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• W3217218045 abstract "Article26 November 2021Open Access Source DataTransparent process SPINK6 inhibits human airway serine proteases and restricts influenza virus activation Dong Wang Dong Wang orcid.org/0000-0003-4402-9756 Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, ChinaThese authors contributed equally to this work Search for more papers by this author Cun Li Cun Li Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, ChinaThese authors contributed equally to this work Search for more papers by this author Man Chun Chiu Man Chun Chiu Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China Search for more papers by this author Yifei Yu Yifei Yu Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China Search for more papers by this author Xiaojuan Liu Xiaojuan Liu Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China Search for more papers by this author Xiaoyu Zhao Xiaoyu Zhao Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China Search for more papers by this author Jingjing Huang Jingjing Huang Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China Search for more papers by this author Zhongshan Cheng Zhongshan Cheng Applied Bioinformatics Center, St Jude Children’s Research Hospital, Memphis, TN, USA Search for more papers by this author Shuofeng Yuan Shuofeng Yuan Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China Search for more papers by this author Vincent Poon Vincent Poon Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China Search for more papers by this author Jian-Piao Cai Jian-Piao Cai Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China Search for more papers by this author Hin Chu Hin Chu Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong, China Search for more papers by this author Jasper Fuk-Woo Chan Jasper Fuk-Woo Chan Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong, China Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong, China Search for more papers by this author Kelvin Kai-Wang To Kelvin Kai-Wang To Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong, China Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong, China Search for more papers by this author Kwok Yung Yuen Kwok Yung Yuen orcid.org/0000-0002-2083-1552 Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong, China Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong, China Search for more papers by this author Jie Zhou Corresponding Author Jie Zhou [email protected] orcid.org/0000-0002-2948-3873 Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong, China Search for more papers by this author Dong Wang Dong Wang orcid.org/0000-0003-4402-9756 Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, ChinaThese authors contributed equally to this work Search for more papers by this author Cun Li Cun Li Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, ChinaThese authors contributed equally to this work Search for more papers by this author Man Chun Chiu Man Chun Chiu Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China Search for more papers by this author Yifei Yu Yifei Yu Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China Search for more papers by this author Xiaojuan Liu Xiaojuan Liu Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China Search for more papers by this author Xiaoyu Zhao Xiaoyu Zhao Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China Search for more papers by this author Jingjing Huang Jingjing Huang Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China Search for more papers by this author Zhongshan Cheng Zhongshan Cheng Applied Bioinformatics Center, St Jude Children’s Research Hospital, Memphis, TN, USA Search for more papers by this author Shuofeng Yuan Shuofeng Yuan Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China Search for more papers by this author Vincent Poon Vincent Poon Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China Search for more papers by this author Jian-Piao Cai Jian-Piao Cai Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China Search for more papers by this author Hin Chu Hin Chu Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong, China Search for more papers by this author Jasper Fuk-Woo Chan Jasper Fuk-Woo Chan Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong, China Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong, China Search for more papers by this author Kelvin Kai-Wang To Kelvin Kai-Wang To Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong, China Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong, China Search for more papers by this author Kwok Yung Yuen Kwok Yung Yuen orcid.org/0000-0002-2083-1552 Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong, China Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong, China Search for more papers by this author Jie Zhou Corresponding Author Jie Zhou [email protected] orcid.org/0000-0002-2948-3873 Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong, China Search for more papers by this author Author Information Dong Wang1, Cun Li1, Man Chun Chiu1, Yifei Yu1, Xiaojuan Liu1, Xiaoyu Zhao1, Jingjing Huang1, Zhongshan Cheng2, Shuofeng Yuan1, Vincent Poon1, Jian-Piao Cai1, Hin Chu1,3, Jasper Fuk-Woo Chan1,3,4, Kelvin Kai-Wang To1,3,4, Kwok Yung Yuen1,3,4 and Jie Zhou *,1,3 1Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China 2Applied Bioinformatics Center, St Jude Children’s Research Hospital, Memphis, TN, USA 3State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong, China 4Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong, China *Corresponding author. Tel: +86 852 22554892; Fax: +86 852 28551241; E-mail: [email protected] EMBO Mol Med (2022)14:e14485https://doi.org/10.15252/emmm.202114485 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 SPINK6 was identified in human skin as a cellular inhibitor of serine proteases of the KLK family. Airway serine proteases are required to cleave hemagglutinin (HA) of influenza A viruses (IAVs) to initiate an infection in the human airway. We hypothesized that SPINK6 may inhibit common airway serine proteases and restrict IAV activation. We demonstrate that SPINK6 specifically suppresses the proteolytic activity of HAT and KLK5, HAT- and KLK5-mediated HA cleavage, and restricts virus maturation and replication. SPINK6 constrains the activation of progeny virions and impairs viral growth; and vice versa, blocking endogenous SPINK6 enhances HA cleavage and viral growth in physiological-relevant human airway organoids where SPINK6 is intrinsically expressed. In IAV-infected mice, SPINK6 significantly suppresses viral growth and improves mouse survival. Notably, individuals carrying the higher SPINK6 expression allele were protected from human H7N9 infection. Collectively, SPINK6 is a novel host inhibitor of serine proteases in the human airway and restricts IAV activation. Synopsis Serine protease inhibitor Kazal-type 6 (SPINK6) restricts influenza A virus (IAV) extracellular maturation via inhibiting proteolytic cleavage of the influenza virus hemagglutinin (HA) by human airway trypsin-like protease (HAT) and kallikrein 5 (KLK5). SPINK6 was identified as a novel host inhibitor of serine proteases in the human airway. SPINK6 restricts IAV activation and replication by inhibiting HA cleavage by HAT and KLK5. Endogenous SPINK6 blocks IAV maturation and viral growth in human airway organoids. SPINK6 ameliorates disease outcome in influenza virus-infected mice. Higher SPINK6 expression allele protects individuals from human H7N9 infection. The paper explained Problem Respiratory protease/antiprotease balance determines susceptibility to viral infections, including influenza. SPINK6 was identified in human skin as a host inhibitor of serine proteases of the KLK family. We sought to address whether SPINK6 can suppress the proteolytic activation of influenza viruses in the human respiratory tract. Results SPINK6 specifically suppresses HAT and KLK5 activation of influenza viruses, and restricts virus maturation and replication. Blocking endogenous SPINK6 enhances HA cleavage and viral growth in physiologically relevant human airway organoids where SPINK6 is intrinsically expressed. In IAV-infected mice, SPINK6 significantly suppresses viral growth and improves mouse survival. Impact SPINK6 is a novel host inhibitor of serine proteases in the human airway and restricts influenza virus activation. SPINK6 can be developed for the prevention and therapy of influenza infections. Introduction Respiratory viral infections are major health threats globally. Seasonal influenza A viruses (IAVs), including pandemic 2009 A(H1N1) (H1N1/pdm), are the major cause of human respiratory infections, affecting 5–15% of the human population with ˜500,000 annual deaths worldwide (Novel Swine-Origin Influenza et al, 2009). Human IAVs normally lead to upper respiratory infection with mild-to-moderate symptoms, and occasionally cause pneumonia