Abstract
ABSTRACTHost 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.
Highlights
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
Infection leads to decreased polymerase II (Pol II) occupancy downstream of transcription start sites (TSSs), and second, interference with 39-end processing leads to marked defects in termination of Pol II transcription at the end of genes
We first investigated whether host transcription may be affected by induction of functional NS1
Summary
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. 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. 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 39 termination of host transcription. 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. Several studies have shown that these sites are important for the process of human adaptation of different animal IAV strains [13]
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