Text S1 - Unraveling a Three-Step Spatiotemporal Mechanism of Triggering of Receptor-Induced Nipah Virus Fusion and Cell Entry

S1) Co-immunoprecipitation between NiV-F and selected headless NiV-G and point mutants from regions 4N and 9. 293T cells were transfected with indicated plasmids. Cell lysates were subjected to immunoprecipitation to pull down (A) wt NiV-G (G) or headless NiV-G mutant 167 using polyclonal rabbit Ab167 antibody (IP: Ab167) or (B) wt NiV-G (G) or selected NiV-G mutants in regions 4N and 9 (V178A, L181A, V182A, C387A), using HA-linked magnetic MicroBeads (IP: αHA). Immunoprecipitated G or G mutants were then detected by immunoblotting using either Ab167 (IB: Ab167) (A) or rabbit anti-HA (IB: αHA) (B) antibodies. Uncleaved(F0) and cleaved version of F (F1) were detected using mouse anti-AU1 (IB: aAU1). Proteins were detected from total (left panels) and from immunoprecipitated (right panels) cell lysates. In agreement with published reports, we observed a doublet of NiV-G when using antiserum 806 or anti-tag antibodies, when the SDS-PAGE bands were sufficiently resolved, likely resulting from variable NiV-G secondary modifications such as glycosylation. S2) Summary of the alanine scan mutants for regions 4N and region 9. Mutants were generated using a QuikChange ™ site directed mutagenesis kit (Stratagene). Fusion indexes = ratio of % cell-cell fusion levels normalized to NiV-G/% CSE levels normalized to those of wt NiV-G. N.D. = non determined because CSE was too low to be reliable. S3) Pymol representation of NiV-G head bound to its ephrinB2 receptor (magenta). Region 9 (orange) and 4N (green) are shown. The crystallized structure was taken from [24] (PDB 2VSM). S4) Cell surface expression and ephrinB2 binding of fusion mutants in regions 4N and 9. Mutants expressing notably hypofusogenic or hyperfusogenic phenotypes were selected and analyzed. CHO cells were transfected with either wt or mutant NiV-G expression plasmids. Binding of soluble ephrinB2 to transfected cells expressing NiV-G, along with cell surface expression using an anti-HA Mab, were measured by flow cytometry. Average ± S.E. are shown. n = 3. (PPTX)