Predominant role of Tax sumoylation in Tax-induced NF-kB activation in T cells

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The Scaffolding Protein Dlg1 Is a Negative Regulator of Cell-Free Virus Infectivity but Not of Cell-to-Cell HIV-1 Transmission in T Cells

Background: Cell-to-cell virus transmission of Human immunodeficiency virus type-1 (HIV-1) is predominantly mediated by cellular structures such as the virological synapse (VS). The VS formed between an HIV-1-infected T cell and a target T cell shares features with the immunological synapse (IS). We have previously identified the human homologue of the Drosophila Discs Large (Dlg1) protein as a new cellular partner for the HIV-1 Gag protein and a negative regulator of HIV-1 infectivity. Dlg1, a scaffolding protein plays a key role in clustering protein complexes in the plasma membrane at cellular contacts. It is implicated in IS formation and T cell signaling, but its role in HIV-1 cell-to-cell transmission was not studied before. Methodology/Principal Findings: Kinetics of HIV-1 infection in Dlg1-depleted Jurkat T cells show that Dlg1 modulates the replication of HIV-1. Single-cycle infectivity tests show that this modulation does not take place during early steps of the HIV-1 life cycle. Immunofluorescence studies of Dlg1-depleted Jurkat T cells show that while Dlg1 depletion affects IS formation, it does not affect HIV-1-induced VS formation. Co-culture assays and quantitative cell-to-cell HIV-1 transfer analyses show that Dlg1 depletion does not modify transfer of HIV-1 material from infected to target T cells, or HIV-1 transmission leading to productive infection via cell contact. Dlg1 depletion results in increased virus yield and infectivity of the viral particles produced. Particles with increased infectivity present an increase in their cholesterol content and during the first hours of T cell infection these particles induce higher accumulation of total HIV-1 DNA

Rôle de Dlg 1 dans le cycle de réplication et la transmission du VIH-1

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Loss of infectivity of HIV-1 particles produced by mobile lymphocytes.

HIV-1 spreads by cell-free particles and through direct cell contacts. To discriminate between these two modes of dissemination, an assay in which the cells are cultured under shaking conditions impairing cell-to-cell transmission has been described. We addressed the impact of shaking on HIV-1 particle infectivity. Kinetics of HIV-1 infection in static or shaking conditions confirmed that HIV-1 replication is reduced in mobile lymphocyte T cells. Strikingly, the infectivity of viruses produced by mobile lymphocytes was dramatically reduced. In parallel, the amount of envelope protein present on these particles showed a continuous decrease over time. We conclude that inefficient HIV-1 replication in mobile lymphocytes in this experimental system is not only due to avoidance of viral cell-to-cell transfer but also to the loss of infectivity of the viral particles due to the alteration of the composition and functionality of the particles produced by these lymphocytes. It is important to take these observations into account when studying viral transmission under shaking conditions

Stability of HIV-1 envelope expressed on the plasma membrane of T cells cultured under shaking conditions.

<p><b>A.</b> Cell-surface level of HIV-1 envelope on CEM-213env1 cells cultured under static or shaking conditions. The T-cell line CEM-213env1 stably expressing the HIV-1 envelope glycoprotein was cultured for up to 7 days under static or shaking conditions. The cell-surface levels of the HIV-1 S envelope were determined by flow cytometry analysis using the anti-Env 5F7 antibodies. Percentage of Env-positive cells are shown in the top pannel and mean of fluorescence intensity (MFI) are shown in the bottom pannel. Two independent experiments were carried out in triplicate. Error bars represent standard deviations. <b>B. Western blot analysis and quantification of protein extracts of static and shaking CEM-213env1 T-cell cultures.</b> Equal amounts of total proteins from cell lysates recovered 1, 2, 4, and 7 days under static or shaking conditions were analyzed using antibodies against the HIV-1 envelope proteins. The envelope levels were estimated from the intensity of the signals on western blots using the ImageJ software and calculated as gp120/tubulin ratios. The results are from one representative experiment out of two independent experiments carried out in duplicate.</p

HIV-1 replication and particle infectivity are drastically reduced in mobile lymphocyte T cells.

<p><b>A.</b> HIV-1 replication in Jurkat T cells cultured under static or shaking conditions. Jurkat T cells infected with the NL4.3 HIV strain were cultured under static or shaking conditions. Viral replication was measured by determining the fraction of HIV-1-infected cells in the two cultures by intracellular Gag labeling and flow cytometry. Cells were labeled with the anti-HIV-p24 mAb KC57-PE, 3, 5 and 7 days post infection (dpi). Data in top pannel are the means of three independent experiments performed in duplicate. FACS profiles in bottom pannel are of one representative experiment. P = 0.01 for 3 dpi. P = 0.006 for 5 dpi. P = 0.005 for 7 dpi. Error bars represent standard error of the mean (SEM). *, P<0.05. **, P<0.01. <b>B. HIV-1 yield of Jurkat T cells cultured under static or shaking conditions.</b> Supernatants were collected at 7 dpi, filtered and the p24 content was measured by ELISA. The values were normalized for protein content of extracts of cultured cells. The data are means of three independent experiments each carried out in duplicate. For each experiment the values were normalized taking as 100% the value obtained for one of the duplicates of cells under static culture at 7 dpi. P = 0.003 at 7 dpi. <b>C. Infectivity of HIV-1 particles produced by Jurkat T cells cultured under static or shaking conditions.</b> Viral supernatants of Jurkat T cells infected with HIV-1 NL4.3 were collected at 7 dpi, filtered and used to infect indicator HeLa P4.2 reporter cells. Equal amounts of virus determined by p24 quantification were used. β-galactosidase production was assessed by a colorimetric assay based on cleavage of CPRG. The data are means of three independent experiments carried out in triplicate. Normalization was performed as in <b>B.</b> P = 0.001. Error bars represent SEM. **, P<0.01.</p

