Suppression of the antiviral response by an influenza histone mimic

Abstract

Viral infection is commonly associated with virus-driven hijacking of host proteins. Here we describe a novel mechanism by which influenza virus affects host cells through the interaction of influenza non-structural protein 1 (NS1) with the infected cell epigenome. We show that the NS1 protein of influenza A H3N2 subtype possesses a histone-like sequence (histone mimic) that is used by the virus to target the human PAF1 transcription elongation complex (hPAF1C). We demonstrate that binding of NS1 to hPAF1C depends on the NS1 histone mimic and results in suppression of hPAF1C-mediated transcriptional elongation. Furthermore, human PAF1 has a crucial role in the antiviral response. Loss of hPAF1C binding by NS1 attenuates influenza infection, whereas hPAF1C deficiency reduces antiviral gene expression and renders cells more susceptible to viruses. We propose that the histone mimic in NS1 enables the influenza virus to affect inducible gene expression selectively, thus contributing to suppression of the antiviral response.

Figure 1. Influenza NS1 contains a histone mimic

a, The homologous carboxyterminal NS1 and the aminoterminal histone H3 sequences are shown (red letters). The table displays C-terminal NS1 sequences of the influenza A subtypes. b, Methylation or acetylation of the NS1 peptide (top panel), the GST–NS1 protein (middle panel) or of the viral NS1 in A549 infected cells (bottom panel) are shown. KR, NS1 substrates where K229 is replaced by arginine. c, Association of the NS1 histone mimic with the hPAF1C subunits and CHD1 in nuclear extracts. In, input material. d, NS1 histone mimic binds to CHD1. Unmodified or methylated NS1 (K229) or methylated H3 (K4) peptides were incubated with the recombinant CHD1 or the CHD1 double-chromodomain. Binding to NS1 was revealed by silver or Coomassie staining (top and bottom panel, respectively). e, Binding of Flag-tagged hPAF1C subunits to NS1 or histone H3 peptides was assessed by western blotting (left and right panels, respectively). IP, immunoprecipitation.

Figure 2. Functional interaction between NS1 and PAF1 in infected cells

a, The ChIP-seq profiles show the distribution of indicated proteins at inducible genes before (black line) and after (red line) infection. The induced genes were revealed by RNA-seq and ChIP-seq analysis of infected A549 cells (Supplementary Tables 1 and 2). TSS and TES, the transcriptional start and end sites, respectively. b, The NS1 levels at gene promoters in PAF1- or CHD1-deficient cells (blue and green lines, respectively). The scrambled siRNA-treated cells (red line) were used as control. The insert shows knockdown of PAF1 or CHD1 in A549 cells. c, PAF1, RNA Pol II and H3K4me3 levels at the TSS and TES of the induced genes in uninfected (ui) cells, cells infected with the wild-type (WT) or PAF1-binding mutant virus (ΔPAF). Data are representative of three independent experiments; error bars show the s.e.m.

Figure 3. NS1 suppresses antiviral gene transcription in infected cells

a, Left: the GRO-seq profile of inducible RNA transcripts in uninfected (ui) A549 cells (black line) or cells infected with wild-type or ΔPAF virus (green and red lines, respectively). Right: GRO-seq profile of IFIT1 and IFI6 genes in uninfected and infected cells. b, GRO-seq profile of A549-expressed genes that are not affected by virus infection (left panel) or of the HPRT1 gene (right panel). Reads from either DNA strands are indicated as +/−. The y axes display reads per million mapped reads per 25 bp.

Figure 4. NS1 inhibits transcriptional elongation in vitro

a, The full-length NS1 protein (NS1) or NS1 lacking the PAF1-binding sequence (NS1(ΔPAF)) (Supplementary Fig. 5c) was added to the RNA elongation reaction as indicated. b, The amount of the 390-nt RNA elongation product was quantified by ImageJ. The results of two independent experiments are shown.

Figure 5. PAF1 controls antiviral response

a, b, The expression levels of mRNAs in influenza infected (a) or IFN-β1-treated (b) control (siCtrl) or PAF1-deficient (siPAF) A549 cells. The tables show the siPAF-affected gene categories. Ut, untreated with siRNA. c, Dynamics of virus replication in control or PAF1-deficient A549 cells. p.f.u. plaque-forming units. Data are representative of three independent experiments. Error bars show the s.e.m.