Fig 1.
VASP is a novel interaction partner of p8.
(A) Scheme of the HTLV-1 p8 protein and its precursor p12. A motif mediating potential interactions with the VASP EVH1 domain is highlighted in blue. (B) 293T cells or (C) Jurkat T-cells were transfected with expression plasmids p8-HA and FLAG-VASP or empty control vectors. After 48 h, cells were lysed and 10% of the lysates were taken as input (IN). Co-immunoprecipitations (IPs) were performed using anti-FLAG antibodies or isotype-matched control antibodies (IgG). Immunoblots are shown. HC, heavy chain. (D) 293T cells were transfected with expression plasmids p8-HA and FLAG-VASP. After 48 h, cells were lysed and 10% of the lysates were taken as input. Lysates were treated without (lane 1) or with 60 mM N-octyl-β-D-glucoside (N-ocytlgluc.; lane 2). IPs were performed using anti-FLAG antibodies. Immunoblots are shown. (E) 293T, Jurkat or (F) HTLV-1-infected MT-2 cells were transfected with expression plasmids p8-HA. After 48 h, cells were lysed and 10% of the lysates were taken as input. Endogenous VASP was precipitated using anti-VASP antibodies. Immunoblots are shown.
Fig 2.
The EVH1-domain and the F- and G-actin binding domains of VASP are required for the interaction with p8.
(A) Domain structure of VASP protein and mutants. (B) 293T cells were transfected with expression plasmids p8-HA and FLAG-VASP, FLAG-VASPΔEVH1, FLAG-VASP-EVH1 or the respective empty control vectors. After 48 h, cells were lysed and 10% of the lysates were taken as input (IN). Co-immunoprecipitations (IPs) were performed using anti-FLAG antibodies. Immunoblots are shown. HC, heavy chain. One representative out of four independent experiments is shown. (C) Densitometry was performed to quantitate the amount of p8-HA after precipitation and values were normalized on the respective FLAG-VASP expression in the input. p8 binding to VASP wildtype was set to 100%. Bars indicate the means of four independent experiments ± SE and values were compared to VASP wildtype using Student’s t-test. * indicates p<0.05. (D) 293T cells were transfected with expression plasmids p8-HA and FLAG-VASP, FLAG-VASPΔPP, FLAG-VASPΔGAB, FLAG-VASPΔFAB, FLAG-VASPΔD, FLAG-VASPΔC or FLAG-EVH2. IPs were performed as described in (A). One representative out of four independent experiments is shown.
Fig 3.
p8-mimicking peptides reduce the interaction between p8 and VASP.
(A) Amino acid sequence of p8. Bars indicate location of competitive p8 inhibitory peptides. (B) p8-HA and FLAG-VASP were expressed individually in 293T cells. After 48 h, cells were lysed and 10% of the cell lysate were taken as input (IN). The remaining lysates were co-incubated with the peptides shown in (A), or with the solvent control DMSO for 1.5 h at 20°C. Thereafter, lysates were mixed for 1.5 h, and co-precipitations (anti-FLAG) were performed (IP). Representative immunoblots are shown. The blot (IP) was cut due to technical reasons. HC, heavy chain. (C) The impact of p8-mimicking peptides on binding of p8-HA after precipitation of FLAG-VASP was assessed by densitometry. DMSO was set as 100%. The means of four independent experiments ± SE were compared to DMSO-treated cells using Student’s t-test. ** indicates p<0.01.
Fig 4.
p8 and VASP partially co-localize at the plasma membrane.
