Fig. 1
Generation of mutant influenza A viruses by use of reverse genetics. a Schematic diagram of a mutant HA vRNA (HAstop) that does not produce HA protein. It contains two stop codons and a FLAG epitope (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) followed by a stop codon, in-frame with the HA reading frame. The numbers indicate the nucleotide positions from the 3′ end of the HA vRNA. Neither the HA protein nor the FLAG protein were detected by western blot analysis in cells transfected with this construct, using anti-HA and anti-FLAG antibodies. b RT-PCR analysis of the mutant viruses. The extracted vRNAs from the supernatants of plasmid-transfected cells were reverse-transcribed using a primer that was complementary to the conserved 12 nucleotides of the 3′ end of all eight vRNAs, followed by amplification by PCR with strand-specific primers for NP or HA vRNA. Reverse transcriptase (RT) reactions were performed with or without reverse transcriptase. c MDCK cells were infected with HA(−), HAstop(+), and wild-type viruses, respectively. At 12 h postinfection, the cell lysates were subjected to western blot analysis using anti-HA and anti-NP monoclonal antibodies. d 293T cells were transfected with plasmids to produce the HA(−), HAstop(+), and wild-type viruses. Virus yields were determined at 48 h post-transfection by use of plaque assays on HA-MDCK cells. The data represent the mean ± SD (n = 3). e HA-MDCK cells were infected with the respective viruses at an MOI of 0.01, and the culture media were harvested at 24 and 48 h postinfection. The virus titers were determined by use of plaque assays on HA-MDCK cells. The data represent the mean ± SD (n = 3)
