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PubMed1989

Multiple functional domains of Tat, the trans-activator of HIV-1, defined by mutational analysis.

The tat gene of HIV-1 is a potent trans-activator of gene expression from the HIV long terminal repeat (LTR). To define the functionally important regions of the product of the tat gene (Tat) of HIV-1, deletion, linker insertion and single amino acid substitution mutants within the Tat coding region of strain SF2 were constructed. The effect of these mutations on trans-activation was assessed by measuring the expression of the bacterial chloramphenicol acetyltransferase (CAT) reporter gene linked to the HIV-LTR. These studies have revealed that four different domains of the protein that map within the N-terminal 56 amino acid region are essential for Tat function. In addition to the essential domains, an auxiliary domain that enhances the activity of the essential region has also been mapped between amino acid residues 58 and 66. One of the essential domains maps in the N-terminal 20 amino acid region. The other three essential domains are highly conserved among the various strains of HIV-1 and HIV-2 as well as simian immunodeficiency virus (SIV). Of the conserved domains, one contains seven Cys residues and single amino acid substitutions for several Cys residues indicate that they are essential for Tat function. The second conserved domain contains a Lys X Leu Gly Ile X Tyr motif in which the Lys residue is essential for trans-activation and the other residues are partially essential. The third conserved domain is strongly basic and appears to play a dual role. Mutants lacking this domain are deficient in trans-activation and in efficient targeting of Tat to the nucleus and nucleolus. The combination of the four essential domains and the auxiliary domain contribute to the near full activity observed with the 101 amino acid Tat protein.

Amino Acid Sequence↗

PubMed1990

Augmentation of cytotoxic effect of tumor necrosis factor on human immunodeficiency virus-infected cells by staurosporine, a potent protein kinase C inhibitor.

We have examined the effect of the protein kinase C (PKC) inhibitor, staurosporine, on tumor necrosis factor (TNF)-induced cytotoxic action and augmentation of human immunodeficiency virus (HIV) expression on the chronically HIV-infected T-cell line, MOLT-4/HIV (HTLV-IIIB strain). Staurosporine enhanced the decrease in the number of viable cells caused by TNF treatment for 3 days (1 ng/ml of TNF, 43% decrease; 1 ng/ml of TNF + 20 nM staurosporine, 94%), whereas the cytotoxic action on that cell line induced by 10 ng/ml of 12-O-tetradecanoylphorbol-13-acetate (TPA), which was known to be an activator of PKC, was partially inhibited by staurosporine. In addition, staurosporine augmented the TNF cytotoxic activity against other cell lines including HIV-uninfected U937 cells(100 ng/ml of TNF, 53% decrease in the number of viable cells; 100 ng/ml of TNF + 5 nM staurosporine, 86%). However, staurosporine did not change the sensitivity of cells to TNF; thus, those insensitive to TNF were not changed to TNF sensitive by staurosporine. Furthermore, staurosporine did not affect the augmentative effect of TNF on HIV expression evaluated by levels of p24 antigen. Moreover, HIV long terminal repeat (LTR)-directed chloramphenicol acetyltransferase assay showed that staurosporine strongly inhibited the TPA-induced activation of HIV LTR, while that caused by TNF was little affected (10 ng/ml of TPA, 98.4% conversion; 10 ng/ml of TPA + 40 nM staurosporine, 22.2%, 1 ng/ml of TNF, 98.5%; 10 ng/ml of TNF + 40 nM staurosporine, 93.9%). These results suggest that TPA and TNF facilitate HIV replication by different pathways and that staurosporine augments TNF cytotoxicity by possible suppression of PKC activity in both HIV-infected and uninfected cells.

Alkaloids↗

PubMedDate not supplied

Herpes simplex virus type-1 can reactivate transcription of latent human immunodeficiency virus.

