Role of the stable signal peptide and cytoplasmic domain of G2 in regulating intracellular transport of the Junín virus envelope glycoprotein complex

Abstract

Enveloped viruses utilize the membranous compartments of the host cell for the assembly and budding of new virion particles. In this report, we have investigated the biogenesis and trafficking of the envelope glycoprotein (GP-C) of the Junín arenavirus. The mature GP-C complex is unusual in that it retains a stable signal peptide (SSP) as an essential component in association with the typical receptor-binding (G1) and transmembrane fusion (G2) subunits. We demonstrate that, in the absence of SSP, the G1-G2 precursor is restricted to the endoplasmic reticulum (ER). This constraint is relieved by coexpression of SSP in trans, allowing transit of the assembled GP-C complex through the Golgi and to the cell surface, the site of arenavirus budding. Transport of a chimeric CD4 glycoprotein bearing the transmembrane and cytoplasmic domains of G2 is similarly regulated by SSP association. Truncations to the cytoplasmic domain of G2 abrogate SSP association yet now permit transport of the G1-G2 precursor to the cell surface. Thus, the cytoplasmic domain of G2 is an important determinant for both ER localization and its control through SSP binding. Alanine mutations to either of two dibasic amino acid motifs in the G2 cytoplasmic domain can also mobilize the G1-G2 precursor for transit through the Golgi. Taken together, our results suggest that SSP binding masks endogenous ER localization signals in the cytoplasmic domain of G2 to ensure that only the fully assembled, tripartite GP-C complex is transported for virion assembly. This quality control process points to an important role of SSP in the structure and function of the arenavirus envelope glycoprotein.

FIG. 1.

Schematic representation of the Junín virus GP-C glycoprotein and G2 cytoplasmic domain 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 SSP 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 G2 cytoplasmic domain sequences among arenavirus species is detailed below the schematic. Sequences include the New World isolates Junín (D10072), Tacaribe (M20304), Pichindé (U77601), Machupo (AY129248), and Sabiá (YP_089665) and Old World isolates Lassa-Nigeria (X52400), Mopeia (M33879), and LCMV-Armstrong (M20869). The sites used to generate truncations in the Junín virus cytoplasmic tail are indicated by angle brackets and dibasic amino acid sequences are underlined.

FIG. 2.

Coexpression of SSP in trans rescues SKI-1/S1P cleavage and cell surface expression of the G1-G2 precursor. (A) Metabolically labeled glycoproteins were immunoprecipitated using the G1-specific MAb BE08 and separated on NuPAGE 4-to-12% bis-Tris gels. The wild-type (GP-C) and SKI-1/S1P cleavage-defective (cd-GPC) glycoproteins are shown for comparison with the CD4sp-GPC construct encoding the conventional signal peptide of human CD4. CD4sp-GPC was expressed alone (−SSP) or with SSP (+SSP). In the bottom panel, the glycoproteins have been treated with PNGase F to resolve G1 and G2 polypeptides. The deglycosylated GP-C polypeptides reveal both the G1-G2 precursor and, in SSP-containing constructs, the pre-GP-C precursor (65); additional species that migrate more slowly than the G1-G2 precursor and with the pre-GP-C precursor are likely products of incomplete deglycosylation. cd-GPC contains a C-terminal S-peptide affinity tag and migrates slightly slower than the other G1-G2 precursors. Known GP-C species are labeled at left; minor unidentified bands are also present. The ¹⁴C-labeled protein markers (Amersham Biosciences) are indicated (in kilodaltons). (B) Cell surface expression of GP-C in Vero cells was determined by flow cytometry using the G1-specific MAb BE08 (54). The cell population was subsequently stained using propidium iodide (1 μg/ml) to exclude dead cells. Cells were fixed using 2% formaldehyde and analyzed using a FACSCalibur flow cytometer (BD Biosciences). The histograms plot cell number (counts) versus the fluorescence intensity of MAb binding. Background staining of mock-transfected cells is shown to identify nonexpressing cells in the transfected cell populations.

FIG. 3.

pH-dependent cell-cell fusion activity. pH-dependent fusion was detected using the recombinant vaccinia virus-based β-galactosidase reporter assay (47) as previously described (64, 65). β-Galactosidase activity was quantitated using the chemiluminescent substrate GalactoLite Plus (Tropix). Relative light unit (RLU) measurements from cultures treated at pH 5.0 are shown after subtraction of background levels from neutral-pH cultures (average background, 1,500 RLU). Control conditions are shown in the underlined bars at left (mock, wild-type GP-C, and cd-GPC). Note that CD4sp-GPC constructs are bracketed in pairs (below the axis) representing the absence (open bars) and presence (gray bars) of SSP. Some bars are not discernible on the scale of the graph. All conclusions were replicated using X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) staining of parallel cocultures.