with fatal outcomes. A novel avian A(H7N9) virus has caused recurrent outbreaks of human infection since 2013 (Chen et al, 2013). Human H7N9 infection commonly manifested as rapidly progressive pneumonia, with a case fatality rate higher than 30%. Influenza virus infection is initiated by the surface glycoprotein hemagglutinin (HA) binding to cellular receptors, followed by the fusion of viral membrane and cellular endosomal membrane. HA is synthesized as a fusion-inactive precursor protein HA0 that requires proteolytic cleavage by cellular proteases into disulfide-linked HA1 and HA2 subunits. The exposed fusion domain in the N terminal of HA2 then undergoes membrane fusion (Klenk & Garten, 1994). HA0 of most human and avian influenza viruses contain a monobasic (a single arginine, or rarely a single lysine) cleavage site that is recognized by trypsin-like serine proteases expressed abundantly in the human respiratory and gastrointestinal tract (Bottcher et al, 2006; Beaulieu et al, 2013); whereas highly pathogenic avian viruses including H5N1 contain a multi-basic cleavage site that is cleaved by ubiquitous intracellular proteases such as Furin. Serine proteases are a predominant class among respiratory proteases. Type II transmembrane serine proteases HAT and TMPRSS2 are the major proteases responsible for HA cleavage in the human airway (Bottcher et al, 2006; Bottcher-Friebertshauser et al, 2010, 2013). The role of these airway serine proteases for influenza virus replication has been extensively studied in vitro and in vivo (Tarnow et al, 2014). A new family of serine proteases, the kallikrein (KLK)-related peptidase family, comprises 15 secreted serine proteases (Prassas et al, 2015). Dysfunction of tissue-specific regulation of KLK activity is linked to several pathologies, including skin barrier dysfunction, psychological disorder, pathological inflammation, and cancer (Fischer et al, 2014; Prassas et al, 2015; Zheng et al, 2017). Interestingly, Hamilton et al reported that KLK5 and KLK12 are secreted from the human respiratory tract, and can cleave the HA of H1, H2, and H3 subtypes (Hamilton & Whittaker, 2013). Magnen et al (2017) recently demonstrated that KLK5 promotes the infectivity of seasonal influenza virus H3N2 in vitro and in vivo. Collectively, both trypsin/chymotrypsin-like serine proteases and some KLK serine proteases contribute to the activation and replication of influenza viruses in the human respiratory tract. Meanwhile, there is a growing recognition that virus activation proteases are not the sole player during viral infection; respiratory protease/antiprotease balance determines susceptibility to viral infections, including influenza (Meyer & Jaspers, 2015). In contrast to an array of HA cleavage proteases mentioned above (Okumura et al, 2010; Baron et al, 2013), only a few antiproteases have been identified. Secretory leukocyte protease inhibitor (SLPI) is highly expressed by epithelial cells and immune cells and present in respiratory secretions; the inducible SLPI was protective during respiratory virus infections (Kido et al, 2004). Plasminogen activator inhibitor 1, encoded by Serpin E1 gene, inhibits HA cleavage and restricts maturation of progeny virions (Dittmann et al, 2015). Serine protease inhibitor Kazal-type 6 (SPINK6) was initially identified in human and mouse skin (Meyer-Hoffert et al, 2010). It acts on most proteases of the KLK family, including KLK2, KLK5, KLK12, KLK13, and KLK14 (Kantyka et al, 2011). As a newly identified protease inhibitor, SPINK6 was reported to be involved in skin barrier function (Fischer et al, 2014) and metastasis of nasopharyngeal carcinoma (Zheng et al, 2017). Given its inhibition of multiple KLKs, more biological functions of SPINK6 are yet to be explored. As aforementioned, KLK5 and KLK12, the protease targets of SPINK6, can activate influenza viruses in the human respiratory tract (Hamilton & Whittaker, 2013). However, there is a gap in whether SPINK6 is indeed expressed in human respiratory cells, in which its inhibition of KLK5 and KLK12 is operational. In addition, SPINK6 inhibition of serine proteases may not be restricted to KLKs exclusively. It was documented that recombinant SPINK6 protein showed a moderate inhibitory effect on the proteolytic activity of bovine trypsin, and it suppressed the activity of trypsin-like proteases in normal mouse keratinocytes (Lu et al, 2012; Fischer et al, 2014). The evidence, collectively, prompted us to elucidate