Confocal laser scanning microscopy of (A) 293T cells (seeded on coverslips), and (B) Jurkat T-cells after co-transfection of p8-HA and FLAG-VASP expression plasmids. (A) Stains of FLAG-VASP (green), p8-HA (red), the cell nucleus (DAPI, blue), the merge of all three stains and transmitted light are shown. Regions of interest (ROI) are shown and highlighted in insets. Solid arrows indicate co-localizations of p8-HA and FLAG-VASP. Graphs show the fluorescence intensities of FLAG-VASP- and p8-HA-specific fluorescence along the ROI. (B) Stains of the plasma membrane marker CD98 (green), FLAG-VASP (blue), p8-HA (red), the merge of all stains and transmitted light are shown. ROIs are shown and highlighted in insets. Solid arrows indicate co-localizations of p8-HA, FLAG-VASP, and CD98. Graphs show the fluorescence intensities of FLAG-VASP-, p8-HA-, and CD98-specific fluorescence along the ROI. (C) Proximity ligation assay (PLA) in 293T cells after co-expression of p8-HA and FLAG-VASP or the control FLAG-N-WASP. PLA was performed following incubation with primary antibodies targeting either HA, FLAG, both FLAG and HA, or the respective isotype controls. Red dots indicate co-localization. Left side: representative stains. Right side: Dots per positive cell were counted in four to ten optical fields per experiment analyzing 28–64 different cells in three independent experiments. Mean values are indicated by a line. Values were compared using Student’s t-test. **, p<0.01.
Fig 5.
p8 and VASP partially co-localize in protrusive structures between cells and p8 is transferred to target T-cells via VASP-containing protrusions.
(A) Confocal laser scanning microscopy of 293T cells upon co-transfection of p8-HA and FLAG-VASP expression plasmids. Stains of FLAG-VASP (green), p8-HA (red), the cell nuclei (DAPI, blue), the merge of all three stains and transmitted light are shown. Regions of interest (ROI) within a protrusive structure are shown and graphs show the fluorescence intensities of FLAG-VASP- and p8-HA-specific fluorescence along the ROI. (B) Jurkat T-cells were co-transfected with expression plasmids p8-HA, FLAG-VASP and pMACS-LNGFR. After 48 h, transfected cells were enriched by magnetic separation using LNGFR-specific microbeads and co-cultured with untransfected Jurkat T-cells pre-stained with the live cell marker Calcein (green) on poly-L-lysine-coated coverslips for 30 min at 37°C. Immunofluorescence stainings of FLAG-VASP (blue), p8-HA (red), the merge of all stainings and transmitted light are depicted. White arrow: p8 co-localizing with VASP in a protrusion; black arrow: p8 in co-cultured target Jurkat T-cell. (C) Two additional examples of merges showing p8 co-localizing with VASP in a protrusion (white arrows) and p8 in co-cultured target Jurkat T-cells (black arrows) as described in (B).
Fig 6.
Repression of endogenous and overexpressed VASP reduces transfer of p8 to target T-cells.
(A) Scheme of experimental setup. (B-C) Jurkat T-cells were transfected with expression plasmids p8-HA and pMACS-LNGFR. Additionally, FLAG-VASP or pEF (mock), shRNAs targeting VASP (shVASP1), or a control shRNA (shNonsense) were co-transfected. After 48 h, transfected cells were enriched by magnetic separation using anti-LNGFR-specific microbeads. Purified Jurkat T-cells were co-cultivated with acceptor Jurkat T-cells pre-stained with CellTracker Blue CMAC Dye (CMAC) on poly-L-lysine coated glass slides for 1 h at 37°C. Thereafter, cells were stained with HA- and FLAG-specific antibodies and the respective secondary antibodies. Slides were covered with ProLong Gold antifade reagent and analyzed by confocal microscopy. The numbers of cells expressing p8 (red) within the acceptor Jurkat T-cells (blue) were counted (see white circle in blow up as example). (C) The mean of five independent experiments (15–20 optical fields each) normalized on the number of p8-donor cells, and on the respective shNonsense ± SE is shown and was compared using Student’s t-test (**, p<0.01). (D) Repression of VASP was controlled by western blot analysis.
Fig 7.
Knockout of VASP impairs p8-transfer between Jurkat T-cells.