FIG. 4.

Intracellular and cell surface visualization of glycoproteins. Confocal images were obtained as described in Materials and Methods. Permeabilized cells were stained in green using either the MAb BF11 (GP-C) or, for CD4ecto, SIM.2 (CD4). Golgi structures were identified using a rabbit polyclonal antiserum and stained in red. Merged images (merge) were created using Lasersharp software. Nonpermeabilized cells (surface) were stained in green using either MAb BF11 or SIM.2. The expressed glycoproteins are indicated in white letters superimposed on the leftmost images. The top row depicts cells expressing native GP-C or mock-transfected cells (all infected with the recombinant vaccinia virus vTF7-3). In subsequent rows, the glycoproteins were expressed either in the absence (−SSP) or presence (+SSP) of SSP. In some images, the Golgi apparatus is vesiculated and dispersed, perhaps due to infection of the cells by vaccinia virus.

FIG. 5.

The chimeric CD4 glycoprotein bearing the transmembrane and cytoplasmic domains of G2 requires SSP for transport to the cell surface. (A) The chimeric CD4ecto construct was expressed alone (−SSP) or with SSP (+SSP) and metabolically labeled. Intact cells were incubated with the anti-CD4 MAb SIM.2 (43, 48) and the cell surface glycoproteins were subsequently isolated from cleared cell lysates using protein A-Sepharose (surface). Intracellular CD4ecto glycoprotein was immunoprecipitated from the post-protein A-Sepharose supernatant using additional SIM.2 MAb (lysate). Mock- and human CD4-transfected cells served as controls. Molecular size markers (in kilodaltons) are as described in the legend to Fig. 2A. (B) Flow cytometry using SIM.2 MAb was otherwise performed as described in the legend to Fig. 2B, and results are plotted similarly. The filled gray (−SSP) and open (+SSP) histograms are overlaid.

FIG. 6.

Truncations to the cytoplasmic domain of G2 ablate SSP binding yet enable transport to the cell surface. (A) The wild-type and truncated CD4sp-GPC glycoproteins (R448Δ, R451Δ, and R460Δ) were expressed alone (−SSP) or with SSP (+SSP). Metabolically labeled glycoproteins were precipitated using the C-terminal Spep affinity tag and S-protein agarose (Novagen) and analyzed as described in the legend to Fig. 2. The G1 and G2 glycoproteins are best resolved following deglycosylation with PNGase F (bottom). Note that the truncated G2 moieties (ΔG2) migrate near the wild-type G1 polypeptide; coassociation between G1 and ΔG2 was formally demonstrated by immunoprecipitation using anti-G1 MAb BF11, which coprecipitated ΔG2 (not shown). Although coprecipitation of SSP was markedly reduced with the truncated glycoproteins, trace amounts could be discerned upon darkening of the image (not shown). This low level of SSP association is judged to be insignificant, as the properties of the truncated glycoproteins are independent of SSP coexpression. Molecular size markers (in kilodaltons) are as described in the legend to Fig. 2A. (B) Cell surface expression of the truncated glycoproteins was determined by flow cytometry as described in the legend to Fig. 2B, and results are plotted similarly. Note that expression of the wild-type CD4sp-GPC glycoprotein (gray histograms in all three panels) is compared with that of the truncations (open histograms), all in the absence of SSP.

FIG. 7.

Alanine mutations to dibasic amino acid motifs enable transport from the ER. (A) CD4sp-GPC constructs containing KK, RR, and double KK/RR mutations were expressed alone (−SSP) or with SSP (+SSP). Metabolically labeled glycoproteins were immunoprecipitated and analyzed as described in the legend to Fig. 2A. Molecular size markers (in kilodaltons) are as described in the legend to Fig. 2A. (B) Flow cytometry was performed as described in the legend to Fig. 2B, and results are plotted similarly. Note that the top panels compare expression of the CD4sp-GPC glycoprotein (gray histograms in all panels) with that of the mutants (open histograms) in the absence of SSP. Expression with SSP is shown in the bottom panels.

FIG. 8.

Cell surface expression of dibasic amino acid motif CD4sp-GPC mutants. Intact cells expressing the constructs shown in Fig. 7 were incubated with the G1-specific MAb BE08, and the cell surface GP-C glycoproteins were isolated from cleared cell lysates using protein A-Sepharose and deglycosylated. The relative amounts of G1, G2, and G1-G2 precursor in each lane were quantitated from the phosphorimage using Image Gauge software (Fuji), and the efficiency of cleavage was determined as the sum of G1 plus G2 relative to total of all forms. Distortion of the SSP band is due to the detergents used in PNGase F treatment. Molecular size markers (in kilodaltons) are as described in the legend to Fig. 2A.