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. 2020 Jan 6;94(2):e01707-19. doi: 10.1128/JVI.01707-19

FIG 6.

FIG 6

FFAR2 is important for the internalization of IAV. (A) A549 cells were treated with FFAR2 siRNA or scrambled siRNA for 48 h, stained with a wheat germ agglutinin (WGA)-Alexa Fluor 488 conjugate, and analyzed by flow cytometry. (B and C) A549 cells treated with FFAR2 siRNA or scrambled siRNA were stained with lectins that have specificity for α-2,3-sialic acids (MAL) (B) or α-2,6-sialic acids (SNA) (C) and analyzed by flow cytometry. (D and E) A549 cells were treated with FFAR2 siRNA or scrambled siRNA for 48 h and then infected with AH05 (H5N1) virus on ice at 4°C for 1 h. Cells were fixed, stained with a mouse anti-HA mAb (D) or a mouse anti-NP mAb (E) and Alexa Fluor 488 goat anti-mouse IgG(H+L), and analyzed by flow cytometry. (F) A549 cells were transfected with FFAR2 siRNA or scrambled siRNA for 48 h and infected with AH05 (H5N1) virus (MOI = 5) on ice at 4°C for 1 h. After unbound virus was washed away, the cells were collected and titrated for infectious virus by means of plaque assays on MDCK cells. (G) A549 cells were infected with AH05 (H5N1) virus (MOI = 5) on ice at 4°C for 1 h, followed by a neutral wash (ice-cold PBS, pH 7.2) or an acidic wash (ice-cold PBS-HCl, pH 1.3) before cell lysis. The amount of internalized virus particles was determined by Western blotting with a rabbit NP pAb. (H) FFAR2 siRNA- or scrambled siRNA-treated A549 cells were infected with AH05 (H5N1) virus (MOI = 5) on ice at 4°C for 1 h, followed by a culture temperature shift to 37°C for 30 min to allow for internalization. The cells were then washed with ice-cold PBS-HCl (pH 1.3) before cell lysis. The amount of internalized virus particles was determined by Western blotting with a rabbit NP pAb. (I) A549 cells were pretreated with 100 μM 4-CMTB, 10 μM Cmp58, or DMSO for 3 h and then infected with AH05 (H5N1) (MOI = 5) at 37°C. The infected cells were washed with ice-cold PBS-HCl (pH 1.3) at the indicated time points before cell lysis. The amount of internalized virus particles was determined by Western blotting with a rabbit NP pAb. A549 cells pretreated with neuraminidase (Neu) were stained with WGA (A), MAL (B), and SNA (C), respectively, and used as negative controls. The band intensities of the Western blots were quantified by using ImageJ software and are expressed as relative NP/GAPDH ratios (G to I).