(A) Experimental setup. (B-D) Stably transduced Jurkat T-cells (guide scramble, VASP-KO) were transfected with p8-HA expression plasmids or a control plasmid (pME) and FLAG-VASP or the respective control (pEF-1α). At 48 h post transfection, cells were either (B-C) co-cultured with pre-stained target Jurkat T-cells (Jurkat-CMAC; ratio 1:1, 37°C) for 0 h, 1 h, or 24 h and subjected to flow cytometry, or (D) cells were lysed for immunoblot analysis (B-C) Flow cytometry. At 48 h post transfection, equal amounts of donor and acceptor cells (1x106 cells each) were either directly fixed in 2% PFA and mixed (time point: 0 h), or they were co-cultured at 37°C for 1 h or 24 h before fixation. After intracellular staining using HA-specific, APC-labeled antibodies or the respective isotype-matched control antibodies, flow cytometry was performed. (B) Representative dot plots at 1 h post co-culture are shown. Upper left: Dot plots display the forward scatter (FSC) plotted against the side scatter (SSC) and living cells are gated (black gate). Upper right: CMAC-specific fluorescence is plotted against the SSC, which allows discrimination between CMAC-negative donor (red gate) and CMAC-positive acceptor (blue gate) cells. Lower plots: HA-specific fluorescence is plotted against the SSC and numbers represent the efficiency of transfection within the CMAC-negative donor cells (lower left) or the transfer of p8 within the CMAC-positive acceptor cells (lower right). (C) The relative transfer of p8 between p8-expressing CMAC-negative Jurkat donor cells (scramble, scramble and FLAG-VASP, VASP-KO, VASP-KO and FLAG-VASP) and CMAC-positive Jurkat acceptor cells is shown over time (0 h, 1 h, 24 h). The means of at least three independent experiments are shown. (D) Western blot analysis depicting p8-HA and VASP in stably transduced Jurkat T-cells (scramble, VASP-KO).
Fig 8.
VASP is crucial for recruitment of p8 to the cell surface.
(A-D) Jurkat scramble or Jurkat VASP-KO cells were transfected with p8-HA, FLAG-VASP or both plasmids. Cells transfected with empty vectors pME and pEF-1α served as control. All samples were replenished with the respective empty vectors to 100 μg. At 48 h post transfection, (A) p8 surface expression, (B-C) cell-cell-protrusion formation, or (D) cell-cell conjugate formation were analyzed in at least three independent experiments. In parallel, protein lysates were isolated and subjected to Western blot analysis. (A) Transfected Jurkat scramble (black bars) and VASP-KO cells (grey bars) were stained without permeabilization using HA-specific, APC-labeled antibodies or the respective isotype-matched control antibodies and flow cytometry was performed. The mean fold change p8-HA surface expression of four independent experiments was compared using Student’s test (*, p<0.05; **, p<0.01). Additionally, a representative Western blot depicting p8-HA, VASP or the housekeeping gene GAPDH is shown. (B) Transfected cells were cultured without fixation in micro-slides for 1 h at 37°C and analyzed with a Leica TCS SP5 confocal laser scanning microscope equipped with a 63x1.4 HCX PL APO CS oil immersion objective lens. Transmitted light is shown. Black arrows indicate cell-cell-protrusions. Insets (dashed lines) are shown as enlargement (solid lines) (C) Quantitative analysis of data exemplified in (B) in Jurkat scramble (upper panel, black bars) and VASP-KO cells upper panel, dark grey bars), and as a control, in Jurkat cells (lower panel,black bars). In the latter case, cells transfected with p12-HA expression plasmids served as control. At least 20 optical fields per experimental condition were analyzed and mean numbers of cell-cell-protrusions per 100 cells were compared using Student’s t-test (*, p<0.05; **, p<0.01; n.s., not significant). (D) Conjugate formation between transfected Jurkat T-cells (scramble: black bars; VASP-KO, grey bars) and co-cultured Raji/CD4+ B-cells was quantitated by flow cytometry. Jurkat cells transfected with the Tax-expression construct pEF1α-Tax served as positive control. After 24h, Jurkat T-cells were co-cultured with Raji/CD4+ B-cells (ratio 1:1) for 1 h at 37°C. Co-cultures were fixed and stained with anti-CD3-AlexaFluor700 (for Jurkat T-cells) and anti-HLA-DR-PacificBlue antibodies (for Raji/CD4+ B-cells) to differentiate between the two cell types. Cell-cell conjugates were identified as double-positive signals (HLA-DR+CD3+) and normalized on the total number of Jurkat T-cells. The means of three independent experiments ± standard deviation are shown and were compared using Student's t-test (**, p<0.01).