The human immunodeficiency virus, HIV (formerly T-cell lymphotropic virus type III, HTLV-III or lymphadenopathy-associated virus, LAV) is the primary cause of AIDS (acquired immune deficiency syndrome). Patients with AIDS have profound immunosuppression as a result of almost complete absence of the OKT4+ cell population and are predisposed to a number of opportunistic infections as well as to certain malignant diseases such as Kaposi's sarcoma and B-cell tumours. The majority of the opportunistic infections observed in AIDS patients are from the herpesvirus group and these are frequently the cause of death in AIDS patients. We have therefore investigated the effect of herpes virus infection on the expression of HIV and we provide evidence that herpes simplex virus type I (HSV-I) can reactivate transcription of latent HIV. In OKT4+ human T-cells HIV replicates to high virus titres, resulting in high level expression of viral RNA. This high level of expression has been attributed to virus-associated trans-acting factors that increase gene expression, directed by the HIV long terminal repeats (LTR), post-transcriptionally. In our studies we have tested whether transcription directed by the LTR of HIV is stimulated by HSV-I.

Antibodies, Monoclonal↗

PubMed1989

Tumor necrosis factor alpha induces expression of human immunodeficiency virus in a chronically infected T-cell clone.

Tumor necrosis factor alpha (TNF-alpha), also known as cachectin, was demonstrated to induce the expression of human immunodeficiency virus (HIV) in a chronically infected T-cell clone (ACH-2). Concentrations of recombinant TNF-alpha as low as 50 pg/ml induced a significant increase over background of HIV expression in the ACH-2 cells as determined by supernatant reverse transcriptase activity. The HIV-inducing effects of TNF-alpha could not be explained by toxic effects on the cells. In addition, both the uninfected parental cell line (A3.01) and the infected ACH-2 cells were shown to have high-affinity receptors for TNF-alpha. Transient-transfection experiments demonstrated that the inductive effects of TNF-alpha were due to specific activation of the HIV long terminal repeat. These studies provide evidence that TNF-alpha may play a role in the mechanisms of pathogenesis of HIV infection.

Cell Line↗

PubMed1992

Regulation of human immunodeficiency virus enhancer function by PRDII-BF1 and c-rel gene products.

The human immunodeficiency virus (HIV) enhancer element is important in the regulation of HIV gene expression. A number of cellular proteins have been demonstrated to bind to the NF-kappa B motifs in this element. The genes encoding several of these proteins, including members of the rel family and PRDII-BF1, have been cloned. We characterized the binding of proteins encoded by the human c-rel and PRDII-BF1 genes to HIV NF-kappa B motifs and related enhancer elements. Both the human c-rel protein and two proteins derived from the PRDII-BF1 gene by alternative splicing bound specifically to the HIV NF-kappa B motif and related enhancer elements found in the immunoglobulin kappa, class I major histocompatibility complex, and interleukin-2 receptor genes. To determine the role of these factors in regulating HIV gene expression, we fused the cDNAs encoding either of the two proteins derived by alternative splicing of the PRDII-BF1 gene or the c-rel gene to the DNA binding region of the yeast transcription factor GAL4. GAL4 binding sites were inserted in place of the native HIV enhancer sequences in an HIV long terminal repeat chloramphenicol acetyltransferase construct. Cotransfection of these constructs revealed that c-rel was a strong activator of basal HIV gene expression but did not result in synergistic effects in the presence of tat. PRDII-BF1-derived cDNAs did not result in stimulation of either basal or tat-induced activated gene expression. These results indicate that multiple enhancer binding proteins may potentially regulate HIV in both a positive and negative manner.

Base Sequence↗

PubMed2005

Human immunodeficiency virus reactivation by phorbol esters or T-cell receptor ligation requires both PKCalpha and PKCtheta.

Latently human immunodeficiency virus (HIV)-infected memory CD4(+) T cells represent the major obstacle to eradicating HIV from infected patients. Antigens, T-cell receptor (TCR) ligation, and phorbol esters can reactivate HIV from latency in a protein kinase C (PKC)-dependent manner; however, it is unknown which specific PKC isoforms are required for this effect. We demonstrate that constitutively active (CA) forms of both PKCtheta, PKCthetaA148E, and PKCalpha, PKCalphaA25E, induce HIV long terminal repeat (LTR)-dependent transcription in Jurkat and primary human CD4(+) T cells and that both PKCthetaA148E and PKCalphaA25E cause HIV reactivation in J1.1 T cells. Suppression of both PKCalpha and PKCtheta with short hairpinned (sh) RNA inhibited CD3/CD28-induced HIV LTR-dependent transcription and HIV reactivation in J1.1 T cells. Both prostratin and phorbol myristate 13-acetate induced HIV LTR-dependent transcription and HIV reactivation in J1.1 T cells that was blocked by shRNA against either PKCalpha or PKCtheta. Since suppression of PKCalpha and PKCtheta together has no greater inhibitory effect on HIV reactivation than inhibition of PKCalpha alone, our data confirm that PKCalpha and PKCtheta act in sequence. The requirement for PKCalpha and PKCtheta for prostratin-induced HIV reactivation and the ability of selective PKCalpha or PKCtheta agonists to induce HIV transcription indicate that these PKC isoforms are important targets for therapeutic drug design.

