FIG 9.

G protein-coupled receptor kinases (GRKs) are required for IAV replication. (A) A549 cells were transfected with siRNA targeting GRK2, GRK3, GRK5, or GRK6 for 48 h. The levels of mRNA of the indicated gene in the cell lysates were determined by qRT-PCR and standardized to those of the scrambled siRNA-treated cells. ***, P < 0.001. (B) A549 cells were treated with siRNA targeting GRK2, GRK3, GRK5, or GRK6 for 48 h, and cell viability was measured by using the CellTiter-Glo assay. (C) HEK293T cells were treated with siRNA targeting GRK2, GRK3, or GRK5 or with scrambled siRNA for 12 h and then transfected with the corresponding Myc-tagged protein expression construct for 36 h. Whole-cell lysates were collected and analyzed by Western blotting with a mouse anti-Myc mAb. Separately, A549 cells were transfected with GRK6 siRNA or scrambled siRNA for 48 h, and whole-cell lysates were collected and analyzed by Western blotting with a rabbit anti-GRK6 pAb. (D) A549 cells were transfected with siRNA targeting GRK2, GRK3, GRK5 or GRK6 or with scrambled siRNA for 48 h and then infected with AH05 (H5N1) virus (MOI = 0.1). Supernatants were collected at the indicated time points, and virus titers were determined by means of plaque assays on MDCK cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (E) BiFC analysis to examine the interaction between FFAR2-VN and GRK2-VC, GRK5-VC, or GRK6-VC in HeLa cells. Cotransfection of vector VN and vector VC served as a negative control.