A human immunodeficiency virus Env inducible transcription system to examine consequences of gp120 expression.

According to several studies, the HIV-1 envelope gp120 protein and the co-receptor CXCR4 play an essential role in HIV-1 induced cell toxicity. Characterisation of the CD4-independent m7NDK isolate provided the opportunity of studying the effects of direct interactions between m7NDK gp120 and CXCR4. Therefore, an inducible expression system was designed enabling synthesis of HIV-1 Env proteins upon doxycycline induction. Analysis of the expression of the env gene of the m7NDK HIV-1 isolate revealed, unexpectedly, that even long-term expression of m7NDK gp120 did not result in cytotoxycity in CXCR4-positive or -negative cell lines. This is the first report of a CD4-independent HIV-1-protein inducible expression regulated through the Tet-On system and by an alternative splicing. Env inducible expression cell lines could constitute a useful cellular tool to undertake analysis of HIV Env protein expression.

Determination of essential amino acids involved in the CD4-independent tropism of the X4 human immunodeficiency virus type 1 m7NDK isolate: role of potential N glycosylations in the C2 and V3 regions of gp120.

Seven mutations in the C2, V3, and C3 regions of gp120 are implicated in the tropism of the first CD4-independent human immunodeficiency virus type 1 isolate, m7NDK. Site-directed mutagenesis revealed that three amino acids are essential to maintain this tropism, one in the C2 region and two in the V3 loop. Two mutations implied N glycosylation modifications.

CXCR4 is down-regulated in cells infected with the CD4-independent X4 human immunodeficiency virus type 1 isolate m7NDK.

Macrophages and T cells infected in vitro with CD4-dependent human immunodeficiency virus type 1 (HIV-1) isolates have reduced levels of CD4 protein, a phenomenon involved in retroviral interference. We have previously characterized the first CD4-independent HIV-1 X4 isolate m7NDK, which directly interacts with CXCR4 through its mutated gp120. We thus investigate CXCR4 expression in cells infected with this m7NDK CXCR4-dependent HIV-1 mutant. We present evidence of the down-regulation of CXCR4 membrane expression in CD4-positive or -negative cells chronically infected with the HIV-1 m7NDK, a phenomenon which is not observed in the CD4-dependent HIV-1 NDK parental strain. This down-regulation of CXCR4 was demonstrated by fluorescence-activated cell sorter analysis and was confirmed by the absence of CXCR4 functionality in m7NDK-infected cells, independently of the presence of CD4 protein. Furthermore, a drastic reduction of the intracellular level of CXCR4 protein was also observed. Reduced levels of CXCR4 mRNA transcripts were found in m7NDK-infected HeLa and CEM cells, reduced levels that could not be attributed to a reduced stability of CXCR4 mRNA. Down-regulation of CXCR4 on m7NDK-infected cells may thus be explained by transcriptional regulation.

Altered expression of tyrosine kinases of the Src and Syk families in human T-cell leukemia virus type 1-infected T-cell lines.

During the late phase of adult T-cell leukemia/lymphoma, a severe lymphoproliferative disorder caused by human T-cell leukemia virus type 1 (HTLV-1), leukemic cells no longer produce interleukin-2. Several studies have reported the lack of the Src-like protein tyrosine kinase Lck and overexpression of Lyn and Fyn in these cells. In this report we demonstrate that, in addition to the downregulation of TCR, CD45, and Lck (which are key components of T-cell activation), HTLV-1-infected cell lines demonstrate a large increase of FynB, a Fyn isoform usually poorly expressed in T cells. Furthermore, similar to anergic T cells, Fyn is hyperactive in one of these HTLV-1-infected T-cell lines, probably as a consequence of Csk downregulation. A second family of two proteins, Zap-70 and Syk, relay the signal of T-cell activation. We demonstrate that in contrast to uninfected T cells, Zap-70 is absent in HTLV-1-infected T cells, whereas Syk is overexpressed. In searching for the mechanism responsible for FynB overexpression and Zap-70 downregulation, we have investigated the ability of the Tax and Rex proteins to modulate Zap-70 expression and the alternative splicing mechanism which gives rise to either FynB or FynT. By using Jurkat T cells stably transfected with the tax and rex genes or inducibly expressing the tax gene, we found that the expression of Rex was necessary to increase fynB expression, suggesting that Rex controls fyn gene splicing. Conversely, with the same Jurkat clones, we found that the expression of Tax but not Rex could downregulate Zap-70 expression. These results suggest that the effect of Tax and Rex must cooperate to deregulate the pathway of T-cell activation in HTLV-1-infected T cells.