Cells, Cultured↗

PubMed1993

A novel mode of human immunodeficiency virus type 1 (HIV-1) activation: ligation of CD28 alone induces HIV-1 replication in naturally infected lymphocytes.

Induction of human immunodeficiency virus (HIV) replication in infected CD4+ T lymphocytes requires cellular activation. The ligation of CD28, a signal-transducing receptor with a natural ligand on activated B cells and antigen-presenting cells, provides a costimulating signal for interleukin 2 production and T-cell proliferation as well as coactivation of the transfected HIV long terminal repeat in Jurkat cells. The aim of the present study was to investigate the ability of CD28 ligation to activate HIV type 1 (HIV-1) replication in naturally infected CD4+ lymphocytes either alone or in combination with immobilized anti-CD3 monoclonal antibody. Our results show that HIV-1 was successfully isolated from 16 of 28 patients. For 5 of these 16, virus was isolated only when anti-CD28 was added in combination with the anti-CD3. Moreover, stimulation by anti-CD28 alone induced HIV-1 replication in 5 of 12 patients tested, in the absence of cell proliferation. We found no correlation between the level of CD3- or CD28-induced proliferative response and induction of HIV-1 replication. Therefore, CD28 ligation, a nonmitogenic CD4+ T-cell activation signal, is sufficient to induce transcription and replication of HIV-1 in naturally infected lymphocytes.

Antigens, CD↗

PubMed1995

Triggering of complement receptors CR1 (CD35) and CR3 (CD11b/CD18) induces nuclear translocation of NF-kappa B (p50/p65) in human monocytes and enhances viral replication in HIV-infected monocytic cells.

Monocyte/macrophages may harbor HIV in a nonproductive fashion for prolonged periods of time. Viral gene expression may be reactivated by stimulation of the cells with LPS or cytokines such as TNF-alpha in vitro. The effect of LPS and TNF-alpha is mediated by their ability to induce nuclear translocation of the DNA-binding heterodimer NF-kappa B (p50/p65), which binds to a specific sequence in the HIV-long terminal repeat. The present study demonstrates that triggering of complement receptors CR1 (CD35) and CR3 (CD11b/CD18) enhances viral replication in HIV-infected human monocytic cells. Monocytic cell lines and normal peripheral blood monocytes were infected with HIV-1 in vitro and cultured in the presence or absence of F(ab')2 fragments of monoclonal anti-CR1 or anti-CR3 Abs or with C3 fragments. Stimulation of CR1 or CR3 induces a two- to fourfold increase in the amount of cell-associated and released p24 Ag in cell cultures that was equivalent to that observed in control cultures triggered with LPS. We further observed that stimulation of CR1 or CR3 induces the nuclear translocation of NF-kappa B p50/p65 in infected cells. Translocation of NF-kappa B p50/p65 was also observed following stimulation of CR1 or CR3 of uninfected peripheral blood monocytes from HIV-seronegative donors. The amount of protein translocated was similar to that observed when cells were stimulated with rhTNF-alpha. TNF-alpha did not mediate the translocation of NF-kappa B p50/p65 induced by triggering of complement receptors. Taken together, these observations suggest that HIV gene expression may be activated in infected monocytes through interaction of the cells with complement-opsonized particles and that enhanced viral replication is associated with C3 receptor-mediated nuclear translocation of the NF-kappa B complex.

Biological Transport↗

PubMed1992

Sequences within the R region of the long terminal repeat activate basal transcription from the HIV-1 promoter.

