Abstract
The influenza A virus (IAV) PA-X protein is a virulence factor that selectively degrades host mRNAs leading to protein shutoff. This function modulates host inflammation, antiviral responses, cell apoptosis, and pathogenesis. In this work we describe a novel approach based on the use of bacteria and plasmid encoding of the PA-X gene under the control of the bacteriophage T7 promoter to identify amino acid residues important for A/Brevig Mission/1/1918 H1N1 PA-X’s shutoff activity. Using this system, we have identified PA-X mutants encoding single or double amino acid changes, which diminish its host shutoff activity, as well as its ability to counteract interferon responses upon viral infection. This novel bacteria-based approach could be used for the identification of viral proteins that inhibit host gene expression as well as the amino acid residues responsible for inhibition of host gene expression.
Highlights
We used a novel approach based on the use of plasmids encoding the 1918 polymerase acidic (PA)-X gene under the control of the T7 bacteriophage promoter to identify amino acid residues important for this shutoff activity (Figure 2)
1918 PA-X expression, which was likely driven by the leaking T7 promoter in bacteria [32], was either deleterious, toxic or both, for bacteria growth, and bacteria were forced to introduce mutations affecting 1918 PA-X translation or its ability to inhibit host gene expression
We identified five different plasmids encoding single amino acid changes in the 1918 PA-X gene, and three different plasmids encoding two amino acid changes each in 1918 PA-X (Figure 2 and Table 1), supporting the hypothesis that bacteria were under selective pressure to introduce changes in the plasmids to abolish the ability of PA-X to inhibit host gene expression
Summary
In this work we describe a novel approach based on the use of bacteria and plasmid encoding of the PA-X gene under the control of the bacteriophage T7 promoter to identify amino acid residues important for A/Brevig Mission/1/1918 H1N1 PA-X’s shutoff activity. Using this system, we have identified PA-X mutants encoding single or double amino acid changes, which diminish its host shutoff activity, as well as its ability to counteract interferon responses upon viral infection. PA-X is translated as a +1 frameshifting from the viral PA mRNA [10], and shares the same first 191 N-terminal amino acids with
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