whether SPINK6 is a novel inhibitor of common respiratory serine proteases and could suppress the activation of IAVs in the human airways. Results SPINK6 restricts proteolytic activity of trypsin, trypsin-mediated HA cleavage, and viral replication SPINK6 was initially identified to be a cellular inhibitor of serine proteases of the KLK family in human skin (Meyer-Hoffert et al, 2010). We asked whether the inhibitory effect of SPINK6 on KLK5 and KLK12 is operational in other common respiratory serine proteases capable of HA activation. We performed a cell-free serine protease activity assay using a fluorogenic substrate of serine proteases. TPCK trypsin, a prototype chymotrypsin-like serine protease, is commonly used as a proxy for HA activation in cell culture-based propagation of most IAVs. We tested whether SPINK6 can inhibit the proteolytic activity of TPCK trypsin. To this end, recombinant wild-type SPINK6 (wtSPINK6) and mutant SPINK6 protein (mutSPINK6) were expressed in E. coli and purified (Fig EV1), the latter carrying loss-of-function mutations in the protease inhibition domain (Zheng et al, 2017). We found that, compared to mutSPINK6 and PBS, wtSPINK6 significantly reduced the proteolytic activity of TPCK trypsin (Fig 1A). SPINK6 is predicted to be a secreted protein. To clarify, we overexpressed SPINK6 in 293T cells, cell lysates and cell-free medium were harvested for Western blot analysis. As shown in Fig 1B, SPINK6 is indeed a secreted protein. Click here to expand this figure. Figure EV1. Coomassie blue staining shows the raw and purified recombinant proteins. Raw recombinant proteins of wtSPINK6 and mutSPINK6, purified wtSPINK6 and mutSPINK6 proteins, and removed poly-His tag are indicated with arrows after SDS-PAGE and Coomassie blue staining. Source data are available online for this figure. Download figure Download PowerPoint Figure 1. SPINK6 constrains proteolytic activity of trypsin, and trypsin-mediated HA cleavage and viral growth. TPCK trypsin is premixed with wtSPINK6 protein or mutSPINK6 protein or PBS in triplicate, incubated with a fluorogenic substrate for 30 min, and then applied to fluorescence assay. Data are presented as mean and SD in a representative experiment performed three times. The fluorescence intensity of TPCK trypsin/PBS mixture is arbitrarily set as 1. ***P < 0.001. At 48 h post-transfection of wtSPINK6 plasmid in 293T cells, cell lysate and concentrated supernatant (50×) were applied to the detection of SPINK6 protein and β-actin by WB. At 48 h after co-transfection of H7 plasmid with wtSPINK6 plasmid or blank vector, the transfectants were incubated with or without TPCK trypsin for 30 min, and then applied to examine HA cleavage by WB. Intensities of HA2 and HA0 bands are quantified with ImageJ. HA2/HA0 ratio of each sample is shown at the bottom. At 48 h after transfection of H7 plasmid, the transfectants were incubated with TPCK trypsin in the presence of recombinant wtSPINK6 protein or PBS or a protease inhibitor AEBSF for 30 min and then applied to examine HA cleavage. At 24 h after transfection of wtSPINK6 plasmid or blank vector in triplicate, A549 cells were inoculated with H7N9/ah virus. At the indicated hours post-infection (hpi), culture media (supernatant) and cell lysate were harvested for viral load detection. Data are presented as mean and SD in a representative experiment performed three times. **P < 0.01; ***P < 0.001. Student’s t-test. Source data are available online for this figure. Source Data for Figure 1 [emmm202114485-sup-0002-SDataFig1.zip] Download figure Download PowerPoint As aforementioned, proteolytic cleavage of HA is a prerequisite for IAV to initiate a productive infection in host cells. We then evaluated the effect of SPINK6 on TPCK trypsin-mediated HA cleavage. To this end, a plasmid encoding the HA of H7N9/ah virus (H7) was co-transfected with SPINK6 plasmid or a blank vector into BHK21 cells, which are devoid of endogenous proteases able to cleave HA. As a low pathogenic strain in domestic poultry, H7N9/ah virus possesses a monobasic cleavage site (Gao et al, 2013). At 48 h post-transfection, the transfected cells treated or mock treated with TPCK trypsin were harvested for detection of HA cleavage by Western blot. As shown in Fig 1C, in the absence of TPCK trypsin, H7 presented as an uncleaved HA0, no matter SPINK6 plasmid or the vector was co-transfected. Upon TPCK trypsin treatment, in both mock-transfected cells and vector-transfected