The Effector Domain of the Influenza A Virus Nonstructural Protein NS1 Triggers Host Shutoff by Mediating Inhibition and Global Deregulation of Host Transcription When Associated with Specific Structures in the Nucleus

mBio. 2021 Oct 26;12(5):e0219621. doi: 10.1128/mBio.02196-21. Epub 2021 Sep 7.


Host shutoff in influenza A virus (IAV) infection is a key process contributing to viral takeover of the cellular machinery and resulting in the downregulation of host gene expression. Analysis of nascently transcribed RNA in a cellular model that allows the functional induction of NS1 demonstrates that NS1 suppresses host transcription. NS1 inhibits the expression of genes driven by RNA polymerase II as well as RNA polymerase I-driven promoters, but not by the noneukaryotic T7 polymerase. Additionally, transcriptional termination is deregulated in cells infected with wild-type IAV. The NS1 effector domain alone is able to mediate both effects, whereas NS1 mutant GLEWN184-188RFKRY (184-188) is not. Overexpression of CPSF30 counteracts NS1-mediated inhibition of RNA polymerase II-driven reporter gene expression, but knockdown of CPSF30 expression does not attenuate gene expression. Although NS1 is associated with nuclear chromatin, superresolution microscopy demonstrates that NS1 does not colocalize with genomic DNA. Moreover, NS1 mutants and NS1 fusion proteins, unable to associate with nuclear chromatin and displaying an altered subcellular distribution are still able to attenuate reporter gene expression. However, tethering NS1 artificially to the cytoskeleton results in the loss of reporter gene inhibition. A NS1 deficient in both native nuclear localization signals (NLS) is able to inhibit gene expression as effective as wild-type NS1 when a synthetic NLS relocates it to specific structures of the nucleus. Colocalization experiments and reporter gene cotransfection experiments with a NS1 fusion guiding it to nuclear speckles suggest that the presence of NS1 in nuclear speckles seems to be essential for host shutoff. IMPORTANCE We investigated the role of IAV nonstructural protein 1 NS1 in host gene shutoff-a central feature of IAV replication. We demonstrate that the effector domain of NS1 alone mediates host gene shutoff by inhibition of host transcription and by deregulation of the polyadenylation (polyA) signal-mediated 3' termination of host transcription. NS1 mutated in amino acids 184 to 188 fails to shut off host gene expression. Knockdown of CPSF30 does not result in transcriptional attenuation. By analyzing the subcellular localization of modified NS1 proteins and relating these data to their ability to inhibit reporter gene expression, we show for the first time that the presence of NS1 in granular structures of the nucleus-representing most likely nuclear speckles-seems to be essential to mediate host gene shutoff. Thus, our data present so far unknown insights into the molecular and spatial requirements needed for IAV-NS1-mediated host shutoff.

Keywords: NS1; host shutoff; influenza virus; transcription; transcriptional repression.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Nucleus / genetics
  • Cell Nucleus / metabolism
  • Cell Nucleus / virology*
  • Cleavage And Polyadenylation Specificity Factor / genetics
  • Cleavage And Polyadenylation Specificity Factor / metabolism
  • Host-Pathogen Interactions
  • Humans
  • Influenza A Virus, H7N7 Subtype / genetics
  • Influenza A Virus, H7N7 Subtype / metabolism*
  • Influenza, Human / genetics*
  • Influenza, Human / metabolism
  • Influenza, Human / virology*
  • Protein Domains
  • RNA Polymerase II / genetics
  • RNA Polymerase II / metabolism
  • Transcription, Genetic*
  • Viral Nonstructural Proteins / chemistry*
  • Viral Nonstructural Proteins / genetics
  • Viral Nonstructural Proteins / metabolism*


  • CPSF4 protein, human
  • Cleavage And Polyadenylation Specificity Factor
  • INS1 protein, influenza virus
  • Viral Nonstructural Proteins
  • RNA Polymerase II