A) Schematic representation of SLPI. SLPI contains an N-terminal signal peptide (black) and two WAP motifs (grey); a.a. are numbered according to Tseng et al. [18]. B) Interaction in the two-hybrid system. L40 yeast strain expressing the indicated Gal4AD and LexA hybrids was analyzed for histidine auxotrophy and β-gal activity. Transformants were patched on medium with histidine (left panels) and then replica-plated on medium without histidine (central panels) and on Whatman filter for β-gal assay (right panels). Growth in the absence of histidine and expression of β-gal activity indicate the interaction between hybrids. C) ELISA interaction analysis. 96-well plates were coated with non-tagged SLPI at a final concentration of 0.25 µM. After washings, GST-PLSCR1 (black curve) or GST (red curves) was incubated at concentrations ranging from 1 nM to 1 µM. Binding was then revealed with an anti-GST antibody and a secondary peroxidase-conjugated anti-mouse IgG. The peroxidase substrate solution was incubated for 10 min and the optical density was measured at 450 nm. D and E) In vitro interactions. Lysates from 293T cells expressing GFP-PLSCR1, GFP-PLSCR3 or GFP-PLSCR4 were incubated with equal amounts of GST fusions (lower panels, Ponceau red) immobilized on GSH-sepharose beads as indicated at the top. Bound proteins were then analyzed by immunoblotting with anti-GFP (upper panels).

A) In vitro interaction. Lysates from Jurkat CD4-positive T cells were incubated with equal amounts of GST or GST-PLSCR1 (upper panel, coomassie blue) immobilized on GSH-sepharose beads. Bound proteins were then analyzed by immunoblotting with anti-CD4 (lower panel). B) Co-immunoprecipitation of endogenous proteins from T lymphocytes. Jurkat T cells were lyzed and CD4 was precipitated with either anti-CD4 (OKT4) or a control isotypic antibody. Precipitates were analyzed by Western blot with anti-CD4 (upper panel) or anti-PLSCR1 (lower panel). C) Co-precipitation of overexpressed CD4 and PLSCR1 proteins. 293T cells expressing HA-tagged PLSCR1 (lower panel, Cell lysate) in combination with wild-type CD4 (WT) or a mutant of CD4 deleted of its cytoplasmic domain (ΔCT) were lyzed and the CD4 forms were precipitated with anti-CD4. Precipitates were then analyzed by Western blot with anti-CD4 (upper panel) or anti-HA (middle panel). D) Co-precipitation of overexpressed CD4 and PLSCR4 proteins. 293T cells expressing GFP- PLSCR1, GFP-PLSCR3 or GFP-PLSCR4 (lower panels, Cell lysate) in combination with CD4 WT (right panels) or CD4 ΔCT (left panels) were lyzed and the CD4 forms were precipitated with anti-CD4. Precipitates were then analyzed as in (C). Of note, the differences in migration observed between PLSCR1 (318 a.a.), PLSCR3 (295 a.a.) and PLSCR4 (329 a.a.) is likely related to the respective amino acid lengths of the GFP fusion proteins [23]. E) Interactions in the two-hybrid system. L40 yeast strain expressing the LexA-CD4c hybrid in combination with the indicated Gal4AD hybrids was analyzed for histidine auxotrophy and β-gal activity. Transformants were patched on medium with histidine (upper panels) and then replica-plated on medium without histidine (middle panels) and on Whatman filter for β-gal assay (lower panels).

A) Primary a.a. sequences of the CD4 long (405–433) and short (405–426) peptides used in the ELISA test. The primary sequence of the entire cytoplasmic domain of CD4 is shown at the top; the 2 Cys residues required for p56Lck binding and mutated in Ala in both peptides are indicated in red. B) ELISA interaction test. 96-well plates were coated with the CD4c long (left panel) or short (right panel) peptides at a final concentration of 0.5 µM. After washings and blocking of non-specific binding sites, GST or GST-PLSCR1 was incubated at concentrations ranging from 1 nM to 300 nM. Binding was then revealed with an anti-GST antibody and a secondary peroxidase-conjugated anti-mouse IgG. The peroxidase substrate solution was incubated for 10 min and the optical density was measured at 450 nm.

