Figure 5.

NS1 is required for robust expression of vtRNAs induced during IAV infection both in vitro and in vivo. (A) A549 cells expressing shRNAs targeting RIG-I or luciferase (luc) were infected with or without WSN for 14 h, and then the expression of vtRNAs was examined by qRT-PCR. (B) A549 cells were transfected with indicated amount of WSN genomic RNA (VG-RNA) using Lipofectamine 2000. Effect of VG-RNA on the expression of vtRNAs was determined by qRT-PCR. (C) Different amounts of total RNA (named as Viral RNA) from A549 cells infected with the IAV were transfected into native A549 cells using Lipofectamine 2000. The expression of vtRNAs in transfected A549 cells was examined by RT-PCR. (D) 293T cells were transfected with empty vector (Mock) or plasmids expressing either NS1, NA, M2, or HA protein of IAV using Vigofect. After 36 h post-transfection, the vtRNAs expression was detected by RT-PCR. (E) A549 cells stably expressing specific shRNAs targeting NS1 or luciferase (control) were infected with or without WSN. Subsequently, the RNA levels of vtRNAs were measured by qRT-PCR. (F) A549 cells were infected with or without PR8 wild-type (WT) or deltaNS1 (delNS1) viruses for 20 h and analyzed by Western blotting and RT-PCR to detect the levels of the indicated genes and proteins. (G) Mice intranasally infected with or without PR8 WT or delNS1 viruses for 2 days were sacrificed, and the lungs were dissected and homogenized, followed by qRT-PCR. Shown are representative RT-PCR data from three independent experiments. Plotted are the average levels from three independent experiments. The error bars represent the SE, **P < 0.01.