cells, H7 was readily cleaved, with a HA2/HA0 ratio of 1.2 and 1.3, respectively. Interestingly, H7 cleavage by TPCK trypsin was restricted upon SPINK6 overexpression, with a notably reduced HA2/HA0 ratio of 0.6. Moreover, the addition of recombinant wtSPINK6 protein remarkably constrained trypsin-mediated H7 cleavage in comparison to PBS, with a HA2/HA0 ratio of 0.6 versus 1.4 (Fig 1D). AEBSF, a commercial inhibitor of serine proteases, was used as a positive control for inhibiting TPCK trypsin-mediated HA cleavage. We also inspected the multiple-cycle replication of H7N9/ah sustained by TPCK trypsin after overexpressing SPINK6 or the blank vector (Fig 1E). We observed an active replication of H7N9/ah upon transfection of the blank vector. In contrast, in SPINK6 overexpression cells, viral loads in culture media (supernatant) and cell lysate barely increased over time, and were significantly lower than those in vector-transfected cells. Collectively, SPINK6 overexpression significantly abolished trypsin-mediated HA cleavage and viral growth. SPINK6 suppresses proteolytic activities of HAT and KLK5, and constrains HAT-/KLK5-mediated HA cleavage To identify the target proteases of SPINK6, we overexpressed common HA activation serine proteases in BHK21 cells. At 48 h post-transfection, the cells were incubated with the fluorogenic substrate for 2 h in the presence of recombinant wtSPINK6 or mutSPINK6 protein or PBS (Fig 2A). Compared to mutSPINK6 and PBS, wtSPINK6 significantly reduced the activities of HAT and KLK5; whereas it showed a negligible effect on TMPRSS2, Matriptase, and Furin. Thus, SPINK6 specifically and significantly inhibits proteolytic activities of serine protease HAT and KLK5. We then assessed whether SPINK6 inhibition of the proteolytic activity of serine proteases could lead to a compromised HA cleavage. To this end, we first tested which serine proteases can activate HA of H7N9/ah and H1N1/pdm. As shown in Fig 2B, H7 is cleaved after overexpression of TMPRSS2, HAT, and Matriptase, while Furin and KLK5 are unable to cleave H7. Besides HAT, TMPRSS2, and Matriptase, KLK5 can also cleave H1, which is consistent with a previous report (Hamilton & Whittaker, 2013). Subsequently, co-transfection of three plasmids, including H7, proteases, and SPINK6 or vector, was performed to evaluate the effect of SPINK6 on HA cleavage by these proteases (Fig 2C). Compared to the blank vector, wtSPINK6 overexpression largely abolished HAT-mediated H7 cleavage (HA2/HA0 ratio of 0.4 vs. 0.1), whereas H7 cleavage by TMPRSS2 or Matriptase was similar no matter wtSPINK6 or vector was overexpressed. We verified the result using mutSPINK6 plasmid to replace the blank vector. wtSPINK6, but not mutSPINK6, specifically restricted H7 cleavage by HAT. In contrast, wtSPINK6 and mutSPINK6 showed a similar effect on H7 cleavage by TMPRSS2 (Fig 2D). Figure 2. SPINK6 inhibits HAT and KLK5, and HAT- and KLK5-mediated HA cleavage. At 48 h post-transfection of the indicated protease plasmids in triplicate, BHK21 cells were incubated with a fluorogenic substrate and wtSPINK6 or loss-of-function mutSPINK6 or PBS for 2 h and then applied to fluorescence assay. Data represent mean and SD of triplicated wells in a representative experiment performed three times. **P < 0.01. Student’s t-test. At 48 h after co-transfection of the indicated protease plasmids and H7 or H1 plasmid, BHK21 cells were lysed to examine HA cleavage with WB. At 48 h after triple transfection of H7, the indicated proteases, and wtSPINK6 or vector, BHK21 cells were lysed for the detection of HA cleavage. At 48 h after triple transfection of H7, HAT or TMPRSS2, and wtSPINK6 or mutSPINK6, BHK21 cells were lysed for the detection of HA cleavage. At 48 h after triple transfection of H1, the indicated proteases, and wtSPINK6 or mutSPINK6, BHK21 cells were lysed for the detection of HA cleavage. Source data are available online for this figure. Source Data for Figure 2 [emmm202114485-sup-0003-SDataFig2.zip] Download figure Download PowerPoint SPINK6-specific inhibition of HA cleavage by HAT, but not TMPRSS2, was reproduced in the cleavage of H1 (Fig 2E). Compared to mutSPINK6 overexpression, wtSPINK6 overexpression largely constrained H1 cleavage by HAT. Interestingly, H1 cleavage by KLK5 was notably attenuated upon wtSPINK6 overexpression. Likewise, wtSPINK6 overexpression had a minimal effect on TMPRSS2- and Matriptase-mediated H1 cleavage. Thus, the role of SPINK6 on HA cleavage by the