The signal peptide of the Junín arenavirus envelope glycoprotein is myristoylated and forms an essential subunit of the mature G1-G2 complex

Abstract

Arenaviruses comprise a diverse family of rodent-borne viruses that are responsible for recurring and emerging outbreaks of viral hemorrhagic fevers worldwide. The Junín virus, a member of the New World arenaviruses, is endemic to the pampas grasslands of Argentina and is the etiologic agent of Argentine hemorrhagic fever. In this study, we have analyzed the assembly and function of the Junín virus envelope glycoproteins. The mature envelope glycoprotein complex is proteolytically processed from the GP-C precursor polypeptide and consists of three noncovalently associated subunits, G1, G2, and a stable 58-amino-acid signal peptide. This tripartite organization is found both on virions of the attenuated Candid 1 strain and in cells expressing the pathogenic MC2 strain GP-C gene. Replacement of the Junín virus GP-C signal peptide with that of human CD4 has little effect on glycoprotein assembly while abolishing the ability of the G1-G2 complex to mediate pH-dependent cell-cell fusion. In addition, we demonstrate that the Junín virus GP-C signal peptide subunit is myristoylated at its N-terminal glycine. Alanine substitution for the modified glycine residue in the GP-C signal peptide does not affect formation of the tripartite envelope glycoprotein complex but markedly reduces its membrane fusion activity. In contrast to the classical view that signal peptides act primarily in targeting nascent polypeptides to the endoplasmic reticulum, we suggest that the signal peptide of the arenavirus GP-C may serve additional functions in envelope glycoprotein structure and trafficking.

FIG. 1.

Schematic representation of the Junín virus GP-C glycoprotein and SP. (A) The Junín virus MC2 GP-C glycoprotein is depicted. Amino acids are numbered from the initiating methionine, and cysteine residues (|) and potential glycosylation sites (Y) are marked. The SP cleavage site (after amino acid 58) and the SKI-1/S1P cleavage site (after amino acid 251) and the resulting SP, G1 and G2 subunits are indicated. Within G2, the C-terminal transmembrane domain (TM) is shown, as are heptad-repeat regions (light gray shading) that may be involved in membrane fusion. The N termini of the arenavirus G1 and G2 glycoproteins have been previously determined experimentally (10, 12, 27, 45). (B) A comparison of the signal peptides of New World (Junín MC2 [accession no. D10072], Tacaribe [accession no. M20304], Pichindé [accession no. M16735], Machupo [accession no. AY129248], and Sabiá [accession no. U41071]) and Old World (Lassa-Nigeria [Lassa-N, accession no. X52400], Lassa-Josiah [Lassa-J, accession no. M15076], Mopeia [accession no. M33879], LCMV-Armstrong [LCMV-A, accession no. M20869] and LCMV-WE [accession no. M22138]) arenaviruses is shown. Identically conserved regions are highlighted, as is the conserved myristoylation (myr) motif G-X₃-S/T (59).

FIG. 2.

pH-dependent fusion mediated by Junín virus envelope glycoproteins. Vero cells expressing either the unmodified Junín virus GP-C glycoprotein (JGPC), the Spep-tagged JGPC/Spep, or the SKI-1/S1P cleavage defective mutant cd JGPC/Spep were tested for their ability to mediate cell-cell fusion. In panel A, cultures of expressing cells were pulsed with medium at pH 5.0 or with neutral medium for 1 h and then incubated for 2 h prior to fixation with cold methanol-acetone (1:1). Cells infected with the T7 polymerase-expressing vaccinia virus vTF7-3 but not transfected with envelope glycoprotein plasmid (vaccinia only) served as negative controls. Cultures were stained with anti-G1 MAb AF03, a secondary antibody conjugated to horseradish peroxidase and diaminobenzidine substrate. Multinucleated syncytia were visualized microscopically. In panel B, parallel assays were performed by using the β-galactosidase fusion reporter gene assay (54). Here, envelope glycoprotein-expressing cells were cocultured with Vero cells infected with a recombinant vaccinia virus bearing the β-galactosidase gene under the control of the T7 promoter (vCB21R-lacZ). Cocultures were then pulsed with medium at pH 5.0 or neutral pH and continued for 5 h. Following fixation with formaldehyde-glutaraldehyde, cell-cell fusion and β-galactosidase expression were detected with X-Gal substrate. Cocultures of cells infected with the respective vaccinia viruses but without an envelope glycoprotein plasmid (vaccinia only) served as negative controls.