A) Inhibition of virus replication. Primary T lymphocytes were isolated from peripheral blood mononuclear cells and used for infection with the HIV-1JR-CSF isolate in the presence of increasing concentrations (0–50 µg/ml) of purified GST-SLPI (see inset, coomassie blue) or GST (50 µg/ml). After washing, infected cells were cultured for 6 days and the viral production was quantified by measuring the p24 production in the cell-culture medium. B) Inhibition of virus transfer from dendritic cells to T lymphocytes. Dentritic cells were derived from purified primary monocytes, and incubated with either HIV-1Bal (R5 strain, upper panel) or HIV-1NDK (×4 strain, lower panel) for 1 h at 37°C in the presence of 50 µg/ml of purified GST-SLPI or GST. After washing, IL-2 activated-lymphocytes were added in a 1/5 ratio and maintained in co-culture for 72 h. Viral production was quantified by measuring the p24 production in the cell culture medium.

A) Schematic representation of PLSCR1. PLSCR1 is a type-II transmembrane protein with a long N-terminal cytoplasmic domain (a.a. 1–290, grey box) containing 3 Cys-rich motifs (red boxes), and a transmembrane domain (a.a. 291–310, black box). Amino acids are numbered according to Zhou et al. [23]. B) Localization of wild type and deleted GFP-PLSCR1 forms. Jurkat CD4-positive T cells expressing the full length (left panel) or deleted forms (1–310 and 1–290, central and right panels, respectively) of GFP-PLSCR1 were fixed and directly examined. Cells were analyzed by epifluorescence microscopy, and images were acquired using a CCD camera. C) Localization of GFP-PLSCR3 and GFP-PLSCR4. Jurkat cells expressing GFP-PLSCR1 (left panel), GFP-PLSCR3 (central panel) or GFP-PLSCR4 (right panel) were fixed and directly examined as in (B). D) Subcellular distribution of CD4 and PLSCR1. Jurkat cells expressing wild type GFP-PLSCR1 (middle panel) were fixed, permeabilized and subsequently stained with an anti-CD4 (left panel). Scale bars, 10 µm.

A) Schematic representation of the human CD4 mutants. The extracellular and transmembrane (TM) domains of CD4 are represented in grey and hatched, respectively. The a.a. sequences of the cytoplasmic tail of the CD4 mutants are aligned with that of the wild-type CD4 (CD4 WT). Dashes (−) indicate a.a. identities with the wild type protein and a.a. substitutions are identified. B) Co-immunoprecipitation assay. Protein extracts were prepared 48 h post transfection from 293T cells co-expressing HA-PLSCR1 together with wild type or mutated CD4 as indicated at the top. Crude extracts were then subjected to CD4 immunoprecipitation followed by Western blot analysis with anti-CD4 (upper panels) and anti-HA (lower panels).

A) In vitro inhibition of the CD4/PLSCR1 interaction by SLPI. GST or GST-PLSCR1 (left panel, coomassie blue) was incubated with equal amounts of lysates from Jurkat CD4-positive T cells in the presence of the indicated concentrations of either GST-SLPI or GST-ARF1 (left panel) used as a control. Bound proteins were analyzed by Western blot with anti-CD4 (right panel). B) Mapping of the PLSCR1 determinants required for binding to CD4 and SLPI. L40 yeast strain expressing either the cytoplasmic domain of CD4 (CD4c) or SLPI fused to LexA in combination with each of the deleted forms of the Gal4AD-PLSCR1 hybrids indicated on the left was analyzed for histidine auxotrophy and beta-gal activity. The interactions between hybrid proteins were scored as follows: (+), cell growth on medium without histidine and development of a β-gal activity; (−), no growth on medium without histidine and no β-gal activity.