Influenza A Virus Virulence Depends on Two Amino Acids in the N-Terminal Domain of Its NS1 Protein To Facilitate Inhibition of the RNA-Dependent Protein Kinase PKR

  1. Thorsten Wolffa

  1. aUnit 17, Influenza and other Respiratory Viruses, Robert Koch Institut, Berlin, Germany

  2. bDepartment of Biology, Molecular Biophysics, IRI Life Sciences, Humboldt Universität zu Berlin, Berlin, Germany

  3. cUniversities Giessen & Marburg Lung Center, Department of Internal Medicine II, Section of Infectious Diseases, German Center
    for Lung Research, Giessen, Germany

  4. dInstitute of Medical Virology, Justus Liebig University Giessen, Giessen, Germany

ABSTRACT

The RNA-dependent protein kinase (PKR) has broad antiviral activity inducing translational shutdown of viral and cellular
genes and is therefore targeted by various viral proteins to facilitate pathogen propagation. The pleiotropic NS1 protein
of influenza A virus acts as silencer of PKR activation and ensures high-level viral replication and virulence. However, the
exact manner of this inhibition remains controversial. To elucidate the structural requirements within the NS1 protein for
PKR inhibition, we generated a set of mutant viruses, identifying highly conserved arginine residues 35 and 46 within the
NS1 N terminus as being most critical not only for binding to and blocking activation of PKR but also for efficient virus
propagation. Biochemical and Förster resonance energy transfer (FRET)-based interaction studies showed that mutation of R35
or R46 allowed formation of NS1 dimers but eliminated any detectable binding to PKR as well as to double-stranded RNA (dsRNA).
Using in vitro and in vivo approaches to phenotypic restoration, we demonstrated the essential role of the NS1 N terminus for blocking PKR. The strong
attenuation conferred by NS1 mutation R35A or R46A was substantially alleviated…

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