Co‐degradation of interferon signaling factor DDX3 by PB1‐F2 as a basis for high virulence of 1918 pandemic influenza
2019; Springer Nature; Volume: 38; Issue: 10 Linguagem: Inglês
10.15252/embj.201899475
ISSN1460-2075
AutoresEun‐Sook Park, Young Ho Byun, Soree Park, Yo Han Jang, Woo‐Ry Han, Juhee Won, Kyung Cho Cho, Doo Hyun Kim, Ah Ram Lee, Gu‐Choul Shin, Yong Kwang Park, Hong Seok Kang, Heewoo Sim, Yea Na Ha, Byeongjune Jae, Ahyun Son, Paul S. Kim, Jieun Yu, Hyemin Lee, S.B. Kwon, Kwang Pyo Kim, Seung‐Hyun Lee, Yeong‐Min Park, Baik Lin Seong, Kyun‐Hwan Kim,
Tópico(s)Immune Response and Inflammation
ResumoArticle12 April 2019free access Source DataTransparent process Co-degradation of interferon signaling factor DDX3 by PB1-F2 as a basis for high virulence of 1918 pandemic influenza Eun-Sook Park Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea KU Open Innovation Center, Konkuk University, Seoul, Korea Search for more papers by this author Young Ho Byun orcid.org/0000-0001-9306-9485 Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea Vaccine Translational Research Center, Yonsei University, Seoul, Korea Search for more papers by this author Soree Park Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea Search for more papers by this author Yo Han Jang Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea Vaccine Translational Research Center, Yonsei University, Seoul, Korea Search for more papers by this author Woo-Ry Han Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea Search for more papers by this author Juhee Won orcid.org/0000-0002-5757-3745 Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea Search for more papers by this author Kyung Cho Cho orcid.org/0000-0003-0483-9543 Department of Applied Chemistry, Kyung Hee University, Yongin, Korea Search for more papers by this author Doo Hyun Kim Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea Search for more papers by this author Ah Ram Lee Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea Search for more papers by this author Gu-Choul Shin orcid.org/0000-0001-8200-9700 Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea Search for more papers by this author Yong Kwang Park Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea Search for more papers by this author Hong Seok Kang Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea Search for more papers by this author Heewoo Sim Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea Search for more papers by this author Yea Na Ha Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea Search for more papers by this author Byeongjune Jae Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea Search for more papers by this author Ahyun Son Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea Search for more papers by this author Paul Kim Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea Vaccine Translational Research Center, Yonsei University, Seoul, Korea Search for more papers by this author Jieun Yu Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea Search for more papers by this author Hye-Min Lee Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea Search for more papers by this author Sun-Bin Kwon Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea Search for more papers by this author Kwang Pyo Kim Department of Applied Chemistry, Kyung Hee University, Yongin, Korea Search for more papers by this author Seung-Hyun Lee Department of Microbiology, School of Medicine, Konkuk University, Seoul, Korea Search for more papers by this author Yeong-Min Park Laboratory of Dendritic Cell Differentiation and Regulation, Department of Immunology, School of Medicine, Konkuk University, Seoul, Korea Search for more papers by this author Baik L Seong Corresponding Author [email protected] orcid.org/0000-0002-7301-082X Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea Vaccine Translational Research Center, Yonsei University, Seoul, Korea Search for more papers by this author Kyun-Hwan Kim Corresponding Author [email protected] orcid.org/0000-0001-5266-072X Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea KU Open Innovation Center, Konkuk University, Seoul, Korea Research Institute of Medical Science, Konkuk University, Seoul, Korea Search for more papers by this author Eun-Sook Park Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea KU Open Innovation Center, Konkuk University, Seoul, Korea Search for more papers by this author