Fusion activity was assessed using a β-galactosidase complementation assay. HEK293T cells were cotransfected with either (A) HN sequence of the indicated patients, lab-adapted HPIV3 F, and the α subunit of β-galactosidase, or (B) F sequence of the indicated patients, lab-adapted HPIV3 HN, and the α subunit of β-galactosidase. Cells were then incubated for 6 hours with HEK293T cells expressing the Ω subunit of β-galactosidase. Fusion is depicted by luminescence relative to WT patient HN (A) or relative to mock-transfected cells (B) containing only lab-adapted HPIV3 HN and the Ω subunit of β-galactosidase. Results depict representative experiments from 3 biological replicates. Data indicate the mean ± SD. A 2-way ANOVA was performed to determine significance; P values are specified as follows: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001.