Influenza virus infection causes global RNAPII termination defects

Viral infection perturbs host cells and can be used to uncover host regulatory mechanisms controlling both cell response and homeostasis. Here, using cell biological, biochemical and genetic tools, we reveal that influenza virus infection induces global transcriptional defects at the 3'-end of active host genes and RNA polymerase II (RNAPII) run-through into extragenic regions. This effect induces the biogenesis of aberrant RNAs (3'-extensions and host gene fusions) which ultimately causes global transcriptional downregulation of physiological transcripts, an effect that impacts antiviral response and virulence. We show that this phenomenon occurs with multiple strains of influenza virus and it is dependent on influenza NS1 protein expression. Mechanistically, pervasive RNAPII run-through can be modulated by SUMOylation of an intrinsically disordered region (IDR) of the NS1 expressed by the 1918 pandemic influenza virus. SUMOylation increases NS1 partitioning in nuclear granules and interference with the host transcriptional apparatus which result in augmentation of termination defects and a concomitant increase in global host gene shut off. Our data identify a general strategy used by influenza virus to suppress host gene expression and indicate that polymorphisms in IDRs of viral proteins, along with human genetic variation in enzymes that metabolize post-translational modifications, can determine the outcome of an infection. We thus propose that analysis of strain-specific determinant of pathogenesis can shed light on the molecular basis of virulence. Overall design: We performed directional RNA-Seq profiling (Illumina TruSeq protocol) of uninfected A549 cells, and at 6 and 12 hours after infection with wild-type H1N1 Influenza A/Puerto Rico/8/1934 (PR8-NS1) or PR8-NS1-SUMO Influenza A virus. In the NS1-SUMO virus we fused a SUMO domain to the C-terminus of the NS1 protein. We also performed RNA-Seq of A549 cells transfected with empty vector, or a vector expressing GFP or SUMO. Additionally, cells transfected with empty vector or a vector expressing SUMO were infected with PR8?NS1 virus and RNA-Seq was performed at 4 hours post-infection. Finally, we performed RNA-Seq of uninfected A549 cells depleted for Ubiquitin Conjugating Enzyme E2 (siUBE2I) and control (siCtrl). All experiments were performed in duplicate.