Young Ho Byun orcid.org/0000-0001-9306-9485 Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea Vaccine Translational Research Center, Yonsei University, Seoul, Korea Search for more papers by this author Soree Park Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea Search for more papers by this author Yo Han Jang Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea Vaccine Translational Research Center, Yonsei University, Seoul, Korea Search for more papers by this author Woo-Ry Han Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea Search for more papers by this author Juhee Won orcid.org/0000-0002-5757-3745 Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea Search for more papers by this author Kyung Cho Cho orcid.org/0000-0003-0483-9543 Department of Applied Chemistry, Kyung Hee University, Yongin, Korea Search for more papers by this author Doo Hyun Kim Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea Search for more papers by this author Ah Ram Lee Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea Search for more papers by this author Gu-Choul Shin orcid.org/0000-0001-8200-9700 Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea Search for more papers by this author Yong Kwang Park Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea Search for more papers by this author Hong Seok Kang Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea Search for more papers by this author Heewoo Sim Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea Search for more papers by this author Yea Na Ha Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea Search for more papers by this author Byeongjune Jae Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea Search for more papers by this author Ahyun Son Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea Search for more papers by this author Paul Kim Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea Vaccine Translational Research Center, Yonsei University, Seoul, Korea Search for more papers by this author Jieun Yu Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea Search for more papers by this author Hye-Min Lee Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea Search for more papers by this author Sun-Bin Kwon Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea Search for more papers by this author Kwang Pyo Kim Department of Applied Chemistry, Kyung Hee University, Yongin, Korea Search for more papers by this author Seung-Hyun Lee Department of Microbiology, School of Medicine, Konkuk University, Seoul, Korea Search for more papers by this author Yeong-Min Park Laboratory of Dendritic Cell Differentiation and Regulation, Department of Immunology, School of Medicine, Konkuk University, Seoul, Korea Search for more papers by this author Baik L Seong Corresponding Author [email protected] orcid.org/0000-0002-7301-082X Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea Vaccine Translational Research Center, Yonsei University, Seoul, Korea Search for more papers by this author Kyun-Hwan Kim Corresponding Author [email protected] orcid.org/0000-0001-5266-072X Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea KU Open Innovation Center, Konkuk University, Seoul, Korea Research Institute of Medical Science, Konkuk University, Seoul, Korea Search for more papers by this author Author Information Eun-Sook Park1,2, Young Ho Byun3,4, Soree Park1, Yo Han Jang3,4, Woo-Ry Han1, Juhee Won1, Kyung Cho Cho5, Doo Hyun Kim1, Ah Ram Lee1, Gu-Choul Shin1, Yong Kwang Park1, Hong Seok Kang1, Heewoo Sim1, Yea Na Ha1, Byeongjune Jae1, Ahyun Son3, Paul Kim3,4, Jieun Yu3, Hye-Min Lee3, Sun-Bin Kwon3, Kwang Pyo Kim5, Seung-Hyun Lee6, Yeong-Min Park7, Baik L Seong *,3,4 and Kyun-Hwan Kim *,1,2,8 1Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea 2KU Open Innovation Center, Konkuk University, Seoul, Korea 3Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea 4Vaccine Translational Research Center, Yonsei University, Seoul, Korea 5Department of Applied Chemistry, Kyung Hee University, Yongin, Korea 6Department of Microbiology, School of Medicine, Konkuk University, Seoul, Korea 7Laboratory of Dendritic Cell Differentiation and Regulation, Department of Immunology, School of Medicine, Konkuk University, Seoul, Korea 8Research Institute of Medical Science, Konkuk University, Seoul, Korea *Corresponding author. Tel: +82 2 2123-2885; E-mail: [email protected] *Corresponding author. Tel: +82 2 2030 7833; Fax: +82 2 2049 6192; E-mail: [email protected] EMBO J (2019)38:e99475https://doi.org/10.15252/embj.201899475 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The multifunctional influenza virus protein PB1-F2 plays several roles in deregulation of host innate immune responses and is a known immunopathology enhancer of the 1918 influenza pandemic. Here, we show that the 1918 PB1-F2 protein not only interferes with the mitochondria-dependent pathway of type I interferon (IFN) signaling, but also acquired a novel IFN antagonist function by targeting the DEAD-box helicase DDX3, a key downstream mediator in antiviral interferon signaling, toward proteasome-dependent degradation. Interactome analysis revealed that 1918 PB1-F2, but not PR8 PB1-F2, binds to DDX3 and causes its co-degradation. Consistent with intrinsic protein instability as basis for this gain-of-function, internal structural disorder is associated with the unique cytotoxic sequences of the 1918 PB1-F2 protein. Infusing mice with recombinant DDX3 protein completely rescued them from lethal infection with the 1918 PB1-F2-producing virus. Alongside NS1 protein, 1918 PB1-F2 therefore constitutes a potent IFN antagonist causative for the severe pathogenicity of the 1918 influenza strain. Our identification of molecular determinants of pathogenesis should be useful for the future design of new antiviral strategies against influenza pandemics. Synopsis The comparative interactome analysis revealed that 1918 PB1-F2 hijacks a key mediator of IFN signaling, DDX3, leading to its proteasomal degradation, and thus shuts off the antiviral response. Influenza PB1-F2 protein plays a multi-functional role in deregulation of innate immune responses and is known to enhance the immunopathology in 1918 pandemic virus. We have showed through the interactome analysis that, DDX3, an essential host protein in the type I IFN signaling pathway, binds to and is co-degraded with 1918 PB1-F2. The hijacking of a key mediator of IFN signaling uncouples the host antiviral responses from the viral infection, resulting in enhanced virulence. Our study reveals a novel molecular basis for the severe pathogenicity of the 1918 strain which will be useful for designing new therapeutic options against influenza pandemics. Introduction The Spanish influenza of 1918 remains the deadliest infectious disease in human history that claimed up to 50 million victims worldwide (Beveridge, 1991; Tumpey et al, 2005). As compared against the 1957 and 1968 pandemic strains, which were reassortants of avian viruses, the genetic origin of the 1918 strain appears to be unique, calling for a different scope and focus of surveillance and preventive efforts (Reid et al, 2004). The extremely high pathogenicity among humans has been ascribed to the host tropism by hemagglutinin (HA) (Tumpey et al, 2007; Zambon, 2007). The non-structural PB1-F2, initially identified as a+1 open reading frame (ORF) of PB1 gene segment (Chen et al, 2001), plays a multifunctional role in the deregulation of innate immune responses, including the inhibition of type I interferon (IFN) induction by targeting mitochondrial MAVS (Varga et al, 2011, 2012). Amino acid substitutions in the PB1-F2 protein contribute to its stability and functions (Alymova et al, 2014; Cheng et al, 2017). Notably, 1918 PB1-F2 is involved in the unique immunopathology of secondary bacterial infections (Lamb & Takeda, 2001; Conenello & Palese, 2007; Kobasa et al, 2007; McAuley et al, 2007, 2010), which contributes significantly to pathogenesis (Zamarin et al, 2006). Being part of the first line of defense against infection, type I interferons are key components of the host antiviral innate immune response and modulators of adaptive immune responses (García-Sastre & Biron, 2006). To foil the host defense system, influenza viruses developed strategies to attenuate IFN responses. The NS1 protein of highly pathogenic influenza viruses, such as the H5N1 avian influenza virus and the 1918 pandemic virus, strongly suppresses type I IFNs (Geiss et al, 2002; Seo et al, 2002). These viruses also lead to the acute recruitment of neutrophils, severe lung injury, and aggressive inflammatory cytokine production in IFNα/β receptor 1-deficient (Ifnar1−/−) mice (Kobasa et al, 2007; Perrone et al, 2008; Cillóniz et al, 2009). Compared to the PB1-F2 proteins of influenza epidemic viruses, that of the 1918 pandemic (1918 PB1-F2) enhances the pathogenesis of