Regulation of human B19 parvovirus promoter expression by hGABP (E4TF1) transcription factor.

The genetic expression of human B19 parvovirus is only dependent on one promoter in vivo and in vitro. This is the P6 promoter, which is located on the left side of the genome and is a single-stranded DNA molecule. This led us to investigate the regulation of the P6 promoter and the possible resulting variability of the nucleotide sequence. After analysis of the promoter region of 17 B19 strains, only 1.5% variability was found. More exciting was the finding of mutations that were clustered around the TATA box and defined a highly conserved region (nucleotides 113-210) in the proximal part of the P6 promoter. HeLa and UT7/Epo cell extracts were found to protect this region, which contained a core motif for Ets family proteins, with YY1 and Sp1 binding sites on either side. Gel mobility shift assays performed with nuclear proteins from HeLa and UT7/Epo cells identified DNA-binding proteins specific for these sites. By supershift analysis, we demonstrated the binding of the hGABP (also named E4TF1) protein to the Ets binding site and the fixation of Sp1 and YY1 proteins on their respective motifs. In Drosophila SL2 cells, hGABPalpha and -beta stimulated P6 promoter activity, and hGABPalpha/hGABPbeta and Sp1 exerted synergistic stimulation of this activity, an effect diminished by YY1.

Spontaneous mutations in the env gene of the human immunodeficiency virus type 1 NDK isolate are associated with a CD4-independent entry phenotype.

Human immunodeficiency virus type 1 (HIV-1) entry into target cells is a multistep process initiated by envelope protein gp120 binding to cell surface CD4. The conformational changes induced by this interaction likely favor a second-step interaction between gp120 and a coreceptor such as CXCR4 or CCR5. Here, we report a spontaneous and stable CD4-independent entry phenotype for the HIV-1 NDK isolate. This mutant strain, which emerged from a population of chronically infected CD4-positive CEM cells, can replicate in CD4-negative human cell lines. The presence of CXCR4 alone renders cells susceptible to infection by the mutant NDK, and infection can be blocked by the CXCR4 natural ligand SDF-1. Furthermore, we have correlated the CD4-independent phenotype with seven mutations in the C2 and C3 regions and the V3 loop. We propose that the mutant gp120 spontaneously acquires a conformation allowing it to interact directly with CXCR4. This virus provides us with a powerful tool to study directly gp120-CXCR4 interactions.

Establishment of a cell line expressing human parvovirus B19 non-structural protein from an inducible promoter.

Human parvovirus B19 non-structural (NS) protein is supposed to play a major role in B19 replication and transcription, and therefore in B19 pathogenicity. Constitutive expression of NS protein in stable cell lines has failed so far, presumably because of its cytotoxicity. To avoid this cytotoxic effect, we have cloned the NS gene in an Epstein-Barr virus episomal vector under the control of a steroid inducible promoter (5xGRE) and transfected this construction into HeLa cells. We obtained stable cell lines inducibly expressing high level of NS protein, with 50% of the cells demonstrating specific nucleo-cytoplasmic staining. In Western blot analysis, three B19 NS proteins (72, 68 and 60 kDa) were found but a unique NS transcript was detected by Northern blotting. The NS protein expressed in HeLa cell lines was demonstrated to be functional as it trans-activates the B19 P6 promoter. These cell lines might be major tools for further study and characterization of B19 NS protein.

Trans-activation of the long terminal repeat of human immunodeficiency virus type 1 by the parvovirus B19 NS1 gene product.

Persistent parvovirus B19 infections in human immunodeficiency virus type 1 (HIV-1)-infected patients have been reported. The two viruses could share common target cells. The NS1 protein of B19 regulates B19 expression and we have investigated its possible effect on the long terminal repeat (LTR) of HIV-1. In transient transfection experiments, NS1 trans-activated the expression of reporter genes under the control of the HIV-1 LTR. The effect of NS1 was apparent only in the presence of the HIV-1 Tat protein, and required intact TAR and TATA box sequences.

Dispensable role of the NF-kappa B sites in the UV-induction of the HIV-1 LTR in transgenic mice.

