigen expression in representative glial cell line TH4-7-5 indicated the presence of all major structural proteins, albeit at low levels, and of Vif, Tat, Rev and Nef. Intracellular levels of Nef exceeded p24-antigen levels by approximately 10-fold. Virus was recovered from TH4-7-5 cells by cocultivation with blood-derived target cells, indicating that low-level virus production is not due to defective provirus. Prominent negative regulatory element (NRE)-mediated suppression of exogenous HIV-LTR activity was observed in TH4-7-5 cells and was unequalled by chronically HIV-producing fibroblast cells or by uninfected fibroblast and glial cells. CONCLUSIONS: Our results suggest that restricted virus production by chronically infected glial cells involves LTR-mediated regulation of virus expression.

Base Sequence↗
PubMed1998

Treponema pallidum, lipoproteins, and synthetic lipoprotein analogues induce human immunodeficiency virus type 1 gene expression in monocytes via NF-kappaB activation.

Syphilitic genital ulcers are cofactors for the bidirectional transmission of human immunodeficiency virus (HIV). U937 human promonocytic cells chronically infected with HIV-1 (U1 cells) or transiently transfected with wild type or mutant HIV long terminal repeat (LTR) reporter constructs were used to examine mechanisms that likely underlie Treponema pallidum-induced immune cell activation and consequent induction of HIV. Virulent T. pallidum, a representative native treponemal lipoprotein (NTp47), or synthetic lipoprotein analogues (lipopeptides) all induced HIV replication in U1 cells. These stimuli also induced HIV gene expression from a wild type HIV LTR. HIV gene expression correlated with the translocation of NF-kappaB, and mutations within the NF-kappaB binding sites of the HIV LTR abrogated HIV gene expression. This study implicates treponemal lipoproteins as key mediators of immune cell activation and provides insights into the cellular and molecular bases for enhanced HIV transmission in syphilitic persons.

Animals↗
PubMed2005

NF-kappaB p50 promotes HIV latency through HDAC recruitment and repression of transcriptional initiation.

Cells latently infected with HIV represent a currently insurmountable barrier to viral eradication in infected patients. Using the J-Lat human T-cell model of HIV latency, we have investigated the role of host factor binding to the kappaB enhancer elements of the HIV long terminal repeat (LTR) in the maintenance of viral latency. We show that NF-kappaB p50-HDAC1 complexes constitutively bind the latent HIV LTR and induce histone deacetylation and repressive changes in chromatin structure of the HIV LTR, changes that impair recruitment of RNA polymerase II and transcriptional initiation. Knockdown of p50 expression with specific small hairpin RNAs reduces HDAC1 binding to the latent HIV LTR and induces RNA polymerase II recruitment. Similarly, inhibition of histone deacetylase (HDAC) activity with trichostatin A promotes binding of RNA polymerase II to the latent HIV LTR. This bound polymerase complex, however, remains non-processive, generating only short viral transcripts. Synthesis of full-length viral transcripts can be rescued under these conditions by expression of Tat. The combination of HDAC inhibitors and Tat merits consideration as a new strategy for purging latent HIV proviruses from their cellular reservoirs.

Cells, Cultured↗
PubMed1999

Recruitment of cyclin T1/P-TEFb to an HIV type 1 long terminal repeat promoter proximal RNA target is both necessary and sufficient for full activation of transcription.

Transcriptional activation of the HIV type 1 (HIV-1) long terminal repeat (LTR) promoter element by the viral Tat protein is an essential step in the HIV-1 life cycle. Tat function is mediated by the TAR RNA target element encoded within the LTR and is known to require the recruitment of a complex consisting of Tat and the cyclin T1 (CycT1) component of positive transcription elongation factor b (P-TEFb) to TAR. Here, we demonstrate that both TAR and Tat become entirely dispensable for activation of the HIV-1 LTR promoter when CycT1/P-TEFb is artificially recruited to a heterologous promoter proximal RNA target. The level of activation observed was indistinguishable from the level induced by Tat and was neither inhibited nor increased when Tat was expressed in trans. Activation by artificially recruited CycT1 depended on the ability to bind the CDK9 component of P-TEFb. In contrast, although binding to both Tat and TAR was essential for the ability of CycT1 to act as a Tat cofactor, these interactions became dispensable when CycT1 was directly recruited to the LTR. Importantly, activation of the LTR both by Tat and by directly recruited CycT1 was found to be at the level of transcription elongation. Together, these data demonstrate that recruitment of CycT1/P-TEFb to the HIV-1 LTR is fully sufficient to activate this promoter element and imply that the sole role of the Tat/TAR axis in viral transcription is to permit the recruitment of CycT1/P-TEFb.