FIG. 3.

Expression and biogenesis of the Junín virus GP-C. Vero cells infected with Candid 1 virus or expressing recombinant forms of the GP-C were metabolically labeled with [³⁵S]methionine and [³⁵S]cysteine, and the envelope glycoproteins were immunoprecipitated with the anti-G1 MAb AF03. Panels A and B represent envelope glycoproteins from cell lysates and cell culture supernatants, respectively. Panels C and D represent the same samples, respectively, treated with PNGase F to generate the deglycosylated polypeptides. Recombinant forms of the envelope glycoprotein gene are unmodified Junín virus GP-C (JGPC), the Spep-tagged JGPC/Spep, and the SKI-1/S1P cleavage defective mutant cd JGPC/Spep. Mock-transfected cells are also represented. Lanes are numbered, and specific comparisons among samples are discussed in detail in the text. The ¹⁴C-labeled-protein markers (Amersham Biosciences) are indicated in kilodaltons and shown by dots. The positions of the GP-C precursors and G2, G1, and SSP subunits are labeled. SSP bands in PNGase F-treated samples are distorted by nonionic detergent. In panel D (left), the source of the diffuse bands (below the G1 polypeptide and at ≈16 kDa) is unclear.

FIG. 4.

Analysis of expression of GP-C bearing CD4 SP. GP-C constructs were expressed in Vero cells and metabolically labeled with [³⁵S]methionine and [³⁵S]cysteine. In the CD4sp-JGPC/Spep construct, the GP-C SP has been replaced with that of CD4. The glycoproteins were immunoprecipitated from cell lysates (cells, left panel) or cell culture supernatants (supt, right panel) with the anti-G1 MAb BF11. All isolated glycoproteins were subsequently deglycosylated with PNGase F to better resolve the G1 and G2 polypeptides. Markers and labels are as described in the legend to Fig. 3.

FIG. 5.

Quantitative analysis of cell-cell fusion by mutant Junín virus envelope glycoproteins. Vero cells infected with vTF7-3 and expressing the Junín virus envelope glycoproteins and cells infected with vCB21R-lacZ were cocultured as described for the β-galactosidase fusion reporter gene assay (see legend to Fig. 2B). β-Galactosidase expression was quantitated by chemiluminescence with Galacto-Lite Plus (Tropix) substrate and is presented in relative light units. Cultures pulsed with medium at pH 5.0 are indicated by dark grey bars, and cultures treated at neutral pH are indicated by light grey bars. Cocultures of cells infected with the respective vaccinia viruses but without the GP-C expression plasmid are shown with the label vLacZ+vT7. Standard deviations among replicate cocultures are shown. The extent of pH-dependent cell-cell fusion is determined after subtracting the relative light unit value of the culture treated at neutral pH.

FIG. 6.

Myristoylation of the Junín virus SSP. Vero cells expressing JGPC/Spep were metabolically labeled with either [³⁵S]methionine and [³⁵S]cysteine or [³H]myristic acid as described in Materials and Methods. Cell lysates were immunoprecipitated with anti-G1 MAb BF11 and analyzed by SDS-PAGE. In this experiment, protein samples were not reduced prior to electrophoresis.

FIG. 7.

Analysis of expression of the nonmyristoylated G2A mutant of GP-C. Wild-type JGPC/Spep and the G2A myristoylation site mutant G2A-JGPC/Spep were expressed in Vero cells and metabolically labeled with [³⁵S]methionine and [³⁵S]cysteine. (A) Glycoproteins from cell culture supernatants (supt) or cell lysates (cells) were analyzed following immunoprecipitation with the anti-G1 MAb BF11 by PAGE with a 10% Bis-Tris NuPAGE gel and morpholineethanesulfonic acid (MES) running buffer. A sample from a mock-transfected culture is shown to identify nonspecific background bands. (B) The immunoprecipitated glycoproteins from cell lysates were deglycosylated with PNGase F prior to electrophoresis. Markers and labels are as described in the legend to Fig. 3.