The importance of the R region in basal human immunodeficiency virus type 1 (HIV-1) transcription was addressed by comparing a panel of HIV-1 R region mutants using in vitro and in vivo assays. Using deletion, base substitution mutants, and compensatory mutants, the precise R region sequences essential for basal HIV-1 promoter activity in vitro were mapped to sequences between +17 to +21. Within this regulatory domain, nucleotides +19 and +21 appear to be critical. The effect of these mutations on steady state RNA levels in transfected cells has been analyzed by S1 nuclease protection assay using uniformly labeled probes. Two main conclusions may be drawn from these studies. First, HIV-1 basal transcription is abundant, with the majority of correctly initiated transcripts truncated between sequences +57 to +70. Second, analysis of the compensatory mutants indicates the secondary structure of the nascent R region RNA is not an obligate requirement for the production of the truncated transcripts. Mutations in R region primary sequence that selectively abolish the production of the truncated transcripts in vivo also exhibit reduced promoter activity in vitro. The appearance of high levels of truncated transcripts raise the interesting possibility that-similar to c-myc, c-myb, and c-fos--basal HIV-1 expression is regulated by transcription elongation.

Gene Expression Regulation, Viral↗

PubMed1993

A recombinant retrovirus carrying a non-producer human immunodeficiency virus (HIV) type 1 variant induces resistance to superinfecting HIV.

A human immunodeficiency virus (HIV) type 1-infected Hut-78 cell clone (F12) shows a peculiar phenotype: it exhibits an altered viral protein pattern, is a nonproducer and is resistant to homologous superinfection. To determine whether this phenotype is dependent upon the expression of the HIV-1 genome integrated therein, the SstI/SstI F12 provirus [deprived of HIV long terminal repeats (LTRs)] was cloned and inserted in the pLj retroviral vector bearing the neomycin (neo) and Geneticin resistance gene. CD4+ HIV-susceptible CEMss cells (a CEM clone able to form large syncytia 2 to 3 days post-HIV infection) were infected with the recombinant retroviruses rescued from the F12/HIV-pLj-transfected (in either sense or antisense orientation) amphotropic packaging cells PA 317. Neo sense resistant gene clones showed approximately 10 copies of viral DNA/cell (without detectable major deletions) only in episomal form, low viral RNA expression and a viral protein pattern characterized by an uncleaved gp160, no gp41 and little, if any, p55 gag precursor (as in F12 cells). Superinfection of these F12/HIV DNA-engineered clones with HIV-1 resulted in a significant reduction in the yield of superinfecting HIV. This effect (more pronounced when the clones were maintained under neo selective pressure) was observed in all five retrovirus-infected clones exhibiting the presence and expression of sense episomal F12/HIV DNA but not in two clones bearing an antisense F12/HIV DNA or in one clone bearing only the pLj vector. These results indicate that bio-engineered human CD4+ cells expressing the F12/HIV genome exhibit a significant resistance to HIV superinfection.

Antigens, Viral↗

PubMed1996

The Ras-Raf pathway is activated in human immunodeficiency virus-infected monocytes and particpates in the activation of NF-kappa B.

Persistent human immunodeficiency virus (HIV) infection of human monocytes and macrophages increases I kappa B alpha degradation, resulting in the activation of NF-kappa B, a key transcription factor in the regulation of the HIV long terminal repeat. The signal transduction pathways leading to NF-kappa B activation in cells of the monocytic lineage, especially those regulated by HIV infection, and their relevance in regulating viral persistence remain unknown. Both p21ras and its downstream Raf-1 kinase participate in the transduction of signals initiated from a variety of cell surface receptors and in the regulation of transcription factors. We have studied whether the Ras-Raf pathway is functional and participates in HIV-mediated NF-kappa B activation in monocytic cells. Constitutively active p21ras (v-H-Ras) activated NF- kappa B-dependent transcription and induces the nuclear translocation of a bona fide p65/p50 heterodimer by targeting I kappa B alpha. In addition, the constitutively active form of Raf (RafBXB) also increases the NF-kappa B-dependent transcriptional activity. Because of the similarity between HIV and Ras-Raf-induced NF-kappa B activation in monocytic cells, we next tested whether HIV-induced NF-kappa B activation was mediated by the Ras-Raf signal transduction pathway. Negative dominant forms of both Ras (Ras N17) and Raf (Raf 301) decreased the HIV- but not lipopolysaccharide-dependent NF-kappa B activation in U937 cells. Moreover, Raf-1 kinase activity was greater in HIV-infected than uninfected monocytic cells in in vitro kinase assays. Altogether, these results indicate that the Ras-Raf pathway is unregulated in HIV monocytic cells and participates in the virus-induced activation of NF-kappa B.

