Volume 3, Issue 1, Pages 23-29 (January 2013) Influenza A Virus Utilizes Suboptimal Splicing to Coordinate the Timing of Infection  Mark A. Chua, Sonja Schmid, Jasmine T. Perez, Ryan A. Langlois, Benjamin R. tenOever  Cell Reports  Volume 3, Issue 1, Pages 23-29 (January 2013) DOI: 10.1016/j.celrep.2012.12.010 Copyright © 2013 The Authors Terms and Conditions

Cell Reports 2013 3, 23-29DOI: (10.1016/j.celrep.2012.12.010) Copyright © 2013 The Authors Terms and Conditions

Figure 1 Silencing of NS1 Does Not Impact Virus Replication In Vitro (A–C) Western blot analysis of whole-cell extract (WCE) derived from (A) Jurkat T cells, (B) murine embryonic fibroblasts (MEFs), and (C) MEFs deficient in Dicer1 (Dicer1−/−) infected with Scbl, 20T, and 142T viruses. Immunoblots depict NP, NS1, NEP, and actin. (D) Multicycle growth curves performed in A549 cells in response to Scbl, 20T, 142T, and delNS1 IAV strains. Error bars represent average ± SD of the mean of samples from independent growth curves analyzed in triplicate. LD, limit of detection. See also Figure S1. Cell Reports 2013 3, 23-29DOI: (10.1016/j.celrep.2012.12.010) Copyright © 2013 The Authors Terms and Conditions

Figure 2 Low Levels of NS1 Are Sufficient for Virus Replication In Vivo (A and B) Western blot analysis of WCE from (A) pLFs or (B) BMDMs from wild-type (WT) C57BL/6 mice infected with Scbl, 20T, or 142T IAV, or mock (M) treated for 24 hr. (C and D) Western blot analysis of (C) Ifnar1−/−/Il28r−/− or (D) WT mouse lungs infected intranasally with either Scbl, 20T, or 142T IAV or mock (M) treated at 48 hpi. Immunoblots depict NP, NS1, and actin. (E) Viral titers from WT C57BL/6 mice infected with Scbl, 20T, or 142T, or mock (PBS) treated. Data points represent individual mice assayed in triplicate. See also Figure S2. Cell Reports 2013 3, 23-29DOI: (10.1016/j.celrep.2012.12.010) Copyright © 2013 The Authors Terms and Conditions

Figure 3 NEP Is Essential in Determining the Levels of Virus Replication (A) Western blot analysis of WCE from fibroblasts infected with either WT or 2A viruses or mock treated for 3, 6, 9, or 12 hr postinfection (HPI). (B) Multicycle growth curve for WT and 2A IAV. (C) Western blot analysis of WCE from fibroblasts infected with VLVs containing either an extragenic scrambled (Scbl) sequence or NEP on segment four. (D) Multicycle growth curve of WT IAV coinfected with VLVs characterized in (C). (A and C) Immunoblots depict NP, NS1, NEP, and actin. (B and D) Error bars represent mean ± SD of the mean of samples from independent multicycle growth curve analyzed in triplicate. LD, limit of detection. See also Figure S3. Cell Reports 2013 3, 23-29DOI: (10.1016/j.celrep.2012.12.010) Copyright © 2013 The Authors Terms and Conditions

Figure 4 NEP Accumulation Is Essential in Coordinating Virus Replication (A) Western blot analysis of WCE from A549 cells infected with an NS encoding an optimal 5′ splice site (NSo), a parental splice NS (NSp) IAV, or mock treated for 3, 6, 9, or 12 hr postinfection (HPI). Immunoblots depict NP, NS1, NEP, and actin. (B) Multicycle growth curves for NSo and NSp IAV in A549 cells. (C) Same as (B) but performed in MDCK-NS1-GFP cells. Error bars represent mean ± SD of the mean of samples from independent multicycle growth curve analyzed in triplicate. LD, limit of detection. (D) Viral titers from WT C57BL/6 mouse lungs infected intranasally with NSo or NSp. (E) A549 cells stained for NP expression from NSo and NSp infection at 5 or 7 hpi and imaged by fluorescence microscopy. Hoechst dye used to visualize nuclei. See also Figure S4. Cell Reports 2013 3, 23-29DOI: (10.1016/j.celrep.2012.12.010) Copyright © 2013 The Authors Terms and Conditions

