Fig. 5: GGCX-mediated Carboxylation modification of viral HA 225E promoted its binding activity to α-2, 6 SA receptors. | Nature Communications

Fig. 5: GGCX-mediated Carboxylation modification of viral HA 225E promoted its binding activity to α-2, 6 SA receptors.

From: GGCX promotes Eurasian avian-like H1N1 swine influenza virus adaption to interspecies receptor binding

Fig. 5

a Flowchart showing the identification of carboxylation modification sites of viral HA by LC-MS. b Venn diagram of identified carboxylation modification sites in viral HA proteins expressed in infected GGCX KO and WT cells. c The Venn diagram analysis of the conservation of glutamic acid (E) at the carboxylation modification sites in EA H1N1 SIV and different AIV subtypes. d Identification of key carboxylation modification sites of viral HA. HEK293T cells were transfected with psPAX2, pLenti-luc, and WT or mutant viral HA plasmids to generate pseudoviruses. The pseudoviruses were then used to infect WT PK-15 cells and luciferase assays were performed. Protein expression levels were evaluated by western blot. e, f Binding activities of HA 225E and mutant viruses. Equal amounts of HA 225E, 225D, and 225 G viruses were each used to infect the WT PK-15 cells and incubated on ice for 1 h, followed by incubation with an anti-influenza virus HA protein antibody. Then, the HA proteins were analyzed by (e) confocal microscopy and (f) average fluorescence intensity analysis (each group had n = 20 cells) as shown in (e). The viral HA was stained red, and the nucleus was stained blue. Scale bars, 10 μm. g Sialic acid receptor binding preferences of HA 225E and mutant viruses. Equal amounts of HA 225E, 225D, and 225 G viruses were used to incubate biotinylated sugar mimics of sialic acid receptors, and ELISA assays were performed to determine the receptor binding activities. The images in panel e are representative of three independent experiments. The error bar in panels (d and g) indicates the standard deviation. The data shown in panels (d, f, and g) are means ± SD (n  =  3 for (d and g), and n = 20 for f biologically independent experiments). Statistical analysis was performed using an unpaired, two-tailed Student’s t test. (ns, P > 0.05; ****P < 0.0001). Exact P-values are available in the Source Data. Source data are provided as a Source Data file.

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