Adaptor Protein Complex alpha Subunits↗

PubMed1998

Enhancement of HIV-1 replication in human macrophages is induced by CD8+ T cell soluble factors.

We previously reported that CD8+ T cell-derived factors enhanced HIV long terminal repeat (LTR)-mediated gene expression and replication in monocytic cell lines. We now report that replication of NSI and SI primary isolates of HIV-1 in human macrophages were significantly enhanced by CD8+ T cell supernatants. The CD8-mediated enhancement of HIV replication was abrogated by pertussis toxin in a dose-dependent manner. The sensitivity to pertussis toxin suggests that the CD8+ T cell-derived enhancing factor is acting through a G protein-coupled signalling pathway. Enhanced HIV replication in macrophages was accompanied by increased levels of HIV-1 mRNA, suggesting that CD8 enhancement was mediated at the transcriptional level. Interestingly, the replication of HIV(Bal), which replicates to high levels in macrophages, was not significantly modulated by culture with CD8+ T cell supernatants. Although direct co-culture of activated CD8+ T cells with HIV(Ada)-infected macrophages did not modulate replication, separation of the CD8+ T cells from macrophages in transwell cultures resulted in significant enhancement of replication. The inability to detect a modulatory effect in direct co-cultures appeared to be due to non-specific lysis of infected macrophages. Thus, soluble factors produced by CD8+ T cells exert strong enhancing effects on HIV-1 replication in human macrophages.

CD8-Positive T-Lymphocytes↗

PubMed1989

Human chromosome 12 is required for elevated HIV-1 expression in human-hamster hybrid cells.

Host cell factors act together with regulatory genes of the human immunodeficiency virus (HIV) to control virus production. Human-Chinese hamster ovary hybrid cell clones were used to probe for human chromosomes involved in regulating HIV gene expression. DNA transfection experiments showed that 4 of 18 clones had high levels of HIV gene expression measured by both extracellular virus production and transactivation of the HIV long terminal repeat in the presence of the trans-activator (tat) gene. Karyotype analyses revealed a 94% concordance (17/18) between human chromosome 12 and HIV gene expression. Other chromosomes had an 11 to 72% concordance with virus production.

Animals↗

PubMed1989

Replication of HIV in human fetal retinal cultures and established pigment epithelial cell lines.

The ability of the human immunodeficiency virus type 1 (HIV-1) to replicate in cells derived from ocular tissue was studied. Primary retinal cultures (containing both glial and neuronal cells) were found to support the replication of HIV upon transfection with molecularly cloned proviral DNA. In addition, established retinal pigment epithelial (RPE) cell lines also produced HIV particles upon transfection. HIV released by these cell lines was able to infect and induce characteristic cytopathic effects in T4+ cells. An indicator plasmid containing the HIV long terminal repeat sequences (LTR) linked to the chloramphenicol acetyltransferase gene showed barely detectable activity in RPE cells and was transactivated by the addition of the HIV "tat" gene. Based on these observations, direct infection of ocular tissue derived cells such as RPE, fetal retinal cells, retinoblastoma cells (Y 79, WER1), choroidal endothelial cells (Chor 55) (mix culture) and corneal fibroblasts (K61) by HIV was attempted. HIV replication in these cells was not detected by reverse transcriptase, antigen and transactivation function assays.

Adult↗

PubMed1990

DNA binding factors that bind to the negative regulatory element of the human immunodeficiency virus-1: regulation by nef.

