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First published online 9 December 2008
doi: 10.1242/jcs.035873

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Moesin is required for HIV-1-induced CD4-CXCR4 interaction, F-actin redistribution, membrane fusion and viral infection in lymphocytes

Marta Barrero-Villar^1,*, José Román Cabrero^1,*, Mónica Gordón-Alonso¹, Jonathan Barroso-González², Susana Álvarez-Losada³, M. Ángeles Muñoz-Fernández³, Francisco Sánchez-Madrid^1, and Agustín Valenzuela-Fernández^1,2

¹ Servicio de Inmunología, Hospital Universitario de La Princesa, 28006 Madrid, Spain
² Departamento de Medicina Física y Farmacología, Facultad de Medicina, Universidad de La Laguna, 38071 Tenerife, Spain
³ Servicio de Inmuno-Biología Molecular, Hospital General Universitario Gregorio Marañón, 28007 Madrid, Spain

View larger version (43K): [in this window] [in a new window]   Fig. 1. HIV-1 phosphorylates endogenous ERM and redistributes F-actin. (A) Western blot analysis of rs-gp120-induced phosphorylation of C-terminal Thr residues of ERM proteins (P-ezrin and P-moesin). Results from untreated control CEM cells are shown in the blot below. Total ERM (ezrin and moesin) is shown for all experimental conditions. A representative experiment of three is shown. The graph shows quantification of Thr phosphorylation of ERMs from three independent experiments. (B) Time course of ERM Thr phosphorylation in PHA-activated PBL, during early infection with the X4-tropic HIV-1NL4.3 viral strain (MOI, 5) compared with non-infected cells. Basal phosphorylation of endogenous ERM-Thr, total ERM and total -tubulin expression are shown for all experimental conditions. (C) Confocal microscopy shows polarized distribution (xy mid-sections) of activated endogenous ERM in CEM cells, 1 hour after HIV-1 infection (MOI, 1; HIV-1NL4.3). Untreated, non-infected cells. Quantification of HIV-1-induced ERM or ERM-P capping is indicated ± s.d. (D) Confocal analysis of ERM-P and endogenous ezrin and moesin localization and redistribution in non-infected or HIV-1-infected CEM cells (1 hour; MOI, 1). (E) Confocal analysis of F-actin and endogenous ERM localization and redistribution in non-infected or HIV-1-infected CEM cells (1 hour; MOI, 1). Quantification of HIV-1-induced ERM actin capping is indicated ± s.d. (F) Western blot analysis of ezrin and moesin phosphorylation induced by rs-gp120IIIB (1 hour exposure) in CEM cells. Blots of total expression of ezrin, moesin and -tubulin are also shown. Rho-K (ROCK) and PKC inhibitors failed to prevent rs-gp120IIIB-induced Thr phosphorylation of ezrin and moesin. A representative of three independent experiments is shown. (G) Blockade of rs-gp120IIIB (5 µg/ml)-induced ERM Thr phosphorylation in CEM cells with neutralizing or non-neutralizing anti-CD4 mAbs (OKT4A or OKT4, respectively), or with the CXCR4 antagonist AMD3100. Blots of total ezrin and moesin proteins are shown. A representative example of three independent experiments is shown.

