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SPINK6 inhibits human airway serine proteases and restricts influenza virus activation.

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.

SUBMITTER: Wang D

PROVIDER: S-EPMC9976594 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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SPINK6 inhibits human airway serine proteases and restricts influenza virus activation.

Wang Dong D Li Cun C Chiu Man Chun MC Yu Yifei Y Liu Xiaojuan X Zhao Xiaoyu X Huang Jingjing J Cheng Zhongshan Z Yuan Shuofeng S Poon Vincent V Cai Jian-Piao JP Chu Hin H Chan Jasper Fuk-Woo JF To Kelvin Kai-Wang KK Yuen Kwok Yung KY Zhou Jie J

EMBO molecular medicine 20211126 1

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 ...[more]

PMID: 34826211

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Project description:UnlabelledCleavage of influenza virus hemagglutinin (HA) by host cell proteases is necessary for viral activation and infectivity. In humans and mice, members of the type II transmembrane protease family (TTSP), e.g., TMPRSS2, TMPRSS4, and TMPRSS11d (HAT), have been shown to cleave influenza virus HA for viral activation and infectivity in vitro Recently, we reported that inactivation of a single HA-activating protease gene,Tmprss2, in knockout mice inhibits the spread of H1N1 influenza viruses. However, after infection of Tmprss2 knockout mice with an H3N2 influenza virus, only a slight increase in survival was observed, and mice still lost body weight. In this study, we investigated an additional trypsin-like protease, TMPRSS4. Both TMPRSS2 and TMPRSS4 are expressed in the same cell types of the mouse lung. Deletion of Tmprss4 alone in knockout mice does not protect them from body weight loss and death upon infection with H3N2 influenza virus. In contrast,Tmprss2(-/-)Tmprss4(-/-)double-knockout mice showed a remarkably reduced virus spread and lung pathology, in addition to reduced body weight loss and mortality. Thus, our results identified TMPRSS4 as a second host cell protease that, in addition to TMPRSS2, is able to activate the HA of H3N2 influenza virus in vivoImportanceInfluenza epidemics and recurring pandemics are responsible for significant global morbidity and mortality. Due to high variability of the virus genome, resistance to available antiviral drugs is frequently observed, and new targets for treatment of influenza are needed. Host cell factors essential for processing of the virus hemagglutinin represent very suitable drug targets because the virus is dependent on these host factors for replication. We reported previously that Tmprss2-deficient mice are protected against H1N1 virus infections, but only marginal protection against H3N2 virus infections was observed. Here we show that deletion of two host protease genes,Tmprss2 and Tmprss4, strongly reduced viral spread as well as lung pathology and resulted in increased survival after H3N2 virus infection. Thus, TMPRSS4 represents another host cell factor that is involved in cleavage activation of H3N2 influenza viruses in vivo.

Project description:Imbalance of airway proteases and antiproteases has been implicated in diseases such as COPD and environmental exposures including cigarette smoke and ozone. To initiate infection, endogenous proteases are commandeered by respiratory viruses upon encountering the airway epithelium. The airway proteolytic environment likely contains redundant antiproteases and proteases with diverse catalytic mechanisms, however a proteomic profile of these enzymes and inhibitors in airway samples has not been reported. The objective of this study was to first profile extracellular proteases and antiproteases using human airway epithelial cell cultures and ex vivo nasal epithelial lining fluid (NELF) samples. Secondly, we present an optimized method for probing the proteolytic environment of airway surface liquid samples (in vitro and ex vivo) using fluorogenic peptides modeling the cleavage sites of respiratory viruses. We detected 48 proteases in the apical wash of cultured human nasal epithelial cells (HNECs) (n = 6) and 57 in NELF (n = 13) samples from healthy human subjects using mass-spectrometry based proteomics. Additionally, we detected 29 and 48 antiproteases in the HNEC apical washes and NELF, respectively. We observed large interindividual variability in rate of cleavage of an Influenza H1 peptide in the ex vivo clinical samples. Since protease and antiprotease levels have been found to be altered in the airways of smokers, we compared proteolytic cleavage in ex vivo nasal lavage samples from male/female smokers and non-smokers. There was a statistically significant increase in proteolysis of Influenza H1 in NLF from male smokers compared to female smokers. Furthermore, we measured cleavage of the S1/S2 site of SARS-CoV, SARS-CoV-2, and SARS-CoV-2 Delta peptides in various airway samples, suggesting the method could be used for other viruses of public health relevance. This assay presents a direct and efficient method of evaluating the proteolytic environment of human airway samples in assessment of therapeutic treatment, exposure, or underlying disease.

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SPINK6 inhibits human airway serine proteases and restricts influenza virus activation.

Publications

SPINK6 inhibits human airway serine proteases and restricts influenza virus activation.

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