FIG. 1.

Identification of cellular proteins that enhance influenza virus replication and downregulation of influenza virus replication by RSK2. (A) Human embryonic kidney 293T cells were transfected with plasmids encoding the components of the influenza viral replication complex (PB2, PB1, PA, and NP). The PB2 protein possesses glutamic acid at position 627 (PB2-627E), which supports efficient replication in avian cells but not in mammalian cells at 33°C. Cells were cotransfected with a plasmid for the synthesis of a virus-like RNA encoding fCD2 (polI-fCD2) and with an avian (quail cell) cDNA library. Cells expressing avian proteins that support efficient replication by PB2-627E virus in mammalian cells at 33°C will produce increased amounts of fCD2. Cells with high levels of fCD2 were selected by immunoaffinity using anti-fCD2 antibody. After a total of four rounds of selection, plasmid DNA was extracted from the cells and sequenced. (B) Schematic diagram of human RSK2 (top) and the N-terminally deleted avian RSK2 protein selected in our screening approach (bottom). The CKD is activated by ERK1/2, resulting in the activation of the NKD. The NKD subsequently phosphorylates target proteins. (C) Influenza viral polymerase activity in 293T cells expressing avian ΔN-RSK2. Cells were transfected with plasmids expressing PB2E, PB1, PA, and NP; a virus-like RNA encoding luciferase (pPolI-luc); and the N-terminally deleted avian RSK2 protein (pCAGGS-ΔN-RSK2) or the “empty” control vector (pCAGGS). Cells were maintained at 33°C for 24 h and were then subjected to luciferase assays. In human cells at 33°C, expression of N-terminally deleted avian RSK2 increased the activity of a replication complex possessing glutamic acid at position 627 in the PB2 protein. The error bars represent standard deviations from three independent experiments.