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Mutant influenza viruses with a defective NS1 protein cannot block the activation of PKR in infected cells
1Department of Biochemistry, Kanazawa University School of Medicine, Kanazawa, Ishikawa 920-0934, Japan.
Abstract:
A short model genome RNA and also the genome RNA of influenza A virus bearing both 5'- and 3'-terminal common sequences activated the interferon-induced double-stranded-RNA-dependent protein kinase, PKR, by stimulating autophosphorylation in vitro. The activated PKR catalyzed phosphorylation of the alpha subunit of eucaryotic translation initiation factor 2 (eIF2alpha). The NS1 protein efficiently eliminated the PKR-activating activity of these RNAs by binding to them. Two mutant NS1 proteins, each harboring a single amino acid substitution at different regions, exhibited temperature sensitivity in their RNA binding activity in the mutant virus-infected cell lysates as well as when they were prepared as fusion proteins expressed in bacteria. The virus strains carrying these mutant NS1 proteins exhibited temperature sensitivity in virus protein synthesis at the translational level, as reported previously, and could not repress the autophosphorylation of PKR developing during the virus growth, which is normally suppressed by a viral function(s). As a result, the level of eIF2alpha phosphorylation was elevated 2.5- to 3-fold. The defect in virus protein synthesis was well correlated with the level of phosphorylation of PKR and eIF2alpha.
Insights
Influenza A virus NS1 protein binds viral RNA, preventing activation of the protein kinase PKR. Mutants show impaired RNA binding, leading to PKR activation and reduced viral protein synthesis.
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- Interferon-induced double-stranded RNA-dependent protein kinase (PKR) plays a crucial role in antiviral defense.
- Influenza A virus employs mechanisms to evade the host immune response, including PKR inhibition.
Purpose of the Study:
- To investigate the role of the influenza A virus NS1 protein in PKR activation and inhibition.
- To characterize the impact of NS1 mutations on RNA binding, PKR activity, and viral protein synthesis.
Main Methods:
- In vitro activation of PKR by viral RNA.
- Assessing NS1 protein's ability to bind viral RNA.
- Analyzing phosphorylation of PKR and eukaryotic translation initiation factor 2 alpha (eIF2alpha) in cells infected with wild-type and mutant viruses.
- Evaluating viral protein synthesis at different temperatures.
Main Results:
- Viral genome RNA activated PKR, leading to eIF2alpha phosphorylation.
- The NS1 protein inhibited PKR activation by binding to viral RNA.
- Mutant NS1 proteins with temperature-sensitive RNA binding failed to repress PKR autophosphorylation.
- This resulted in elevated eIF2alpha phosphorylation and temperature-sensitive viral protein synthesis.
Conclusions:
- The NS1 protein's RNA-binding activity is essential for suppressing PKR-mediated antiviral responses.
- Impaired NS1 function leads to increased PKR and eIF2alpha phosphorylation, consequently hindering viral replication.