Affinity thresholds for membrane fusion triggering by viral glycoproteins

Abstract

Enveloped viruses trigger membrane fusion to gain entry into cells. The receptor affinities of their attachment proteins vary greatly, from 10(-4) M to 10(-9) M, but the significance of this is unknown. Using six retargeted measles viruses that bind to Her-2/neu with a 5-log range in affinity, we show that receptor affinity has little impact on viral attachment but is nevertheless a key determinant of infectivity and intercellular fusion. For a given cell surface receptor density, there is an affinity threshold above which cell-cell fusion proceeds efficiently. Suprathreshold affinities do not further enhance the efficiency of membrane fusion.

FIG. 1.

Characterization of the HER2/neu-retargeted measles viruses. (A) Schematic representation of the HER2-retargeted viruses. The Y481A and R533A mutations ablate CD46 and SLAM interactions. The scFv was inserted as a SfiI/NotI fragment and displayed as a C-terminal extension on mutated HA proteins with a His₆ tag. (B) Immunoblotting results of the parental and HER2-retargeted virions using anti-HA and anti-N antibodies. Equal titers of each virus were loaded. (C) Specificity of the receptor usage of parental and fully retargeted anti-HER2 measles viruses. CHO cells and CHO cells stably expressing CD46, SLAM, HER2, or αHis were infected with the parental MV-eGFP, the CD38-retargeted, or the HER2-retargeted measles viruses at an MOI of 1.0. Cells were photographed with fluorescence microscopy after 48 h.

FIG. 2.

The impact of receptor affinity on virus binding and infectivity. (A) There is no correlation between receptor affinity and virus binding with SKOV3ip.1 cells. HER2-retargeted viruses were allowed to bind to SKOV3ip.1 cells on ice for various times, after which unbound viruses were removed. Numbers of GFP-positive cells were counted at 48 h postinfection and plotted. The negative control virus MV-αCD38 displays a CD38-specific scFv and does not bind HER2/neu. (B, C, and D) We next determined the impact of receptor affinity on viral infectivity with high-HER2-expressing SKOV3ip.1 cells or low-HER2-expressing TE671 cells (40-fold fewer HER2 receptors). CHO cells expressing the pseudoreceptor αHis were used as a positive control for virus infectivity. Cells were infected at an MOI of 1.0 or 4.0, and the numbers of GFP-positive cells were counted by flow cytometry at 48 h postinfection. Error bars represent standard deviations. P value was determined by one-way analysis of variance at an MOI of 4.0. Asterisks (**) indicate a significant difference (P < 0.01) between the test group and the MV-αHER-8-infected cells, using Dunnett's multiple comparison test.

FIG. 3.

Influence of MOI on syncytial formation of low- and high-affinity viruses. (A) HER2-overexpressing SKOV3ip.1 cells were infected at MOIs of 1.0 and 4.0 with retargeted viruses. Photographs were taken at 72 h postinfection, and syncytial formations were compared. Increasing the numbers of infectious centers by increasing MOI did not result in syncytial formation for the low-affinity viruses. (B) SKOV3ip.1 cells were infected with each member of the HER2-retargeted virus panel at MOIs of 0.02, 0.1, and 0.5. Cells were fixed and stained with Hoechst 33342 at 72 h postinfection. Numbers of nuclei in each syncytium were counted (10 to 20 syncytia were counted). Sizes of the syncytia were not affected by MOI and were comparable at all MOIs.

FIG. 4.

Cytopathic potential of the HER2-retargeted measles viruses. HER2-overexpressing SKOV3ip.1 cells, low-HER2-expressing TE671 cells, and Vero-αHis cells were infected with HER2-retargeted viruses at an MOI of 1.0. Virus-induced apoptosis (A) and antiproliferative effects (B) were analyzed by enzyme-linked immunosorbent assay and by clonogenic assay, respectively. Error bars represent ± standard deviations. The P value was determined by one-way analysis of variance. Asterisks (**) indicate a significant difference (P < 0.01) between the test group and the MV-αHER-8-infected group, using Dunnett's multiple comparison test.

FIG. 5.

The impact of HER2 receptor density and affinity with virus-induced cell-to-cell fusion. (A) A panel of tumor cell lines with various amounts of HER2 receptors was infected with each of the HER2-retargeted viruses at an MOI of 1.0. Asterisks (*) denote receptor levels below the levels of detection (10³). Photographs were taken at 72 h postinfection. (B) CHO cells or CHO clones expressing low and high HER2 receptors and CHO-αHis cells were treated as shown in panel A.