viral infection to a much greater extent (McAuley et al, 2007). However, the host target(s) and the molecular mechanism underlying this high pathogenicity remain elusive (Conenello & Palese, 2007). The reconstruction of the 1918 virus (Reid et al, 2004; Tumpey et al, 2005, 2007) and subsequent landmark discoveries on PB1-F2 as a virulence factor (Zamarin et al, 2006; Conenello & Palese, 2007; McAuley et al, 2007, 2010) prompted us to address this unresolved issue associated with the most devastating infection in human history. Through comparative interactome analysis between the two isogenic viruses differing only in PB1-F2, we showed that 1918 PB1-F2 hijacks the key mediator of IFN signaling, acquiring a new gain-of-function of shutting off the antiviral response (García-Sastre & Biron, 2006). Our results reveal a novel molecular mechanism behind the unparalleled high mortality of the 1918 pandemic and may help to design novel antiviral strategies. Results The 1918 PB1-F2 protein is very unstable and is degraded by the ubiquitin–proteasome pathway We first compared the expression profiles of the PB1-F2 proteins in infected cells. A549 and U937 cells were infected with either IAV (PR8) or IAV (1918), two isogenic viruses with the PR8 backbone that differ only in their PB1-F2 and the corresponding PB1 region. IAV (PB1-F2[-]), a mutant virus that does not express PB1-F2, was used as a negative control. In both cell lines, the expression level of 1918 PB1-F2 was very low compared to that of PR8 PB1-F2 (Fig 1A). However, the level of the 1918 PB1-F2 transcript was similar to that of PR8 PB1-F2 (Fig 1B). When treated with the proteasome inhibitor MG132, the level of the 1918 PB1-F2 protein recovered significantly. We obtained similar results using an immunofluorescence assay (Fig 1C). Both 1918 and PR8 PB1-F2 proteins were heavily ubiquitinated in infected cells (Fig 1D), demonstrating that the very low steady-state level of the 1918 PB1-F2 protein in cells was due to its degradation by the ubiquitin–proteasome pathway. Figure 1. The stability of the 1918 PB1-F2 protein is very low, and it is degraded by the Ub-dependent proteasome pathway A549 and U937 cells were infected with each IAV at MOI 1 or 5 for 24 h, and lysates were analyzed by Western blotting using polyclonal anti-PB1-F2 antibody. A549 cells were transfected with a plasmid encoding Flag-tagged PB1-F2 derived from the PR8 or 1918 strain and treated with the proteasome inhibitor MG132 (25 μM) or vehicle for 6 h before harvesting. PB1-F2 transcripts were analyzed by semi-quantitative RT–PCR (top panel), and the PB1-F2 protein was analyzed by Western blotting with monoclonal anti-Flag antibody (bottom panel). Representative immunofluorescence images showing expression of PB1-F2 (red). A549 cells were transfected with plasmids encoding indicated PB1-F2. At 18 h after transfection, cells were treated with MG132 for 6 h and subjected to immunofluorescence analysis. Magnification, ×100; scale bar, 100 μm. Each HA-tagged PB1-F2 was transfected into 293T cells with or without Flag-ubiquitin. At 18 h after transfection, cells were treated with MG132 for 6 h. The lysates were immunoprecipitated with anti-HA antibody and analyzed by Western blotting with anti-Flag antibody. PR8, A/Puerto Rico/8/34 (H1N1); 1918, A/Brevig Mission/1/1918 (H1N1). Data information: Results shown are representative of three independent experiments. Source data are available online for this figure. Source Data for Figure 1 [embj201899475-sup-0007-SDataFig1.pdf] Download figure Download PowerPoint Amino acid residues 68 and 69 determine the characteristics of the 1918 PB1-F2 protein The 1918 PB1-F2 protein is highly divergent in comparison with PR8 PB1-F2 (12 amino acid substitutions; Fig 2A). To narrow down the molecular determinants of its instability, we constructed various mutants containing the sequences of PR8 or 1918 PB1-F2 (Fig 2A). Comparing the chimeric PB1-F2 mutants, the C-terminus of 1918 PB1-F2 (1918-C) was found responsible for the low stability (Fig 2B). Site-specific mutations within the C-terminus showed that both positions 68 and 69 of the 1918 strain crucially reduced the stability of PB1-F2 (Fig 2C). Conversely, if a PR8 amino acid was introduced into the 1918 backbone at the corresponding positions, the stability was restored (Fig 2D). Moreover, interesting changes in cellular localization was noted; PR8 PB1-F2 was localized predominantly in mitochondria