The nef gene has been reported to be a silencer of human immunodeficiency virus (HIV) transcription that requires the presence of the negative regulatory element (NRE) located at the 5' end of the HIV long terminal repeat (LTR) to exert its negative effect. We have examined nuclear extracts from human, nontransformed T cells for factors that bind to the NRE of HIV-1 and to determine whether binding of factors to this region can be affected by the nef gene. Using gel retardation and methylation interference assays, we have observed several DNA binding factors that bind to a region between nucleotides -315 and -240 upstream of the cap site, within the NRE segment of the 5' LTR. Furthermore, the precise locations of the binding sites for two of these factors, termed here A1 and R, were determined. Factor A1 appears to belong to a family of cellular activation associated factors (called here A1-A4), but it is distinct in that it is the only DNA binding factor so far observed that appears to be downregulated by the nef gene or its product and that it has been found only in cells undergoing lymphokine-driven cell division. In contrast to the A factors, factor R appears to be associated with cellular quiescence and binds to a nearby but distinct site in the NRE. Experiments in which extracts were mixed before gel retardation suggest that the binding of factors R and A1 are mutually exclusive. Based on these observations we propose a model in which the nef gene aids in the maintenance of HIV latency by downregulating the binding of proliferation associated DNA binding factor, which we have called A1.

Base Sequence↗

PubMed1988

Alterations in binding characteristics of the human immunodeficiency virus enhancer factor.

Five regions of the human immunodeficiency virus (HIV) long terminal repeat (LTR) serve as binding sites for cellular proteins as demonstrated by DNase I footprinting. These include the negative regulatory, enhancer, SP1, TATA, and untranslated regions. The HIV enhancer region contains two direct repeats of a sequence, GGGACTTTCC, which is also found in the enhancer sequences of simian virus 40, cytomegalovirus, and the immunoglobulin kappa gene. To further characterize binding to the enhancer sequences in the HIV LTR, DNase I footprinting was performed using extracts prepared from several different cell lines. Extracts prepared from lymphoid cells gave altered binding over the enhancer region as compared with extracts prepared from either monocytes or HeLa cells. This altered binding in extracts prepared from lymphoid cells resulted in protection of both direct repeats in the HIV LTR in contrast to complete protection of only one direct repeat with HeLa cell extracts. When HeLa cells were treated with phorbol esters in either the presence or absence of the protein synthesis inhibitor cycloheximide, the binding characteristics over the enhancer element became similar to those seen in extracts prepared from lymphoid cells. These results suggest that phorbol esters may induce posttranslational modifications of cellular transcription factors that alter their DNA-binding characteristics.

Cells, Cultured↗

PubMedDate not supplied

Induction of NF-kappa B during monocyte differentiation is associated with activation of HIV-gene expression.

Cells of the monocyte-macrophage lineage are important targets of HIV infection. We report here that the phenotypic differentiation of monocyte cell lines induced by phorbol esters or tumour necrosis factor alpha (TNF alpha) is associated with expression of nuclear factor kappa B (NF-kappa B). In parallel with such differentiation, HIV transcription, monitored using an HIV long terminal repeat reporter gene construct, is activated in such cells under the influence of enhanced NF-kappa B expression. Also, in a promonocyte cell line chronically infected with HIV, NF-kappa B expression and HIV transcription were enhanced on stimulation with phorbol ester or TNF alpha. Thus, stimulation of monocyte cell lines by phorbol esters or TNF alpha induces cell differentiation and activates HIV transcription. Such a process may have fundamental implications in AIDS pathogenesis in vivo and may be important in disease progression induced by opportunistic infections directly or indirectly involving macrophages.

Cell Differentiation↗

PubMed1990

Development of RD-tat cell lines: use in HIV recombination studies.

The transactivator (tat) gene of human immunodeficiency virus (HIV) plays an essential role in the replication cycle of HIV. Previous studies have evaluated the extent and mechanistic aspects of tat-mediated transactivation using lymphoid and adherent non-lymphoid cells. We have exploited the transactivation property of the tat gene to achieve high levels of hybrid HIV resulting from recombination between HIV DNAs. For this purpose, we have generated stably transformed human rhabdomyosarcoma (RD) cell lines expressing tat gene product of HIV-1. Functional analysis of the cell lines for the presence of tat protein by transfecting HIV-long terminal repeat (LTR) linked to chloramphenicol acetyl transferase (CAT) revealed low, moderate, and high tat producer cell lines. RD-tat cell lines also showed enhanced virus production upon transfection of HIV-1 proviral DNA. Further, tat producer cell lines showed a high amount of hybrid virus in comparison to the control RD cells upon transfection of truncated viral DNAs. Thus, RD-tat cell lines would be valuable target cells for generating homogeneous viruses upon transfection of viral DNA.

Base Sequence↗