Low nuclear body formation and tax SUMOylation do not prevent NF-kappaB promoter activation

Abstract Background The Tax protein encoded by Human T-lymphotropic virus type 1 (HTLV-1) is a powerful activator of the NF-κB pathway, a property critical for HTLV-1-induced immortalization of CD4+ T lymphocytes. Tax permanently stimulates this pathway at a cytoplasmic level by activating the IκB kinase (IKK) complex and at a nuclear level by enhancing the binding of the NF-κB factor RelA to its cognate promoters and by forming nuclear bodies, believed to represent transcriptionally active structures. In previous studies, we reported that Tax ubiquitination and SUMOylation play a critical role in Tax localization and NF-κB activation. Indeed, analysis of lysine Tax mutants fused or not to ubiquitin or SUMO led us to propose a two-step model in which Tax ubiquitination first intervenes to activate IKK while Tax SUMOylation is subsequently required for promoter activation within Tax nuclear bodies. However, recent studies showing that ubiquitin or SUMO can modulate Tax activities in either the nucleus or the cytoplasm and that SUMOylated Tax can serve as substrate for ubiquitination suggested that Tax ubiquitination and SUMOylation may mediate redundant rather than successive functions. Results In this study, we analyzed the properties of a new Tax mutant that is properly ubiquitinated, but defective for both nuclear body formation and SUMOylation. We report that reducing Tax SUMOylation and nuclear body formation do not alter the ability of Tax to activate IKK, induce RelA nuclear translocation, and trigger gene expression from a NF-κB promoter. Importantly, potent NF-κB promoter activation by Tax despite low SUMOylation and nuclear body formation is also observed in T cells, including CD4+ primary T lymphocytes. Moreover, we show that Tax nuclear bodies are hardly observed in HTLV-1-infected T cells. Finally, we provide direct evidence that the degree of NF-κB activation by Tax correlates with the level of Tax ubiquitination, but not SUMOylation. Conclusions These data reveal that the formation of Tax nuclear bodies, previously associated to transcriptional activities in Tax-transfected cells, is dispensable for NF-κB promoter activation, notably in CD4+ T cells. They also provide the first evidence that Tax SUMOylation is not a key determinant for Tax-induced NF-κB activation.</p

Low nuclear body formation and tax SUMOylation do not prevent NF-kappaB promoter activation.

International audienceUNLABELLED: ABSTRACT: BACKGROUND: The Tax protein encoded by Human T-lymphotropic virus type 1 (HTLV-1) is a powerful activator of the NF-κB pathway, a property critical for HTLV-1-induced immortalization of CD4+ T lymphocytes. Tax permanently stimulates this pathway at a cytoplasmic level by activating the IκB kinase (IKK) complex and at a nuclear level by enhancing the binding of the NF-κB factor RelA to its cognate promoters and by forming nuclear bodies, believed to represent transcriptionally active structures. In previous studies, we reported that Tax ubiquitination and SUMOylation play a critical role in Tax localization and NF-κB activation. Indeed, analysis of lysine Tax mutants fused or not to ubiquitin or SUMO led us to propose a two-step model in which Tax ubiquitination first intervenes to activate IKK while Tax SUMOylation is subsequently required for promoter activation within Tax nuclear bodies. However, recent studies showing that ubiquitin or SUMO can modulate Tax activities in either the nucleus or the cytoplasm and that SUMOylated Tax can serve as substrate for ubiquitination suggested that Tax ubiquitination and SUMOylation may mediate redundant rather than successive functions. RESULTS: In this study, we analyzed the properties of a new Tax mutant that is properly ubiquitinated, but defective for both nuclear body formation and SUMOylation. We report that reducing Tax SUMOylation and nuclear body formation do not alter the ability of Tax to activate IKK, induce RelA nuclear translocation, and trigger gene expression from a NF-κB promoter. Importantly, potent NF-κB promoter activation by Tax despite low SUMOylation and nuclear body formation is also observed in T cells, including CD4+ primary T lymphocytes. Moreover, we show that Tax nuclear bodies are hardly observed in HTLV-1-infected T cells. Finally, we provide direct evidence that the degree of NF-κB activation by Tax correlates with the level of Tax ubiquitination, but not SUMOylation. CONCLUSIONS: These data reveal that the formation of Tax nuclear bodies, previously associated to transcriptional activities in Tax-transfected cells, is dispensable for NF-κB promoter activation, notably in CD4+ T cells. They also provide the first evidence that Tax SUMOylation is not a key determinant for Tax-induced NF-κB activation