Figure S1 miRNA-Mediated Targeting of NS1, Related to Figure 1 (A) Northern blot analysis of total RNA probed for miR-20 and miR-142 in indicated cell lines. U6 levels used as a loading control. 293, human embryonic kidney cells; 293 + miR-142, 293 cells transfected with a vector expressing miR-142; A549, human alveolar epithelial cells; Jurkat, human T lymphocyte; Dicer1−/−, Dicer1 knockout mouse embryonic fibroblasts; MEF, mouse embryonic fibroblasts; pLF, primary lung murine fibroblast; BMDM, bone marrow–derived murine macrophages. (B) Diagram of recombinant NS segment containing target sites in the 3′ untranslated region (UTR). Target sites are either a scrambled sequence (Scbl) or four identical target sites for miR-20a (20T) or miR-142 (142T). T indicates position of target sites. (C) (Top two panels) Western blots of WCE derived from fibroblasts expressing Scbl, 20T, or 142T vectors with either empty vector or vector producing miR-142 (miR-142). (Middle two panels) Northern blot of total RNA from samples above, probed for miR-142 and U6. (Bottom two panels) Semiquantitative RT-PCR on total oligo-dT RNA using NS1- and beta tubulin specific primers. (D) Western blot analysis of WCE derived from samples in Figure 1D. Immunoblots depict Mx1, NP, NS1, NEP, or Actin. Cell Reports 2013 3, 23-29DOI: (10.1016/j.celrep.2012.12.010) Copyright © 2013 The Authors Terms and Conditions

Figure S2 Silencing of NS1 Does Not Impact ISG15 or IRF7 Levels In Vivo, Related to Figure 2 (A and B) qRT-PCR of (A) ISG15 and (B) IRF7 from mouse lungs from Figure 2E normalized to tubulin. Data points represent individual mice assayed in triplicate. Cell Reports 2013 3, 23-29DOI: (10.1016/j.celrep.2012.12.010) Copyright © 2013 The Authors Terms and Conditions

Figure S3 NS1/NEP 2A IAV, siRNAs, and VLVs, Related to Figure 3 (A) Diagram of segment 8 of NS1/NEP 2A IAV which is a non-splicing segment 8 expressing NS1-NEP as a fusion separated by a porcine teschovirus-1 (PTV-1) 2A sequence. (B) Western blot analysis of WCE from fibroblast transfected with different NEP siRNAs or scbl siRNA for 16 hr then infected with WT IAV for 8 hr. Immunoblots depict NP, NS1, NEP, or Actin. (C) Multicycle growth curves of WT IAV infection for 0, 12, 24, 36, and 48 hpi in fibroblast cells transfected with NEP1/2 siRNA or scbl siRNA for 16 hr prior to infection. P-values for 24, 36, and 48 hr are 0.01, 0.001, and 0.01 respectively. (D) Diagram of how VLVs are constructed. In brief, wt segments 1,2,3,5,6,7, and 8 were combined with a recombinant segment 4 expressing either NEP or scrambled (Scbl) RNA. These eight segment are used to generate virus-like vectors (VLVs) in a MDCK cell line that complements the loss of wild-type segment four by expressing exogenous HA protein. Cell Reports 2013 3, 23-29DOI: (10.1016/j.celrep.2012.12.010) Copyright © 2013 The Authors Terms and Conditions

Figure S4 Characterization of the NSo Virus, Related to Figure 4 (A) Semiquantitative RT-PCR on total oligo-dT RNA from fibroblast infected with WT, NSo, or NSp viruses for 3, 6, or 9 hpi. cDNA treated using primers to amplify NS1 and NEP as depicted. Tubulin was used as a loading control. (B) Western blot analysis of WCE from A549 cells infected with NSp, NSo, and 20T IAV strains or mock treated for 8 hr. (C) Western blot analysis of WCE from MDCK and MDCK-NS1-GFP cells infected with delNS1 virus for 24 hr. (D) Western blot analysis of WCE derived from samples in Figure 4B. Immunoblots depict Mx1, NP, NS1, or Actin. (E) qRT-PCR of viral NP levels from mouse lungs in Figure 4D normalized to tubulin. Data points represent individual mice assayed in triplicate. (F) A549 cells were fixed following infection with either WT or 2A IAV at 8 hpi. Cells were stained with the NP antibody prior to imaging by fluorescence microscopy. Hoechst dye used to visualize nuclei. Cell Reports 2013 3, 23-29DOI: (10.1016/j.celrep.2012.12.010) Copyright © 2013 The Authors Terms and Conditions