serine proteases exactly recapitulated its inhibition of proteolytic activities of these proteases as shown in Fig 2A. SPINK6 attenuates IAV replication by restricting virus maturation To assess the effect of SPINK6 on protease-driven IAV replication, we inoculated BHK21 cells with H7N9/ah at 36 h after co-transfection of HAT, or TMPRSS2 or Matriptase, together with SPINK6 or vector. Cell-free culture media were harvested at 24 h post-infection for detecting viral growth (Fig 3A). SPINK6 overexpression significantly reduced HAT-mediated viral growth, whereas it exerted a minimal effect on viral replication sustained by TMPRSS2 and Matriptase. We further verified SPINK6 inhibition of protease-driven viral growth in more susceptible A549 cells upon infection with H1N1/pdm. IAVs with a monobasic HA cleavage site, including H1N1/pdm, barely replicate in A549 cells in the absence of exogenous proteases such as TPCK trypsin. At 24 h after transfection of HAT or KLK5, A549 cells inoculated with H1N1/pdm were incubated in the presence of wtSPINK6 or mutSPINK6 for 24 h. The culture medium in each well was harvested and stored in two aliquots; one aliquot was applied to the conventional plaque assay for viral titration (Fig 3B). wtSPINK6 treatment in HAT overexpression cells resulted in a significantly decreased viral titer of more than 1 log10 unit than mutSPINK6 (HAT, black bars in wtSPINK6 vs. mutSPINK6). A significant viral reduction resulted from wtSPINK6 treatment was reproduced in KLK5-driven viral growth (KLK5, black bars in wtSPINK6 vs. mutSPINK6). Figure 3. SPINK6 inhibits virus growth and maturation-mediated HAT and KLK5. At 36 h after transfection of the indicted protease and wtSPINK6 plasmids or blank vector in triplicate, BHK21 cells were inoculated with H7N9/ah at a MOI of 0.5. Cell-free culture media were harvested at 24 hpi for viral titration. Data represent the mean and SD of the triplicated wells in a representative experiment performed three times. **P < 0.01. Student’s t-test. At 24 h after transfection of HAT or KLK5 plasmid in sextuplicate, A549 cells were inoculated with H1N1/pdm at MOI of 0.25. The infected cells were incubated with recombinant wtSPINK6 or mutSPINK6 protein in triplicate for 24 h. Culture media in each well were stored in two aliquots, one was applied to viral titration with conventional plaque assay (black bars) and the other was pre-treated with TPCK trypsin for 1 h prior to plaque assay (grey bars). Data represent the mean and SD of the triplicated wells in a representative experiment performed three times. *P < 0.05; **P < 0.01; ***P < 0.001. Student’s t-test. Download figure Download PowerPoint The other aliquots of the same medium samples were simultaneously applied to a modified plaque assay in which the media were treated with TPCK trypsin prior to inoculation onto MDCK monolayers. Notably, TPCK trypsin pretreatment significantly rescued wtSPINK6-induced viral reduction in both HAT and KLK5 overexpression cells (black vs. grey bars in wtSPINK6 treatment), suggesting that the compromised viral growth in wtSPINK6-treated cells was indeed attributed to the accumulation of non-infectious progeny virions with uncleaved HA0. In stark contrast, trypsin pretreatment had a minimal effect on the infectivity of progeny virions released from mutSPINK6-treated cells (black vs. grey bars in mutSPINK6 treatment). Collectively, SPINK6 suppressed viral growth by restricting virus maturation. SPINK6 suppression of virus activation and growth occurs in human airway organoids We performed immunofluorescence staining and found that SPINK6 is indeed expressed in the airway epithelial cells in human lung tissues (Fig EV2). We have reported the establishment of human airway organoids from adult stem cells in primary lung tissues. The non-differentiated airway organoids can be consecutively and stably expanded in vitro for over 1 year; upon induction of maturation, the differentiated airway organoids can morphologically and functionally simulate human airway epithelium to a near physiological level. Furthermore, the differentiated airway organoids sustain robust replication of IAVs in the absence of TPCK trypsin since they possess endogenous HA activation serine proteases (Zhou et" @default.
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• W3217218045 title "SPINK6 inhibits human airway serine proteases and restricts influenza virus activation" @default.
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