FIG 2.

Schematic representation of the bacterium-based assay to identify PA-X mutants affected in inhibition of host gene expression and identified mutants. (A) A bacterium-based assay for the identification of amino acid residues of A/Viet Nam/1203/2004 H5N1 PA-X involved in inhibition of host gene expression: steps involved in the molecular cloning of A/Viet Nam/1203/2004 H5N1 PA-X into pGBKT7 plasmid, under the control of T7 polymerase promoter (blue arrow). After ligation of PA-X into pGBT7, the mixture is transformed in bacteria, and individual colonies are isolated and sequenced. Leaked PA-X expression from the pGBKT7 promoter results in inhibition of host gene expression in bacteria and therefore no bacterial growth. PA-X mutants affected in inhibition of host gene expression allow bacteria to grow. Sequencing bacterial clones identify A/Viet Nam/1203/2004 H5N1 PA-X mutants affected in host shutoff. (B) A/Viet Nam/1203/2004 H5N1 PA-X and PA ORFs and amino acid mutations in PA-X identified in the bacterial-based assay: the +1 frameshift motif (UCC UUU CGU C) is indicated by a striped bar. The amino acid substitutions identified are indicated with black (affecting both PA-X and PA proteins) or gray (affecting only PA-X protein) lines. The superscript 1 indicates that mutations E101G and R125K were found in the same clone; the superscript 2 indicates that mutations D111N and E196G were found in the same clone. “##” symbols indicate that the clones were found twice. (C) Deletions and/or insertions of nucleotides (NT) leading to no protein expression (i), translation of short peptides (ii to v), and translation of a protein encoding the first 250 residues of WT PA-X and a short tail (vi). The #, ##, and ### symbols indicate that the clones were found one, two, and three times, respectively. Other mutations were found in one clone. *, Stop codons. Amino acids in boldface indicate WT PA-X sequence.