Bitopic membrane topology of the stable signal peptide in the tripartite Junín virus GP-C envelope glycoprotein complex

Abstract

The stable signal peptide (SSP) of the GP-C envelope glycoprotein of the Junín arenavirus plays a critical role in trafficking of the GP-C complex to the cell surface and in its membrane fusion activity. SSP therefore may function on both sides of the lipid membrane. In this study, we have investigated the membrane topology of SSP by confocal microscopy of cells treated with the detergent digitonin to selectively permeabilize the plasma membrane. By using an affinity tag to mark the termini of SSP in the properly assembled GP-C complex, we find that both the N and C termini reside in the cytosol. Thus, SSP adopts a bitopic topology in which the C terminus is translocated from the lumen of the endoplasmic reticulum to the cytoplasm. This model is supported by (i) the presence of two conserved hydrophobic regions in SSP (hphi1 and hphi2) and (ii) our previous demonstration that lysine-33 in the ectodomain loop is essential for pH-dependent membrane fusion. Moreover, we demonstrate that the introduction of a charged side chain or single amino acid deletion in the membrane-spanning hphi2 region significantly diminishes SSP association in the GP-C complex and abolishes membrane fusion activity. Taken together, our results suggest that bitopic membrane insertion of SSP is centrally important in the assembly and function of the tripartite GP-C complex.

FIG. 1.

Schematic representation of the Junín virus GP-C glycoprotein and SSP sequences. Amino acids of the Junín virus envelope glycoprotein are numbered from the initiating methionine, and cysteine residues (|) and potential glycosylation sites (Y) are marked. The SPase and SKI-1/S1P cleavage sites and the resulting SSP, G1, and G2 subunits are indicated. Within G2, the C-terminal transmembrane (TM) and cytoplasmic (cyto) domains are shown, as are the N- and C-terminal heptad-repeat regions (light-gray shading). A comparison of SSP sequences among arenavirus species is detailed below. Sequences include the New World isolates Junín (D10072), Tacaribe (M20304), Pichindé (U77601), Machupo (AY129248), and Sabiá (YP_089665) and the Old World isolates Lassa-Nigeria (Lassa-N) (X52400), Mopeia (M33879), and LCMV-Armstrong (LCMV-A) (M20869). The two hydrophobic regions (hφ1 and hφ2) are highlighted in gray, and critical K33 (50) and C57 (49) residues are boxed. The N- and C-terminal sites for the insertion of the 15-amino-acid Spep are indicated.

FIG. 2.

Confocal microscopy of digitonin-permeabilized cells. Vero cells on two-well chambered coverglasses (Lab Tek II) were infected with the recombinant vaccinia virus vTF7-3 expressing T7 polymerase (23), transfected to express the indicated GP-C proteins, and grown for 6 h in growth medium containing 10 μM araC (1). Intact cells (Int) were incubated in the cold with anti-G1 MAb BE08 (anti-G1) or anti-Spep MAb MA1-198 (anti-Spep) and an Alexa Fluor 488-conjugated (green) anti-mouse immunoglobulin secondary F(ab′)₂ fragment (Molecular Probes) prior to fixation with 2% formaldehyde. For staining of cells treated with 0.1% Triton X-100 (Tx), cultures were fixed prior to permeabilization. Selective permeabilization with 5 μg/ml digitonin (Dig) was done in the cold using live cells, prior to incubation with primary and secondary antibodies and fixation. Intact and digitonin-treated cells were also incubated with a rabbit polyclonal antibody directed against the cytoplasmic domain of giantin (PRB-114C; Covance Research Products) and an Alexa Fluor 568-conjugated (red) secondary antibody (Molecular Probes) in parallel with the respective anti-G1 and anti-Spep antibodies to detect permeabilization of the plasma membrane. Chambers were covered with Slow Fade Gold (Molecular Probes) and visualized using an inverted Nikon TE-300 microscope. Fluorescence was examined using a Bio-Rad Radiance 2000 confocal laser scanning microscope, and images were merged using LaserSharp software (Bio-Rad). Note that the leftmost image in panel F was captured at a greater laser power than the others to enhance visibility; the intensity of cell surface anti-G1 staining in the F49K mutant was approximately 25% of wild-type levels. The images omitted in the layout of panel F were all unremarkable.

FIG. 3.

Expression of the GP-C complex containing terminally tagged SSP. (A) Vero cells were transfected to express CD4sp-GPC alone or in trans with wild-type (wt) SSP, C-term SSP-Spep, or N-term SSP-Spep (50). In all cases, transcription was directed by the T7 polymerase of vTF7-3 (23, 51). Metabolically labeled glycoproteins were immunoprecipitated using a G1-specific MAb, BF11 (42), and separated on NuPAGE (Invitrogen) 4 to 12% bis-Tris gels under denaturing and reducing conditions (top panel). The G1 glycoprotein migrates heterogeneously with the discrete G2 subunit, and together, they are labeled G1,G2. In the bottom panel, the glycoproteins were first treated with PNGase F to resolve the G1 and G2 polypeptides (51). [¹⁴C]-labeled protein markers (Amersham Biosciences) are indicated (in kilodaltons). (B) Cell surface expression of the GP-C complex was determined by flow cytometry using MAb BE08 (1, 50). The cell population was subsequently stained using propidium iodide (1 μg/ml) to exclude dead cells. Formaldehyde-fixed cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences).

FIG. 4.

Model for bitopic topology of SSP in the GP-C complex. In this drawing, the insertion of the hφ1 and hφ2 regions of SSP in the membrane results in both N and C termini of SSP residing in the cytosol (cyto). The intervening ectodomain of SSP includes the K33 side chain that is critical for pH-dependent membrane fusion (50), perhaps through interaction with the membrane-proximal or heptad repeat (thicker lines) region of the G2 ectodomain. The drawing is not to scale.

FIG. 5.

Genetic analysis of the hφ2 region of SSP. (A) SSP mutants without Spep tags were expressed in trans with CD4sp-GPC, and the radiolabeled GP-C complex was immunoprecipitated using the anti-G1 MAb BF11 as described in the legend of Fig. 3. The stable association of SSP in the GP-C complex is demonstrated by coprecipitation of the SSP subunit. The right and left panels were imaged at comparable settings; excessive darkening of the right panel reveals low levels of SSP (see text). (B) pH-dependent cell-cell fusion by the trans-complemented GP-C complex was initiated by a pulse of medium at pH 5.0 and detected using a recombinant vaccinia virus-based β-galactosidase reporter assay (36) as previously described (50, 51). The β-galactosidase expression induced upon syncytium formation was quantitated using the chemiluminescence substrate GalactoLite Plus (Tropix), and the percentage of pH-dependent fusion relative to that of the wild-type (wt) GP-C complex is indicated. Error bars (±1 standard deviation) are drawn where discernible on the scale of the graph.