Animals↗
PubMed2000

Transcriptional regulation of HIV-1 LTR during antigen-dependent activation of primary T cells by dendritic cells.

Numerous factors are known to bind human immunodeficiency virus (HIV) long terminal repeat (LTR) and activate viral transcription, but little is known as to how they function in naturally activated T cells and to what extent their binding is relevant to HIV replication in vivo. To characterize the HIV LTR-binding factors responsible for antigen-dependent activation of HIV, we examined replication of LTR mutant viruses in CD4+ T cells activated by different stimuli. NF-kappaB or Sp1 mutant virus replicated well in CD4+ T cells activated by phorbol ester and calcium ionophore. When they were activated by antigen-pulsed dendritic cells, the replication of the Sp1-deleted virus was severely impaired in CD45RA+, but not in CD45RO+ T cell subsets that dominantly produce interleukin-2 (IL-2). Stimulation via CD3/CD28 induced a high level of IL-2 production in both T cell subsets, but Sp1-deleted virus poorly replicated in CD45RA+ subset. The level of NF-kappaB and Sp1-binding factors did not differ between these subsets. Our results suggest that additional cofactors distinct from IL-2-inducing signaling molecules are important for LTR activation during antigen-dependent T cell activation.

Antigen Presentation↗
PubMed2002

Inhibition of endosomal/lysosomal degradation increases the infectivity of human immunodeficiency virus.

Productive entry of human immunodeficiency virus type 1 (HIV-1) into a host cell is believed to proceed via fusion of the viral envelope with the host cell's plasma membrane. Interestingly, the majority of HIV-1 particles that bind to the cell surface are taken up by the host cell via endocytosis; however, this mode of internalization generally does not result in infection. Presumably, virus particles remain trapped in the endocytic pathway and are eventually degraded. Here, we demonstrate that treatment of cells with various pharmacological agents known to elevate the pH of endosomes and lysosomes allows HIV-1 to efficiently enter and infect the host cell. Pretreatment of cells with bafilomycin A1 results in up to a 50-fold increase in the infectivity of HIV-1(SF2). Similarly, pretreatment of target cells with amantadine, concanamycin A, concanamycin B, chloroquine, and ammonium chloride resulted in increases in HIV-1 infectivity ranging between 2- and 15-fold. Analysis of receptor and coreceptor expression, HIV-long terminal repeat (LTR) transactivation, and transduction with amphotropic-pseudotyped murine leukemia virus (MLV)-based vectors suggests that the increase in infectivity is not artifactual. The increased infectivity under these conditions appears to be due to the ability of HIV-1 and MLV particles to enter via the endocytic pathway when spared from degradation in the late endosomes and lysosomes. These results could have significant implications for the administration of current and future lysosmotropic agents to patients with HIV disease.

Amantadine↗
PubMed1997

A potent activator of HIV-1 replication is present in the genital tract of a subset of HIV-1-infected and uninfected women.

OBJECTIVE AND DESIGN: To determine whether the female genital tract contains factors that affect HIV-1 replication. Cervicovaginal lavage (CVL) samples were collected from HIV-1-seropositive and seronegative women and added to cell cultures. METHODS: HIV p24 production was used to measure the effects of CVL on replication of HIVMN in a T-cell line, of a primary isolate in peripheral blood mononuclear cells, or on HIV expression by the latently-infected monocytic U1 cell line. The effects of CVL on the HIV long terminal repeat (LTR) were determined in 1G5 T cells by measuring luciferase activity. RESULTS: Increased replication of HIVMN and a primary isolate were observed in T cells cultured with CVL samples from three out of 38 HIV-infected women, one out of four uninfected high-risk women, and none of 12 low-risk women. The CVL factor increased replication by enhancing virus expression via activation of the HIV LTR. The HIV-inducing activity was highly stable to heat but was sensitive to proteases, indicating that the activity was distinct from heat-labile cytokines including tumour necrosis factor-alpha. CONCLUSIONS: This is the first study to show that a factor which can stimulate HIV-1 replication is present at biologically active levels in the reproductive tract of women. This factor could potentially affect sexual or vertical transmission of HIV-1 by altering genital tract virus load or virus expression.

Cells, Cultured↗