Influenza virus infection causes global RNAPII termination defects

Abstract

Viral infection perturbs host cells and can be used to uncover regulatory mechanisms controlling cellular responses and susceptibility to infections. Using cell biological, biochemical, and genetic tools, we reveal that influenza A virus (IAV) infection induces global transcriptional defects at the 3' ends of active host genes and RNA polymerase II (RNAPII) run-through into extragenic regions. Deregulated RNAPII leads to expression of aberrant RNAs (3' extensions and host-gene fusions) that ultimately cause global transcriptional downregulation of physiological transcripts, an effect influencing antiviral response and virulence. This phenomenon occurs with multiple strains of IAV, is dependent on influenza NS1 protein, and can be modulated by SUMOylation of an intrinsically disordered region (IDR) of NS1 expressed by the 1918 pandemic IAV strain. Our data identify a strategy used by IAV to suppress host gene expression and indicate that polymorphisms in IDRs of viral proteins can affect the outcome of an infection.

Fig. 1.. NS1 from 1918 pandemic influenza virus is SUMOylated in its unique C-terminal domain.

a, Venn diagram of influenza virus NS1s bearing a SUMO site in the C-terminal domain (tail). Bottom: amino acidic sequence of the NS1 tail from A/Brevig Mission/1/1918 (H1N1) bearing SUMOylation site and PDZ domain ligand site. 1918 NS1 C-termini is unique (1 out 1341 H1N1 from human isolates). b, Conservation plot of amino acidic sequences of NS1 among different viral isolates and hosts. The color-coding is the difference between the conservation score and average value of the score across the protein. Protein domains of NS1 are shown at the bottom. c, Ectopic expression of SUMO2 and the indicated NS1 (WT NS1 and the SUMO-consensus mutants at positions K70 and K227) in A549 cells. Immunoprecipitation (IP) with anti-Flag antibody, and Western Blotting (WB) analysis with anti-NS1 and anti-SUMO2/3 antibodies are shown. d, IP with anti-NS1 antibody and WB with anti-NS1 and anti-SUMO2/3 antibodies in A549 cells transfected with the indicated NS1s. The tail swapping of H3N2 NS1 was performed using the sequences from the indicated viruses, all of human origins but Hong Kong 1992 and Indonesia 2005 which are avian viruses. e, IP and WB with anti NS1- antibody of whole cellular extract from A549 cells infected with the reassortant NS1 virus bearing the segment 8 encoding for 1918 NS1 (see Figure 2B). NEM: N-Ethylmaleimide, a SUMO peptidase inhibitor

Fig. 2.. 1918 NS1 wt and mutant viruses.

a, Top: schematic representations of the recombinant NS1, NS1-KR and NS1-SUMO proteins used for Bio-layer interferometry (BLI). Bottom: spectra showing BLI measurements of the interactions between recombinant PSD95 and NS1, NS1-KR and NS1-SUMO. Kd and R² are shown. b, Schematic representation of WT PR8, the reassortant virus (7+1) PR8 NS1 bearing 7 segments of WT PR8 and the Segment 8 of the 1918 Influenza virus, and the reassortant virus (7+1) PR8 NS1-SUMO bearing the Segment 8 of the 1918 Influenza virus encoding a fusion protein between NS1 and SUMO2. c, Growth curves of the indicated viruses after infection of MDCK cells at MOI=1 (n=2). Plotted values represent one of two independent experiments. Plaque size from a representative experiment is shown (n=2). Error bars correspond to mean ± s.e.m. d, Hierarchical clustering of genes with significant changes in expression (FDR q<0.001) between A549 cells infected with NS1 or NS1-SUMO virus at indicated time (hpi). Rows show the log-fold change in expression (IDs not shown and color key at bottom), and columns represent different experimental conditions (labeled at the top and bottom of the panel). e, Scatterplot of cellular gene expression changes in A549 cells at 6 hours (upper panel) or 12 hours (lower panel) post-infection with PR8 NS1 or PR8 NS1-SUMO virus, relative to mock- infected (Mock) cells (log2 ratio). Solid and dotted lines correspond to the regression line and 95% confidence interval, respectively.

Fig. 3.. SUMOylation of NS1 induces oligomeric assembly and increases pervasive RNAPII termination defects at host transcripts.

a, SDD-AGE (top) and WB (bottom) analysis on NS1 and NS1-SUMO virus infected lysates of A549. Mock: uninfected control. b, B-isox enrichment of NS1 from A549 lysate infected by NS1 and NS1-SUMO virus as analyzed by mass-spectrometry. ***, p value: 1.98*10⁻³. c, GO enrichment analysis of biological processes of B-isox enriched proteins during NS1 and NS1-SUMO infection (left panel), WB of the input and B-isox precipitates from NS1 and NS1-SUMO virus infected A549 lysates by the indicated antibodies (right panel). FUS and EWS are controls for resident RNA granule proteins. d, Schematic of the approach used to detect a relative increase in transcript levels in 3’ end gene-flanking regions. For each gene, a 3-prime transcript ratio (Termination ratio, TR) was calculated as the average number of total RNA-Seq reads per bp in 5-kilobase 3’ gene-flanking regions, divided by that in exonic regions. A global relative increase in 3’ transcript levels results in a horizontal transformation of the cumulative TR plot, as indicated. e-f, TR plots at termination region of active genes (RPKM > 1 in 50% of samples) in uninfected A549 cells and 6 hpi (e) or 12 hpi (f) with NS1 or NS1-SUMO virus. Data is shown for two replicates in each condition (n=2). g, TR plots at termination region of active genes (RPKM > 1 in 50% of samples) in uninfected A549 cells and 6 hpi with PR8 or ∆NS1 virus. Data is shown for two replicates in each condition. h-j, TR plots at termination region of uninfected A549 cells depleted for Ubiquitin Conjugating Enzyme E2 (siUBE2I) and control (siCtrl) (h), transfected with either empty vector (EV), or a vector expressing GFP or SUMO (i), and infected with PR8ΔNS1 A459 cells transfected with an empty vector (EV) or a vector expressing SUMO (j). The insets show the RNA expression levels of UBE2I in siCtrl or siUBE2I treated cells (h) and of GFP and/or SUMO in each condition (i-j) as counts per million sequenced reads (CPM). Results are shown for two biological replicates.

Fig. 4.. Relationship between RNAPII run-through and splicing defect.

a, TR plots at termination region of uninfected cells depleted for CPSF73, one subunit of the CPSF complex, (siCPSF73) and control (siLuc). b, TR plots at termination region of uninfected cells treated with the indicated inhibitors and control (DMSO). c, Schematic of the approach used to detect a relative accumulation of unprocessed intronic transcripts. Analogous to the TR plots, an intron/exon transcript ratio was calculated by dividing the average read coverage per basepair in 5,000 bp intronic regions directly flanking an upstream exon, by the average read coverage of the upstream exon. An increase in unprocessed intronic transcripts results in horizontal transformation of the cumulative intron/exon ratio plots, as indicated. d-e, Cumulative Intron/Exon ratio distributions of active genes (RPKM > 1 in 50% of samples) in uninfected A549 cells and 6 hpi with NS1 or NS1-SUMO virus (d), and at 12 hpi with NS1 or NS1-SUMO virus (e). Data is shown for two replicates in each condition. (f) Classification of all IsoSeq circular consensus (CCS) reads based on comparisons with GENCODE v38 transcript annotations using cuffcompare. (g) Percentage of reads containing full length RNA from the segment 8 in cells infected with NS1-SUMO virus.