An antibody directed against the fusion peptide of Junin virus envelope glycoprotein GPC inhibits pH-induced membrane fusion

Abstract

The arenavirus envelope glycoprotein (GPC) initiates infection in the host cell through pH-induced fusion of the viral and endosomal membranes. As in other class I viral fusion proteins, this process proceeds through a structural reorganization in GPC in which the ectodomain of the transmembrane fusion subunit (G2) engages the host cell membrane and subsequently refolds to form a highly stable six-helix bundle structure that brings the two membranes into apposition for fusion. Here, we describe a G2-directed monoclonal antibody, F100G5, that prevents membrane fusion by binding to an intermediate form of the protein on the fusion pathway. Inhibition of syncytium formation requires that F100G5 be present concomitant with exposure of GPC to acidic pH. We show that F100G5 recognizes neither the six-helix bundle nor the larger trimer-of-hairpins structure in the postfusion form of G2. Rather, Western blot analysis using recombinant proteins and a panel of alanine-scanning GPC mutants revealed that F100G5 binding is dependent on an invariant lysine residue (K283) near the N terminus of G2, in the so-called fusion peptide that inserts into the host cell membrane during the fusion process. The F100G5 epitope is located in the internal segment of the bipartite GPC fusion peptide, which also contains four conserved cysteine residues, raising the possibility that this fusion peptide may be highly structured. Collectively, our studies indicate that F100G5 identifies an on-path intermediate form of GPC. Binding to the transiently exposed fusion peptide may interfere with G2 insertion into the host cell membrane. Strategies to effectively target fusion peptide function in the endosome may lead to novel classes of antiviral agents.

FIG. 1.

Schematic representation of the JUNV G2 ectodomain and fusion peptide region. The amino acid sequence of the JUNV G2 ectodomain is shown on top, in text, and as a line drawing (residues 252 to 428; MC2 strain, accession number D10072). Small dots above the text are spaced 10 amino acids apart, starting with position 260. The boxed sequences comprise peptides G2*, G2-151, and G2-114 (see text). The nominal start of the G2 transmembrane domain is indicated at residues 425 to 428. Cysteines are marked by vertical lines in the schematic, and gray boxes represent HR1 and HR2, as defined by the N29 and C30 peptides (see text). The X marks the division between N- and C-terminal regions in MBP fusion proteins, and arrowheads indicate alanine mutations used in this work. The positions chosen for mutation are identically conserved or invariant in charge among arenaviruses. Below, fusion peptide sequences are compared among arenaviruses (JUNV residues 252 to 309). N-terminal and internal fusion peptide regions (N-FPS and I-FPS, respectively) are indicated and are based on the work of Klewitz et al. (50). Conserved cysteines are highlighted in gray, and arrowheads represent the alanine mutations studied. Accession numbers for other arenavirus glycoproteins are as follows: Machupo virus (MACV), AAT40455; Guanarito virus (GUAV), AAS55656; Tacaribe virus (TCRV), NP_694849; Sabiá virus (SABV), YP_089665; Chapare virus (CHPV), YP_001816782; LASV-Jos, NP_694870; lymphocytic choriomeningitis virus ([LCMV] LCMV-Arm), NP_694851; Mopeia virus (MOPV), YP_170709; and Pichinde virus (PICV), AAB58484.

FIG. 2.

Solution properties of the N29/C30 complex. (A) CD spectra in PBS (pH 7.0) at 4°C and 100 μM peptide concentration. (B) Thermal melts monitored by CD at 222 nm. (C) Sedimentation equilibrium data for the N29/C30 complex (100 μM) at 20°C and 22 krpm in PBS (pH 7.0). The data fit closely to a trimeric complex. Curves expected for dimeric and tetrameric models are indicated for comparison. The deviation in the data from the linear fit for a trimer is plotted (upper). (D) Helical wheel diagrams of the six-helix bundle. The antiparallel N and C helices are drawn looking down toward the membrane. The register of the respective coils was assigned to maximize hydrophobicity at interhelical a and d positions.

FIG. 3.

Flow cytometric analysis of MAb binding to cell surface GPC. (A) Selected MAbs were incubated with Vero cells expressing wild-type (wt) JUNV GPC that had been exposed to neutral pH (gray histogram) or to pH 5.0 (black line histogram). The MAb (and secondary antibody) used is shown below each histogram. Abbreviations: GAMFITC, fluorescein isothiocyanate-conjugated goat anti-mouse secondary antibody; BE08, MAb GB03-BE08. (B) MAb F100G5 was incubated with cells expressing wt or mutant GPC that had been exposed to neutral pH (gray histogram) or to pH 5.0 (black line histogram). Mutants are cleavage-defective (cd GPC [81]), K33A GPC (80), and K283A GPC (see text). + ST-294 indicates that cells expressing wt GPC were first incubated with a 50 μM concentration of the small-molecule fusion inhibitor ST-294 (8, 76) prior to exposure to neutral or acidic pH.

FIG. 4.

MAb F100G5 inhibits cell-cell fusion when added on exposure to acidic pH. Cells expressing wild-type GPC were incubated with MAb GB03-BE08 (left; 10 μg/ml) or F100G5 (right; ∼30 μg/ml) either during the initial coculture with target cells at neutral medium (pre), during acidification at pH 5.0 (pH 5), or upon the return of the culture to neutral medium, in which cell-cell fusion becomes manifest (post) (81). Syncytium formation was reported by expression of β-galactosidase and quantitated by chemiluminescence (in relative light units [RLU]). Error bars represent 1 standard deviation among quadruplicate wells. Missing bars were not rendered at the scale of the graph. The experiment shown is representative of five independent repetitions.

FIG. 5.

Western blot analysis of F100G5 binding to MBP fusion proteins. Sequences encoding the entire ectodomain of JUNV G2 (G2ecto; residues 252 to 424), the N-terminal region (N-term; residues 252 to 316), or the C-terminal region (C-term; residues 317 to 424) were appended to the C terminus of MBP, and the recombinant fusion proteins were expressed in E. coli. Affinity-purified proteins were resolved by SDS-PAGE and detected by using Sypro Red protein stain (left) or by Western blot analysis using MAb F100G5 (right). The fusion proteins were proteolytically unstable, and the major band detected by protein staining is the stable MBP core (MBP*); the full-length fusion proteins are visible in decreasing order of molecular mass (62, 55, and 50 kDa). F100G5 binds only to the full-length proteins and to proteolytic fragments of G2-ecto. Molecular mass markers are indicated in kilodaltons.

FIG. 6.

Mapping the F100G5 epitope using GPC mutants. Vero cells expressing the indicated wild-type (wt) or alanine mutant GPC were solubilized using 1% Triton X-100, and proteins were resolved by SDS-PAGE and detected by Western blot analysis using the indicated MAbs. MAb F100G1 recognizes the C-terminal trimer-of-hairpins structure and thus serves to control for potential differences in expression among the mutants. Only the uncleaved G1-G2 precursor is shown, to simplify the analysis of MAb binding; the mature G2 subunit is less abundant, and staining intensity varies depending on the extent of proteolytic maturation in each mutant.