Fig 9.
Repression of VASP impairs p8-transfer between chronically-infected MT-2 cells and uninfected T-cells.
(A) Experimental setup. (B-D) Stably transduced MT-2 cells (shNonsense, shVASP2, shVASP3) were nucleofected with p8-HA expression plasmids or a control plasmid together with pMACS-LNGFR. At 24 h post transfection, transfected MT-2 cells were enriched by magnetic separation using anti-LNGFR microbeads. Separated MT-2 cells were either (B-C) co-cultured with target Jurkat T-cells (ratio 1:1) for 1 h at 37°C and subjected to flow cytometry or (D) MT-2 cells were cultured for another 48 h and lysed for western blot analysis. (B) Flow cytometry of co-cultures after use of primary mouse anti-HA-APC conjugated specific antibodies. Representative dot plots are shown. Upper part: Jurkat acceptor cells (blue) and MT-2 donor cells (red) were gated by forward scatter (FSC) and side scatter (SSC) analysis. Lower part: HA-APC-specific fluorescence is plotted against the SSC and numbers represent the efficiency of transfection within the MT-2 donor cells (red, left side) or the transfer of p8 within the Jurkat acceptor T-cells (blue, right side). (C) The relative transfer of p8 from MT-2 cell lines (shNonsense, shVASP2, shVASP3) transfected with p8-HA or p12-HA to co-cultured Jurkat T-cells was calculated and values were normalized on MT-2/shNonsense. The means of four independent experiments ± SE were compared using Student’s t-test (**, p<0.01). (D) Western blot analysis depicting p8-HA or p12-HA and VASP in stably transduced MT-2 cells (shNonsense, shVASP2, shVASP3). Numbers indicate densitometric analysis of VASP normalized on GAPDH and shNonsense.
Fig 10.
Repression of VASP impairs virus transmission to uninfected T-cells, but not Gag processing and virus release in chronically infected MT-2 cells.
(A) Jurkat T-cells were co-cultured with stably transduced MT-2 cells (shNonsense, shVASP2, shVASP3) at a ratio of 1:1 for 1 h. The transfer of Gag p19 to Jurkat acceptor cells was measured by flow cytometry using the primary antibodies mouse anti-Gag p19 and the secondary antibodies anti-mouse Alexa Fluor 647. Upper part: Jurkat acceptor cells were gated by forward scatter (FSC) and side scatter (SSC) analysis; lower part: Gag p19-specific fluorescence (indicated by numbers) in Jurkat acceptor T-cells. (B) The amount of Gag p19 positive Jurkat acceptor cells normalized on the MT-2/shNonsense control cells of four independent experiments ± SE is depicted and was compared using Student’s t-test (**, p<0.01). (C) The amount of Gag p19 protein in the supernatant of transduced MT-2 cell lines was assessed by Gag p19 ELISA. MT-2 cells treated with 5 μM cytochalasin D, an inhibitor of actin-polymerization, in comparison to the DMSO solvent control served as positive control for an impaired Gag p19 release. The mean of four independent experiments ± SE is depicted and was compared using Student’s t-test (**, p<0.01). (D) Western blot analysis depicting VASP, Tax and Gag p55 precursor and processed Gag p19 matrix protein in stably transduced MT-2 cells (shNonsense, shVASP2, shVASP3) and the indicated controls. Numbers indicate densitometric analysis of VASP normalized on Hsp90 and shNonsense.
Fig 11.
Model of the VASP:p8 interaction and its role in p8 transfer and HTLV-1 transmission.
Our data suggest a weak interaction between the EVH1 domain of VASP and p8, which may be important for the recruitment of VASP, and a strong interaction of the G- and F-actin binding site of VASP with p8. Transfer of p8 and of Gag p19, and thus, presumably of HTLV-1 to target cells depends on VASP and may be driven by formation of a VASP- and actin-containing cellular conduit.