as punctate structures, whereas 1918 PB1-F2 was detected both in the cytoplasm and the nucleus as diffused expression pattern (Figs 2E and EV1; summarized in Table EV1). The changes in subcellular localizations by mutations are consistent with recent studies on H5N1 and further extend to 1918 PB1-F2 with relevance to enhanced virulence (Cheng et al, 2017). Thus, biochemical and functional characteristics of 1918 PB1-F2-stability, cellular localization, expression pattern, and inhibition of IFN induction (see below) all correlated with the I68T and L69P mutations (Table EV1). Figure 2. Ile68 and Leu69 in the C-terminal part of PB1-F2 determine its stability A. Amino acid sequences of PR8 PB1-F2, 1918 PB1-F2, and the cloned PB1-F2 mutants. B–D. A549 cells were transfected with the indicated plasmids encoding Flag-tagged PB1-F2 derived from the PR8 or 1918 strain and chimeric PB1-F2 mutants. At 18 h after transfection, cells were treated with or without MG132 for 6 h. The expression of PB1-F2 and control genes was detected by semi-quantitative RT–PCR (upper panel) or Western blotting (lower panel). E. Representative immunofluorescence images of PB1-F2 expression in A549 cells. Cells transfected with the Flag-tagged PB1-F2 plasmid were stained with anti-Flag (red) antibody and analyzed by immunofluorescence assay. Magnification, ×400; scale bar, 50 μm. Data information: Results shown are representative of three independent experiments. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Cellular localization and expression patterns of various PB1-F2 proteins A–C. A549 cells were transfected with plasmids encoding Flag-tagged PB1-F2 derived from the PR8 or 1918 strain or chimeric PB1-F2 mutants. The PB1-F2 mutants shown in figure were also analyzed. At 24 h after transfection, the cellular localization of PB1-F2 was determined by immunofluorescence assay. PB1-F2, red; MitoTracker, green; DAPI, blue. Magnification, ×400; scale bar, 50 μm. Download figure Download PowerPoint The 1918 PB1-F2 protein strongly inhibits IFNβ induction We then examined potential effects of PB1-F2 on key molecules in the signaling pathways of IFNβ. We observed only marginal effects on NF-κB, pro-inflammatory cytokines (Fig 3A), and IFNα expression (Fig EV2). However, the induction of IFNβ, a critical element of the innate immune system, was strongly inhibited by the expression of 1918 PB1-F2 in two different cell lines; in contrast, PR8 PB1-F2 had no effect (Fig 3B). Furthermore, the expression of 1918 PB1-F2 but not that of PR8 PB1-F2 strongly suppressed polyI:C-induced IFNβ activation in the reporter assay (Fig 3C). Consistent with the reporter system in vitro, infection of mice with IAV (1918) resulted in a strong suppression of IFNβ, whereas infection with either IAV (PR8) or IAV (PB1-F2[-]) did not (Fig 3D). The suppression of IFNβ was accompanied by a low level of PB1-F2 expression (Fig 3D). Of note, IFNβ inhibition by 1918 PB1-F2 was more prominent than by NS1 (Fig 3C), a previously established IFN antagonist in influenza viruses (Geiss et al, 2002; García-Sastre & Biron, 2006). These data suggest that 1918 virus acquired an ability to inhibit the type I IFN response uniquely associated with the in-cell stability of PB1-F2. Figure 3. Inhibition of IFNβ induction and its association with proteasomal degradation of 1918 PB1-F2 Relative activity of NF-κB luciferase reporter was measured in A549 cells transfected with plasmids encoding PB1-F2. The mRNA levels of pro-inflammatory cytokines were determined by semi-quantitative RT–PCR. Cells were transfected with PB1-F2-expressing plasmids. Relative IFNβ mRNA levels were determined by semi-quantitative RT–PCR (top) and qPCR (bottom). U937 cells were co-transfected with the IFNβ luciferase reporter plasmid and the PB1-F2 or NS1 expressing plasmid and treated with polyI:C for 12 h before harvesting. IFNβ promoter activity was determined by luciferase reporter assay. Cells were infected for 24 h with each virus at MOI 1. The mRNA level of IFNβ was determined by semi-quantitative RT–PCR, and PB1-F2 protein expression was assessed by Western blotting. Cells were transfected with each plasmid encoding PB1-F2. 