We have previously reported the epidermis-specific expression of the HIV-1 LTR in transgenic mice and its induction by UV-B rays. To dissect the underlying mechanism of the UV induction of the LTR in mice, we developed two approaches. We first demonstrated by gel mobility shift analysis, using mice epidermal extracts, that the NF-kappa B sites of the HIV-1 LTR were one of the targets of the UV induction. The Sp-1 sites and the potential AP-1 sites of the LTR were not involved in this phenomenon. The transient transfection assays of modified LTR in HeLa cells also demonstrated the involvement of the NF-kappa B sites in the UV induction and were consistent with previously published data. Secondly, to study the regulation acting on an integrated gene, we generated transgenic mice carrying the lacZ gene under the control of the partially deleted LTR. All the transgenic lines and unexpectedly those carrying the LTR deleted for the kappa B sites displayed a UV-inducible epidermal expression. This suggests that, in mice, the UV induction might be mediated through other sites than the kappa B sites and may also depend on changes of the chromatin state.

Stimulation of a human T-cell clone with anti-CD3 or tumor necrosis factor induces NF-kappa B translocation but not human immunodeficiency virus 1 enhancer-dependent transcription.

The expression of transiently transfected expression vectors under the control of the long terminal repeat (LTR) of the human immunodeficiency virus (HIV) or its enhancer sequence and the translocation of the HIV enhancer-binding protein NF-kappa B were analyzed in two human T-cell clones stimulated through their T-cell receptor complex or by tumor necrosis factor or phorbol 12-myristate 13-acetate. We found a dissociation of NF-kappa B translocation from transactivation of either the HIV LTR or the HIV enhancer. Interleukin 2 induced proliferation but not NF-kappa B translocation or LTR transactivation. Phorbol ester or specific antigen recognition induced HIV LTR transactivation, whereas stimulation with tumor necrosis factor or antibody to CD3 did not. The two latter signals were nevertheless able to induce NF-kappa B translocation with a pattern in the band-shift assay indistinguishable from that observed using phorbol ester. Our finding that induction of NF-kappa B by tumor necrosis factor or antibody to CD3 is not sufficient to induce HIV enhancer-dependent transcription in cloned T cells contrasts with results obtained in most lymphoblastoid T-cell lines and indicates that normal T lymphocytes differ from tumoral T cells in terms of requirements for HIV LTR activation. Furthermore, our results suggest that events linked to T-cell activation, in addition to NF-kappa B translocation per se, induce functional interactions of the NF-kappa B complex with the HIV enhancer.

Constitutive expression of human immunodeficiency virus (HIV) nef protein in human astrocytes does not influence basal or induced HIV long terminal repeat activity.

Since human immunodeficiency virus (HIV) nef has been suggested to exert regulatory effects on HIV long terminal repeat (LTR) activity, we transiently transfected HIV LTR chloramphenicol acetyltransferase or luciferase expression vectors into a human astrocytoma clone (U-373nef) that constitutively expresses the HIV nef gene. In these cells, basal HIV LTR activity, as well as tumor necrosis factor-induced or tat-driven activity, was similar to that in control cells. Lack of any detectable effect of HIV nef on LTR activity was not the result of mutations in integrated nef DNA, as was shown by polymerase chain reaction. These data suggest that the role of nef in HIV genome transcription does not necessarily involve a direct influence on HIV LTR activity.

A microtransfection method using the luciferase-encoding reporter gene for the assay of human immunodeficiency virus LTR promoter activity.

A microtransfection method, using either the DEAE-dextran or the Ca.phosphate procedure has been developed. A plasmid expressing the luciferase-encoding gene under the control of the human immunodeficiency virus (HIV) LTR promoter was constructed. Transfections were performed in 96-well plates, allowing statistical evaluation of the results. This microtransfection method requires the use of 100- to 1000-fold less plasmid and cells than in a conventional chloramphenicol acetyl transferase (CAT) assay. A Luciferase index which takes into account cell viability after transfection has been defined using a semi-automated absorbance assay. A 20-h incubation period post-transfection is sufficient for optimal results. Basal long terminal repeat activity and autologous Tat transactivation were studied in various lymphoid, monocytic and adherent human cell lines. Infection of microtransfected cells by HIV activated luc expression. This assay can thus also be used for rapid detection and quantitation of HIV. Antiviral activities of drugs can be assessed in a two-day test.

