Features of a spatially constrained cystine loop in the p10 FAST protein ectodomain define a new class of viral fusion peptides

Abstract

The reovirus fusion-associated small transmembrane (FAST) proteins are the smallest known viral membrane fusion proteins. With ectodomains of only approximately 20-40 residues, it is unclear how such diminutive fusion proteins can mediate cell-cell fusion and syncytium formation. Contained within the 40-residue ectodomain of the p10 FAST protein resides an 11-residue sequence of moderately apolar residues, termed the hydrophobic patch (HP). Previous studies indicate the p10 HP shares operational features with the fusion peptide motifs found within the enveloped virus membrane fusion proteins. Using biotinylation assays, we now report that two highly conserved cysteine residues flanking the p10 HP form an essential intramolecular disulfide bond to create a cystine loop. Mutagenic analyses revealed that both formation of the cystine loop and p10 membrane fusion activity are highly sensitive to changes in the size and spatial arrangement of amino acids within the loop. The p10 cystine loop may therefore function as a cystine noose, where fusion peptide activity is dependent on structural constraints within the noose that force solvent exposure of key hydrophobic residues. Moreover, inhibitors of cell surface thioreductase activity indicate that disruption of the disulfide bridge is important for p10-mediated membrane fusion. This is the first example of a viral fusion peptide composed of a small, spatially constrained cystine loop whose function is dependent on altered loop formation, and it suggests the p10 cystine loop represents a new class of viral fusion peptides.

FIGURE 1.

ARVp10 ectodomain contains two essential cysteine residues. A, aligned sequences of the ARV and NBV p10 ectodomains and the consensus sequence (Con.) indicating identical residues (top panel). The locations of the cysteine-flanked 11-residue HP and adjacent 9-residue conserved region (CR) are indicated. QM5 cells were transfected with either authentic ARV p10 or with the C9S or C21S substitution constructs. Cells were fixed and stained with Wright-Giemsa at 32 h post-transfection to detect syncytium formation by bright field microscopy at ×100 (bottom panel). B, cells were transfected as in A, and p10 protein expression was analyzed by Western blotting at 24 h post-transfection with α p10 antiserum or α actin (Ac) as a loading control. Vec, vector. C, surface expression was determined by live cell-labeling transfected QM5 cells with α p10 antibodies and fluorescently conjugated secondary antibodies at 24 h post-transfection, followed by flow cytometry analysis. Results are presented as the mean ± S.D. of the percent expression relative to authentic p10 from one of two experiments conducted in triplicate. D, pore formation activity was analyzed by the transfer of fluorescent calcein red-orange from target Vero cells to QM5 donor cells co-transfected with the indicated constructs and pEGFP as a marker for transfection. EGFP-positive QM5 cells were gated, and the acquisition of the calcein red-orange dye is shown versus forward scatter (FSC). Representational dot plots are from one of two experiments conducted in triplicate.

FIGURE 2.

Cysteine residues in the p10 ectodomain form an intramolecular disulfide bond. A, at 24 h post-transfection with the indicated p10 constructs, QM5 cells were incubated for 5 min in HBSS with or without 0.1 mm DTT. Free thiols were then labeled with maleimide-PEG2-biotin, and biotinylated proteins were isolated from cell lysates using immobilized neutravidin beads and resolved by SDS-PAGE, and Western blots were probed with ARV p10-specific antiserum. Vec, vector. B, QM5 cells were treated with DTT followed by maleimide-PEG2-biotin as in A, and cell lysates (L) were fractionated into membrane pellets (P) and soluble (S) fractions. Gels were then either Coomassie-stained or processed for Western blotting using HRP-conjugated neutravidin to detect biotinylated proteins.

FIGURE 3.

p10 fusion activity and intramolecular disulfide bond formation are highly sensitive to the location and context of the cysteine residues. A, point substitutions were introduced in the p10 ectodomain to create cysteine shift constructs (S1–S8). Substituted residues are underlined, and arrows depict the relative shift of the Cys-9 and Cys-21 residues. B, surface expression of each shift construct (S1–S8) was determined by live cell staining transfected cells followed by flow cytometric analysis at 24 h post-transfection. C, syncytium-inducing capacity of the p10 shift constructs at 32 h post-transfection was determined using Wright-Giemsa-stained monolayers to quantify the average numbers of syncytial nuclei in five random microscopic fields at ×200, and results are presented relative to authentic p10 set at 100%. Results in B and C are presented as the mean ± S.D. relative to authentic p10 from one of two experiments conducted in triplicate. D, pore formation was quantified based on the transfer of the calcein red-orange dye from target Vero cells to donor QM5 cells co-transfected with the indicated constructs and pEGFP as a marker for transfection. Results shown are representational dot plots of calcein fluorescence versus forward scatter (FSC) of EGFP-gated cells from one of two experiments conducted in triplicate. E, at 24 h post-transfection with the indicated p10 plasmid constructs, cells were incubated in HBSS with or without 0.1 mm DTT; free thiol groups were labeled with maleimide-PEG2-biotin; biotinylated proteins were isolated with immobilized neutravidin beads and fractionated by SDS-PAGE, and Western blots were probed with p10-specific antiserum. V, vector.

FIGURE 4.

Fusion activity and intramolecular disulfide bond formation are dependent on the size, location, and residue context of the cysteine residues. A, alanine residues were inserted in the ARV p10 ectodomain adjacent to Cys-9 and Cys-21, either individually or in combination (Cys-9 + Ala, Cys-20 + Ala, and Cys-9/20 + Ala, respectively) to change the size and geometry of the cystine loop. Hydrophobic, apolar, and polar residues are indicated on black, gray, or white backgrounds, respectively. B, surface expression of cells transfected with the indicated p10 constructs was determined by FACS analysis following live cell labeling with p10-specific antiserum and fluorescently conjugated secondary antibodies. Results represent the mean ± S.D. relative to authentic p10 surface expression from one of two experiments conducted in triplicate. C, calcein red-orange-labeled Vero cells were incubated with donor QM5 cells co-transfected with the indicated p10 constructs and pEGFP as a transfection marker. Pore formation was detected using FACS analysis to quantify transfer of the calcein dye from target Vero to donor QM5 cells. Results are presented as representational dot plots of EGFP-gated QM5 cells versus forward scatter (FSC) from one of two experiments conducted in triplicate. D, QM5 cells transfected with the indicated p10 plasmids were treated with or without DTT; free thiols were labeled with maleimide-PEG2-biotin, and p10-biotinylation was detected by precipitating biotinylated proteins with immobilized neutravidin beads, followed by Western blotting with p10-specific antiserum. Vec, vector.

FIGURE 5.

Reduction of the intramolecular disulfide bond by cell surface oxidoreductases is essential for efficient p10-mediated membrane fusion. A, at 3 h post-transfection with RRV p14, QM5 cells were incubated with the indicated concentrations of the cell surface thiol:disulfide oxidoreductase inhibitors Bacitracin (Bac) or DTNB. Cells were fixed 4 h later and Wright-Giemsa-stained, and syncytium formation was quantified by counting syncytial nuclei from five random microscopic fields at ×200 magnification. B, QM5 cells were transfected with either p14, ARV p10, or NBV p10, and 3 h later were incubated with 5 mm Bacitracin or 2.5 mm DTNB. At 7 h post-transfection with p14 or NBV p10, or 30 h post-transfection with ARV p10, cells were fixed and Wright-Giemsa-stained, and fusion was quantified by syncytial indexing as described in A. All results are presented as the mean ± S.D. relative to authentic p10 fusion activity from one of two experiments conducted in triplicate.