Table S2.

Hendra F protein expression and fusion activity

Construct	Total protein expression* (normalized to WT)	Cell surface protein† expression (normalized to WT)	Fusion activity‡ (normalized to WT)
65B	0.49	0.25	0.04
68B	0.64	0.61	1.00
70B	0.70	0.74	1.01
K70A	1.00	0.74	0.91
D188A	0.54	0.44	0.95
V68A	0.49	0.37	0.44
T164L	0.42	0.25	0.18
WT	1.00	1.00	1.00
Vector	0.09	0.12	0.02
K60C-T173C	0.35	0.22	0.02
P63C-V175C	0.28	0.12	0.03
Y97C-G131C	0.81	0.87	0.02
N100C-A116C	0.33	0.15	0.02
N100C-A119C	1.34	1.03	0.03
A137C-V267C	0.28	0.16	0.03

^*Total protein expression was assessed by radioimmunoprecipitation of cell lysates with anti-HeVF 527–540 antiserum. In brief, Vero cells were transfected with HeV F expression plasmid metabolically protein-labeled with S35 and cell surface biotinylated. Cells were lysed with radioimmunoprecipitation assay (RIPA) buffer, and anti-HeVF 527–540 antiserum was incubated with the lysate, followed by Ab pull-down with protein A beads. Ten percent of the protein A bead eluate was saved for total protein fraction analysis.

^†Cell surface protein expression was assessed by streptavidin pull-down of cell surface biotinylated HeV F from the total HeV pulled down by anti-HeV F 527–540 antiserum. In brief, 90% of the total fraction was incubated with streptavidin-Sepharose beads, and all of the eluate was saved for cell surface protein fraction analysis.

^‡Fusion activity was assessed by luciferase reporter gene assay. In brief, Vero cells transfected with HeV F, HeV G, and luciferase expression plasmids were overlaid with BSR-T7 cells expressing T7 polymerase. Because luciferase expression is under control of the T7 promoter, cell fusion results in luciferase protein production, which is then measured by a luminescent substrate activity assay. Reported values are the means of four measurements for single-site mutants, and the means of three measurements for double-Cys mutants.