Fig 2: Influenza Virus Infection Causes a Host Pol II Termination Defect Independently of NS1-CPSF30 Interaction(A) The closely related H1N1 strains (A/WSN/33 and A/PR/8/34) encode NS1 proteins that differ in the ability of their C-terminal effector domains to bind CPSF30. Evolutionarily distinct influenza B viruses, including B/Florida/04/2006 (B/FL/04), encode an unrelated effector domain that does not bind CPSF30. Meta-profiles of Pol II occupancy at the 3′ end of expressed protein-coding genes with a single poly(A) site as well as statistical analysis of Pol II read-through on each gene during viral infection show that all three viruses induce a significant failure of Pol II to terminate downstream of poly(A) sites.(B) mNET-seq profiles of Pol II occupancy at the 3′ end of a protein-coding gene, KRT7. The unexpressed downstream gene, KRT87P, is not shown in the annotation.(C) The H3N2 influenza virus (A/Udorn/72) encodes an NS1 protein with an effector domain that binds CPSF30. Infections were performed in parallel with a mutant virus of the same strain in which the NS1 protein is truncated to remove the effector domain (NS1Δ99). Meta-profiles of Pol II occupancy at the 3′ end of expressed protein-coding genes with a single poly(A) site as well as statistical analysis of Pol II read-through on each gene during viral infection show that both the wild-type and the mutant viruses induce a strong termination defect.(D) mNET-seq profiles of Pol II occupancy during H3N2 viral infection at the 3′ end of a protein-coding gene, KRT7. The unexpressed downstream gene, KRT87P, is not shown in the annotation.Data shown are from a single mNET-seq biological replicate. See also Figure S3.

Fig 3: Influenza Virus Infection Dramatically Alters Transcription Dynamics of Host RNA Polymerase II(A) (i) Influenza virus interferes with the host Pol II transcription complex, both at the start of genes where the viral polymerase (FluPol) binds to the Ser5P form of the Pol II CTD to carry out cap snatching, generating capped RNA primers for viral mRNA synthesis, and at the end of genes where the viral NS1 interferes with host 3′ end processing by inhibiting CPSF30. pAS, poly(A) site. (ii) Pol II behavior was profiled genome-wide using mNET-seq (see STAR Methods for details and Figure S1).(B) Meta-profile of Pol II occupancy on all non-overlapping protein-coding genes (n = 13,851) as measured by mNET-seq shows that influenza virus infection simultaneously results in decreased Pol II occupancy downstream of the transcription start site (TSS), as well as a failure of Pol II to terminate downstream of the poly(A) site. FPKM, fragments per kilobase of transcript per million mapped reads.(C) Heatmap of Pol II occupancy of non-overlapping protein-coding genes, with the TSS and poly(A) site of each gene aligned to each other, as illustrated at the top of the panel. Each horizontal line represents a single gene.(D and E) mNET-seq profiles of Pol II occupancy along protein-coding genes KRT7 (D) and MAZ (E). Downstream unexpressed genes (KRT87P and PRRT2) are not shown in the annotation.In (B)–(E), data shown are from a single, representative biological replicate (see Figures S1B–S1D and Table S1 for comparison). See also Figure S1.

Fig 4: (A) Diagram of the human MYC gene with exons (black boxes) and the pA site indicated. Regions assayed by ChIP are underlined and labeled, and a scale marker is shown. (B) ChIP experiment assaying the distribution of Pol II (N20), Ser5p, Ser2p, and Ser7p across the MYC gene. For each antibody, values are normalized to the maximum value (see the Materials and Methods for details). (NTS) A negative control region. (C) ChIP experiment assaying the distribution of CPSF30 and CstF77 across the MYC gene. For each antibody, values are normalized to the maximum value. (NTS) A negative control region. (D) Native ChIP analyzing Ser2p across the MYC gene. Values are normalized to the maximum value. (NTS) A negative control region. All error bars represent standard deviation from at least three biological replicates.