View larger version (40K): [in this window] [in a new window]   Fig. 2. F-actin and activated exogenous moesin polarize during early HIV-1 infection. (A) Western blot analysis of HIV-1 Env-induced phosphorylation of endogenous moesin in CEM cells overexpressing GFP fusions of full-length moesin (FL-moesin), the N-terminal FERM domain (N-moesin), or the C-terminal domain (C-moesin). A representative experiment is shown. ERM-P/ERM band density ratios from three independent experiments are shown beneath lanes. (B) ERM phosphorylation and subcellular localization of nucleofected moesin-GFP proteins in CEM cells either without infection (untreated, left panels) or 1 hour after HIV-1 infection (MOI, 1; right panels). Localization of exogenous moesin-GFP fusions was tracked by GFP fluorescence (GFP). Active ERM-P (Thr-P) was monitored with a specific Thr-P antibody and Alexa Fluor 568-labeled secondary antibody. The yz and xz planes are shown for each xy mid-section presented (arrows in the right panel). Data are from three independent experiments, presented as means ± s.e.m. Quantification of basal (untreated) and HIV-1 Env-mediated co-distribution of ERM-P and ERM is shown in parentheses; the percentages represent the number of cells showing co-distribution per 200 cells counted. (C) Distribution of F-actin and nucleofected moesin-GFP proteins in non-infected or HIV-1-infected (1 hour; MOI, 1) CEM cells. Quantification of HIV-1 Env-mediated co-distribution of endogenous moesin, nucleofected moesin-GFP constructs and F-actin are shown in parentheses. Scale bars: 10 µm. (D) Quantified flow cytometry of F-actin in non-infected CEM T cells that were either untransfected (Control) or nucleofected with GFP or FL-, N- or C-moesin-GFP. Data are from three independent experiments, presented as mean ± s.e.m.

View larger version (32K): [in this window] [in a new window]   Fig. 3. Effect of overexpressed moesin-GFP proteins and moesin knockdown on CD4 and CXCR4 cell-surface levels and F-actin distribution in primary lymphocytes. (A) Western blot analysis of specific silencing of endogenous expression of ezrin (oligo 1E) and/or moesin (oligo 2M) 72 hours after siRNA nucleofection of CEM cells. Silencing of endogenous ezrin and/or moesin expression is quantified as the band intensity ratios to -tubulin. A representative experiment of four is shown. (B) Confocal analysis of HIV-1 Env-induced F-actin redistribution in CEM cells in which endogenous moesin is suppressed by knockdown (siRNA moesin-2M). Scrambled indicates the control. xy mid-sections are shown. Quantification of HIV-1-induced moesin or Actin capping is indicated ± s.d. (C) CD4 and CXCR4 cell-surface expression levels (red histograms) in PHA-activated PBLs overexpressing GFP or FL-, N- or C-moesin-GFP fusion proteins. A representative flow cytometry analysis of three is shown. Blue histograms indicate IgG negative control. (D) Flow cytometry analysis of the effect of specific moesin-silencing on CD4 and CXCR4 cell surface expression in primary lymphocytes. Data are the means ± s.e.m. of four independent experiments carried out in triplicate.

View larger version (39K): [in this window] [in a new window]   Fig. 4. Effect of overexpressed moesin-GFP proteins and moesin knockdown on HIV-1 infection and entry. (A) Effect of FL-, N- and C-moesin-GFP fusions on HIV-1 infection (MOI, 1) in CEM cells. GFP indicates transfection control for virus infection. As further controls, non-transfected cells were either not infected (non-infected) or infection was blocked with neutralizing anti-CD4 Ab (1 µg/ml) or AZT (5 µM). Data are the means ± s.e.m. of four independent experiments carried out in triplicate. (B) Effect of moesin-GFP fusions on HIV-1 infection of PHA-activated PBL (MOI, 1). GFP indicates transfection control for virus infection (defined as 100% infection). Data are the means ± s.e.m. of three independent experiments carried out in triplicate. (C) Effect of silencing endogenous ezrin and/or moesin expression on HIV-1 viral infection in CEM cells. Data are from three independent experiments carried out in triplicate, presented as means ± s.e.m. Scrambled indicates the control. When indicated, infection was inhibited with neutralizing anti-CD4 mAb. (D) Luciferase-based assay of viral entry by non-replicative HIV-1 particles in CEM T cells (left) or primary T cell blasts (right) overexpressing GFP (control, defined as 100% viral entry) or FL- or N-moesin-GFP fusions as indicated. Data are means ± s.e.m. of three independent experiments carried out in triplicate. (E) β-lactamase-based assay of viral entry by non-replicative HIV-1 particles in CEM T cells silenced for moesin (control, defined as 100% viral entry) with X4-tropic HIV-1 envelope (left panel) or VSV-G envelope (right panel). Data are means ± s.e.m. of three independent experiments carried out in triplicate.

