Mutation
number
1
2
3
4
5
6
7
8
9
10
11
Mutation in
Nipah G (aa
residues)
72-111
112-145
146-182
196-212
213-247
248-277
278-307
337-371
372-393
400-436
437-464
+
+
+
+
+
+
Cell surface
binding to 293T
cells
+
+
+
+
-
+
-
Protein
Produced
(28 NiV-G-Fc)
12
13
14
508-532
533-558
559-587
Supplementary Table I. Deletion analysis of NiV-G. Based on the predicted folding
patterns of the extracellular domain of Hendra G protein1, systematic deletions were
made to the ectodomain of Nipah G to remove predicted units of secondary structures.
The indicated amino acid residues were deleted using the QuickChange™ site-directed
mutagenesis kit according to the manufacturer’s directions (Stratagene, San Diego, CA).
Protein production was measure by an Fc ELISA (see Supp. Methods 1) and values that
were greater than 0.1ug/ml of unconcentrated supernatant were considered positive. Each
mutant was also analyzed for its ability to bind 293T in a flow cytometry assay. Deletion
number 11 (residues 437-464) produced protein comparable to wild type NiV-G-Fc and
did not bind to 293T cells. This construct, named 28 Niv-G-Fc, was chosen to serve as a
negative control for immunoprecipation experiments.
1.
Yu, M., Hansson, E., Langedijk, J. P., Eaton, B. T. & Wang, L. F. The
attachment protein of Hendra virus has high structural similarity but limited
primary sequence homology compared with viruses in the genus
Paramyxovirus. Virology 251, 227-33 (1998).