Tumor necrosis factor stimulates transcription of HIV-1 in human T lymphocytes, independently and synergistically with mitogens.

We have investigated the effect of TNF, a cytokine produced during most immunologic and inflammatory reactions, on HIV genome expression in human T lymphocytes. A CD4+ human T cell line (J.Jhan) was transfected with vectors permitting the expression of the chloramphenicol acetyl transferase (CAT) gene under the control of the long terminal repeat (LTR) of HIV-1. rTNF was found to induce HIV LTR transactivation as intensely as PHA or phorbol esters. PHA enhanced TNF receptor expression in J.Jhan cells and acted synergistically with TNF on HIV LTR induction. TNF was also shown to induce well expression of a whole HIV provirus genome transfected into J.Jhan cells. The use of various CAT constructs carrying fragments of the HIV LTR, combined with bandshift assays, showed that TNF stimulates HIV transcription by acting on the kB-like enhancer element of the LTR through induction and/or activation of an NF-kB-like protein factor. Such findings are compatible with the hypothesis that TNF production participates in the pathogenesis of AIDS by enhancing HIV replication in T lymphocytes.

c-Ha-ras transfection induces human immunodeficiency virus (HIV) transcription through the HIV-enhancer in human fibroblasts and astrocytes.

Transient transfection of ras expression vectors into human fibroblasts and astrocytes has been used to test the hypothesis that p21 ras, a known membrane signal transductor, may participate in pathways linking cellular activation and human immunodeficiency virus (HIV) reactivation. Expression vectors carrying the chloramphenicol acetyl transferase coding sequence under the control of various fragments of the long terminal repeat (LTR) of HIV were co-transfected with expression vectors of the mutated (val 12) c-Ha-ras gene or of its normal counterpart. Both forms of the ras gene induced transactivation of the HIV-LTR via the two direct repeat sequences which constitute the HIV enhancer. This repeat sequence was shown to be sufficient for ras-induced LTR transactivation. Other LTR sequences tested were not found to be responsive to co-transfected ras expression vectors. Deletion of the TAR sequence impaired the response to tat, but not to ras co-transfection. The mutated ras gene was more efficient than the proto-oncogene in activating the HIV enhancer. Transfection of ras was shown to enhance transcription of a complete provirus DNA clone of HIV-1. Such findings may shed new light on the mechanisms through which cell membrane activation signals result in HIV reactivation.

Cytomegalovirus infection and trans-activation of HIV-1 and HIV-2 LTRs in human astrocytoma cells.

Susceptibility of a human astrocytoma cell line to human cytomegalovirus (HCMV) infection was investigated. Infection of U-373MG astrocytoma cells with two strains of HCMV resulted in both production of extracellular, infectious virus and expression of immediate early and early antigens within 18 hours and late antigens after 72 hours of infection. The kinetics of infection in U-373MG cells were the same as in human diploid fibroblasts (MRC-5). Since HCMV and human immunodeficiency virus (HIV) have reportedly been found in astrocytic cells in vivo, we studied the possible interaction between HCMV and HIV long terminal repeat (LTR) elements in this cellular environment. HCMV infection transactivated the LTR of HIV-1 and HIV-2 to similar levels. Interestingly, transfection of these cells with infectious HIV-1 provirus did not result in expression of gag, env, or F proteins detectable by immunofluorescence. However, provirus gene expression was not completely silent, since it transactivated HIV-1 LTR. The level of this transactivation was similar to that seen following cotransfection with a tat expression vector. These results suggest that opportunistic infection with HCMV may reactivate latent HIV genomes in glial cells.

[Infection of insect cell lines by the HIV virus, an agent of AIDS, and a demonstration of insects of African origin infected by this virus]. / Infection de cellules d'insectes en culture par le virus HIV, agent du SIDA, et mise en évidence d'insectes d'origine africaine contaminés par ce virus.

The etiological agent of AIDS known as HIV has been shown to bind on different insect cell lines including Drosophila, Mosquito, Ceratitis; and his DNA to be integrated in the cellular genome, but no expression of the viral genome was detected in those cells. None of the human lymphocytes markers is expressed at the surface of the insect cells. HIV proviral DNA has been also found in various insects from Central Africa (Zaïre and Central Africa Republic) but not similar insects from the Paris area. These data suggest that insects could be a reservoir or a vector for the AIDS virus.