Point mutations in the paramyxovirus F protein that enhance fusion activity shift the mechanism of complement-mediated virus neutralization

Abstract

Parainfluenza virus 5 (PIV5) activates and is neutralized by the alternative pathway (AP) in normal human serum (NHS) but not by heat-inactivated (HI) serum. We have tested the relationship between the fusion activity within the PIV5 F protein, the activation of complement pathways, and subsequent complement-mediated virus neutralization. Recombinant PIV5 viruses with enhanced fusion activity were generated by introducing point mutations in the F fusogenic peptide (G3A) or at a distal site near the F transmembrane domain (S443P). In contrast to wild-type (WT) PIV5, the mutant G3A and S443P viruses were neutralized by both NHS and HI serum. Unlike WT PIV5, hyperfusogenic G3A and S443P viruses were potent C4 activators, C4 was deposited on NHS-treated mutant virions, and the mutants were neutralized by factor B-depleted serum but not by C4-depleted serum. Antibodies purified from HI human serum were sufficient to neutralize both G3A and S443P viruses in vitro but were ineffective against WT PIV5. Electron microscopy data showed greater deposition of purified human antibodies on G3A and S443P virions than on WT PIV5 particles. These data indicate that single amino acid changes that enhance the fusion activity of the PIV5 F protein shift the mechanism of complement activation in the context of viral particles or on the surface of virus-infected cells, due to enhanced binding of antibodies. We present general models for the relationship between enhanced fusion activity in the paramyxovirus F protein and increased susceptibility to antibody-mediated neutralization.

Fig 1

Schematic diagram of PIV5 F protein and syncytia formed by PIV5 harboring the F protein mutants G3A and S443P. (A) The structure of the PIV5 F protein is shown schematically as a box with F2 and F1 subunits separated by an Arg-rich cleavage site. The fusion peptide (FP) (residues 103 to 128), heptad repeat A (HRA) (residues 129 to 204), HRB (residues 446 to 477), and transmembrane domain (TM) (residues 478 to 555) are shown by crosshatched, black, and white boxes, respectively. The locations of G3A and S443P mutations which enhance fusion activity are indicated (26). (B) CV-1 cells were infected with the indicated viruses, and pictures were taken at 24 or 30 hpi. Arrows indicate representative sites of cell-cell fusion.

Fig 2

Differential neutralization of WT PIV5 and the G3A mutant by NHS and HI serum. (A) One hundred PFU of WT PIV5 (left panel) or fusion mutant G3A (right panel) was incubated with the indicated dilutions (x axis) of NHS (black bars) or HI serum (hatched bars). After 1 h of incubation, remaining infectivity was determined by plaque assay. As a control, virus was incubated for 1 h with PBS alone (gray bars). (B and C) One hundred PFU of WT PIV5 or the G3A fusion mutant was incubated with PBS or a 1:20 dilution of NHS, HI serum, fB-depleted serum or fB-depleted serum supplemented with purified fB (B), or C4-depleted serum or C4-depleted serum supplemented with purified C4 (C). After 1 h of incubation, remaining infectivity was determined by plaque assay. (D) One hundred PFU of the S443P F fusion mutant was incubated for 1 h with the indicated dilutions of NHS, and remaining infectivity was determined by plaque assay. For all experiments, data are from six independent reactions and error bars represent standard deviation. *, P < 0.05 for values compared to PBS controls.

Fig 3

C4 is activated by G3A but not WT PIV5 virions. (A) Equal amounts of gradient-purified WT PIV5 and the G3A and S443P mutants were analyzed by Western blotting for levels of NP, M, HN, and F proteins. (B and C) The indicated amounts of purified virus were incubated for 1 h with a 1:20 dilution of NHS, and levels of C3a (B) and C4a (C) were determined by ELISA. # and ^, P < 0.005 and P < 0.05, respectively, for comparison against data with WT PIV5.

Fig 4

Time course of C4 activation and deposition by mutant but not WT PIV5 virions. (A) A 0.1-μg amount of purified WT PIV5 or the G3A or S443P fusion mutant was incubated for the indicated times with a 1:20 dilution of NHS, and levels of C4a were determined by ELISA. NHS, a control sample that lacked virus. # and ^, P < 0.005 and P < 0.05, respectively, for comparison against data with WT PIV5. (B) Purified virus was treated with NHS followed by anti-C4 antibody and 12-nm-colloidal-gold-labeled goat anti-mouse antibody. Control samples were treated with secondary antibody alone. Samples were analyzed by EM at a magnification of ×55,000 (bar, 0.1 μm).

Fig 5

Purified hAb neutralizes the hyperfusogenic G3A and S443P mutants but not WT PIV5. (A and B) One hundred PFU of WT PIV5 or the G3A or S443P fusion mutant was incubated with PBS, the indicated dilutions of pooled commercial NHS or HI serum (A), or the indicated amount (μg) of purified hAb (B) as described in Materials and Methods. Remaining infectivity was determined by plaque assay. Data are from six independent reactions, and error bars represent standard deviation. *, P < 0.05 for values from HI compared to NHS samples (A) or from G3A and S443P compared to PIV5 samples (B). The data in panel B are from two separate experiments. (C) Purified virus was treated with purified hAb followed by 6-nm-colloidal-gold-labeled goat anti-human Ig. Control samples were treated with secondary antibody alone. Samples were analyzed by EM at a magnification of ×55,000 (bar, 0.1 μm).

Fig 6

Enhanced binding of human antibodies to heat-treated WT virions. Purified WT PIV5 was deposited on preheated EM grids and held for 10 min in a humidified chamber at either 37°C or 50°C. After cooling to room temperature, purified human antibody was added and incubated for 10 min at 37°C before analysis as described in the legend to Fig. 5.

Fig 7

Differential sensitivity of WT PIV5 and G3A mutant derived from human A549 cells to NHS and HI serum. (A) WT and G3A viruses were grown in A549 cells and analyzed for neutralization as described in the legend to Fig. 5A. (B) HI serum was mock treated or treated with 10 mg/ml soluble Galα1-3Gal before use in neutralization of WT and G3A viruses that were derived from A549 cells as described for panel A. For both panels, data are from six independent reactions and error bars represent standard deviation. *, P < 0.05 for G3A compared to WT PIV5 values.

Fig 8

C4 and hAb are deposited to a greater extent on the surface of cells infected with the G3A and S443P fusogenic mutants than on those infected with WT PIV5. CHO cells were mock infected or infected at an MOI of 10 with WT, G3A, or S443P virus. At 20 hpi, cells were treated with NHS (A, B, and D) or with purified human antibody (C) as described in Materials and Methods. Samples were analyzed for levels of cell-associated C4 and Ig by flow cytometry (A, B, and C) or by Western blotting for Ig and the C4 beta chain (D). The positions of heavy and light chains are indicated as H and L, respectively. Panels A and B show results from two experiments. Panel C shows mean fluorescence intensity of C4 (hatched bars) and Ig (black bars) as an average from two experiments.