View larger version (41K): [in this window] [in a new window]   Fig. 5. Effect of moesin-GFP fusions on HIV-1 infection and HIV-1 Env-mediated cell-to-cell fusion. (A) Flow cytometry histograms show fusion (F) after 12 hours co-culture of Env+Jurkat-Hxbc2 cells and CEM cells overexpressing GFP (control) or FL-, N- or C-moesin-GFP fusions. A representative experiment of six is shown. (B) Quantification of six independent cell-to-cell fusion experiments as shown in A; bars show means ± s.e.m. *P<0.01 using the Mann-Whitney U test. (C) A series of X-Gal staining showing Env-mediated membrane fusion of HeLa P4 cells, 24 hours after their lipotransfection with moesin-GFP constructs, and X4-tropic Env-HeLa 243 cells. A representative experiment of four is shown. GFP indicates transfection control for Env-induced cell fusion (100%). Quantification of three independent experiments (mean ± s.e.m., n=3) is shown in parentheses. (D) Effect of silencing endogenous ezrin and/or moesin on HIV-1 Env-mediated CEM-Hxbc2 cell fusion. When indicated, cell-to-cell fusion was inhibited by a neutralizing anti-CD4 mAb. Values are means ± s.e.m., n=4. Oligos are as indicated in Fig. 3; scrambled, non-specific RNA control sequence. (E) Confocal microscopy and quantification of Hxbc2-Env-mediated membrane fusion after 30 minutes. Localization of F-actin and endogenous ERM, and CMAC diffusion from Jurkat-derived Hxbc2 cells (blue cell to right) to the target CEM T cell (left); FL-moesin-GFP concentrates at cell-cell contacts where there is a strong accumulation of F-actin and CMAC diffuses better; N-moesin does not localize to cell contacts, impairing the redistribution of F-actin and cell-cell diffusion of CMAC; C-moesin-GFP concentrates at cell-cell contacts along with F-actin and CMAC cell-diffusion was observed. Histograms show quantification of CMAC fluorescence along the white lines (see merged pictures); segments between points 1 and 2 correspond to the cytoplasm of the target cell. The percentages represent the number of contacting cells showing redistribution per 200 cell-cell contacts counted.

View larger version (40K): [in this window] [in a new window]   Fig. 6. Moesin drives HIV-1 Env-mediated CD4-CXCR4 interaction and polarized redistribution, during early viral infection. (A) Effect of FL- and N-moesin constructs on rs-gp120IIIB (10 µg/mL)-induced CD4/CXCR4 direct association. Immunoprecipitation assays were performed with anti-CD4 OKT4 mAb. Inducible co-immunoprecipitation of both receptors was measured as the ratio between the intensities of CXCR4 and CD4 immunoblotted bands. A representative experiment of three is shown. (B) Effect of the silencing of endogenous moesin on HIV-1 Env-induced direct CD4-CXCR4 interaction, in CEM cells. One representative experiment of three is shown. (C) Confocal microscopy of CD4 (anti-CD4 mAb HP2/6) and CXCR4 (biotin-conjugated mAb anti-CXCR4 12G5) in non-infected (Untreated) or 1 hour HIV-1-infected CEM cells (MOI, 1). The yz and xz planes are shown for each xy mid-section presented (arrows). Scale bars: 10 µm. (D) Percentage of colocalization of ERM and CD4, Actin or CXCR4, and of CD4 and CXCR4 in the presence of HIV-1 (MOI, 3) during a tim-course assay. (E) CD4 and CXCR4 redistribution in 1 hour HIV-1-infected cells overexpressing the different moesin-GFP proteins. xy maximal projections (left) and xy mid-sections (right) are shown. Scale bars: 10 µm. (F) Quantification of HIV-1-induced CD4-CXCR4 co-distribution in CEM T cells overexpressing FL-moesin-GFP and N-moesin-GFP constructs. (G) Quantification of HIV-1-induced CD4-CXCR4 capping in untransfected cells (Control), cells overexpressing the scrambled siRNA or lacking endogenous moesin (siRNA-moesin oligo 2). In F and G, quantification of HIV-1-induced CD4-CXCR4 capping is indicated as a percentage of each 200 and 100 cells counted, respectively, under each experimental condition.

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