18 h after transfection, cells were treated with MG132 for 6 h and the level of IFNβ mRNA was determined by semi-quantitative RT–PCR (left) and qPCR (right). PB1-F2 protein expression was assessed by Western blotting. A549 cells were transfected with PB1-F2 clones mutated at positions 68 and 69; 24 h after transfection, the IFNβ mRNA level and PB1-F2 protein expression were determined by semi-quantitative RT–PCR and Western blotting, respectively (left). The relative expression level of IFNβ mRNA was also analyzed by qPCR (right). Data information: All data are shown as mean (±SEM) from at least three independent experiments (**P < 0.01, ***P < 0.001). Source data are available online for this figure. Source Data for Figure 3 [embj201899475-sup-0008-SDataFig3.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Effect of PB1-F2 on IFNα expressionA549 cells were transfected with PB1-F2-expressing plasmids. Relative IFNα mRNA levels were determined by semi-quantitative RT–PCR (top) and qPCR (bottom). Download figure Download PowerPoint Proteasome-dependent degradation of 1918 PB1-F2 is linked to the potent inhibition of type I IFN induction The inhibition of IFNβ induction by 1918 PB1-F2 was abrogated by MG132 treatment (Fig 3E). Thus, a strong association between the inhibition of IFNβ induction (Fig 3B) and the instability of PB1-F2 (Fig 2) was confirmed in two independent cell lines. When the 1918-specific mutations (I68T and L69P) were introduced into PR8 PB1-F2, IFNβ induction was dramatically inhibited, and the inhibition was associated with the instability of PB1-F2 (Fig 3F). Likewise, when the PR8 sequence (T68I and P69L) was introduced back into 1918 PB1-F2, IFNβ induction was restored, along with a concomitant increase in PB1-F2 stability. These results confirm that the instability of 1918 PB1-F2 is invariably associated with a potent inhibition of IFNβ. Amino acids 68 and 69 of 1918 PB1-F2 contribute to viral pathogenicity We next compared the pathogenicity of recombinant viruses in mice. Higher mortality was observed in mice infected with IAV (1918) than in mice infected with IAV (PR8), suggesting that 1918 PB1-F2 is indeed responsible for the increased virulence (Fig 4A). All mice infected with 500 pfu of IAV (PR8) or IAV (PB1-F2[-]) recovered from weight loss and survived after 2 weeks post-infection (Fig 4B). However, the virulence of IAV (I68T), IAV (L69P), or IAV (I68T/L69P) was much higher and comparable to that of IAV (1918) (Fig 4B). The virulence at different infection doses showed that both I68T and L69P mutations, in particular, among sequence variations between PR8 and 1918 PB1-F2 (Fig 2A), contributed to the high pathogenicity of IAV (1918) (Figs EV3A and B, and 4C), consistent with the definition of cytotoxic sequences (Conenello & Palese, 2007; Alymova et al, 2014; Cheng et al, 2017). Structural prediction in silico (Obradovic et al, 2005; Xue et al, 2010) revealed that PB1-F2 belongs to a family of intrinsically disordered proteins (IDPs) and has a high propensity for disorder at the N-terminus and extreme C-terminus (Fig EV3C). Interestingly, in contrast to PR8, 1918 PB1-F2 had an additional disordered region which was affected by substitutions at positions 68 and 69. Of note, the elements identified in 1918 PB1-F2 that are crucial for increased pathogenicity mapped within the internal intrinsically disordered region (IDR) that encompasses the positions previously identified as cytotoxic sequences (Conenello & Palese, 2007; Alymova et al, 2014; Cheng et al, 2017). The results suggest that the biochemical instabilities that lead to vulnerability to proteasomal degradation (Fig 2) and the consequent suppression of IFN responses (Figs 3 and 4) are associated with the unique structural perturbation within the 1918 PB1-F2 protein. Figure 4. IAV (1918) PB1-F2 increases viral pathogenicity in Balb/c mice and its amino acid residues 68 and 69 contribute to virulence Mice (n = 5 or 6 per group) were infected with IAV with indicated pfu. Survival was monitored daily for 2 weeks. Mice (n = 6–9 per group) were infected with each IAV as indicated. Body weight was recorded daily after infection for 2 weeks. Mice (n = 5 or 6 per group) were infected with viruses (1,000 pfu) carrying PR8 PB1-F2 with the I68T, L69P, or I68T+L69P mutation. Body weight changes and survival rate were monitored daily. Detailed analyses on the virulence necessitated the monitoring of body weight below the standard level (80%) of mortality. Three cohorts of mice (5 or 6 per group) were infected with 500 pfu of one of the three IAVs. Viral protein expression in mouse lung was determined by Western blotting 2 days after
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