The Host Protease TMPRSS2 Plays a Major Role in In Vivo Replication of Emerging H7N9 and Seasonal Influenza Viruses

Influenza A virus (IAV) affects the upper and lower respiratory tracts and rapidly induces the expression of mucins, which are common O-glycosylated proteins, on the epithelial surfaces of the respiratory tract. Although mucin production is associated with the inhibition of virus transmission as well as characteristic clinical symptoms, little is known regarding how mucins are produced on the surfaces of respiratory epithelial cells and how they affect IAV replication. In this study, we found that two microRNAs (miRNAs), miR-17-3p and miR-221, which target GalNAc transferase 3 (GALNT3) mRNA, are rapidly downregulated in human alveolar basal epithelial cells during the early stage of IAV infection. We demonstrated that the expression of GALNT3 mRNA is upregulated in an IAV replication-dependent fashion and leads to mucin production in bronchial epithelial cells. A lectin microarray analysis revealed that the stable expression of GALNT3 by human alveolar basal epithelial cells induces mucin-type O-glycosylation modifications similar to those present in IAV-infected cells, suggesting that GALNT3 promotes mucin-type O-linked glycosylation in IAV-infected cells. Notably, analyses using short interfering RNAs and miRNA mimics showed that GALNT3 knockdown significantly reduces IAV replication. Furthermore, IAV replication was markedly decreased in embryonic fibroblast cells obtained from galnt3-knockout mice. Interestingly, IAV-infected galnt3-knockout mice exhibited high mortality and severe pathological alterations in the lungs compared to those of wild-type mice. Our results demonstrate not only the molecular mechanism underlying rapid mucin production during IAV infection but also the contribution of O-linked glycosylation to the replication and propagation of IAV in lung cells. IMPORTANCEViral infections that affect the upper or lower respiratory tracts, such as IAV, rapidly induce mucin production on the epithelial surfaces of respiratory cells. However, the details of how mucin-type O-linked glycosylation is initiated by IAV infection and how mucin production affects viral replication have not yet been elucidated. In this study, we show that levels of two miRNAs that target the UDP-GalNAc transferase GALNT3 are markedly decreased during the early stage of IAV infection, resulting in the upregulation of GALNT3 mRNA. We also demonstrate that the expression of GALNT3 initiates mucin production and affects IAV replication in infected cells. This is the first report demonstrating the mechanism underlying the miRNA-mediated initiation of mucin-type O-glycosylation in IAV-infected cells and its role in viral replication. Our results have broad implications for understanding IAV replication and suggest a strategy for the development of novel anti-influenza approaches.

“…Histological evaluations were carried out by the New Histo Science Laboratory Company. The inflammation symptoms of the lungs were scored based on the method of a previous study (22).…”

Section: Cells and Viruses A549 Beas-2b (Crl-9609mentioning

confidence: 99%

Influenza A Virus-Induced Expression of a GalNAc Transferase, GALNT3, via MicroRNAs Is Required for Enhanced Viral Replication

Nakamura

Horie

Daidoji

et al. 2016

“…Although TMPRSS2 is a marker of susceptibility to severe A (H1N1)pdm09 influenza virus (8), there is no clinical data indicating the involvement of a specific protease in the activation of seasonal IAV. However, in mice, TMPRSS2 has been identified as being essential for the spread of H1N1 IAV (9)(10)(11) and of H3N2 IAV in association with TMPRSS4 (12).…”

mentioning

confidence: 99%

Kallikrein-Related Peptidase 5 Contributes to H3N2 Influenza Virus Infection in Human Lungs

Magnen

Gueugnon

Guillon

et al. 2017

Hemagglutinin (HA) of influenza virus must be activated by proteolysis before the virus can become infectious. Previous studies indicated that HA cleavage is driven by membrane-bound or extracellular serine proteases in the respiratory tract. However, there is still uncertainty as to which proteases are critical for activating HAs of seasonal influenza A viruses (IAVs) in humans. This study focuses on human KLK1 and KLK5, 2 of the 15 serine proteases known as the kallikrein-related peptidases (KLKs). We find that their mRNA expression in primary human bronchial cells is stimulated by IAV infection. Both enzymes cleaved recombinant HA from several strains of the H1 and/or H3 virus subtype in vitro, but only KLK5 promoted the infectivity of A/Puerto Rico/8/34 (H1N1) and A/Scotland/20/74 (H3N2) virions in MDCK cells. We assessed the ability of treated viruses to initiate influenza in mice. The nasal instillation of only the KLK5-treated virus resulted in weight loss and lethal outcomes. The secretion of this protease in the human lower respiratory tract is enhanced during influenza. Moreover, we show that pretreatment of airway secretions with a KLK5-selective inhibitor significantly reduced the activation of influenza A/Scotland/20/74 virions, providing further evidence of its importance. Differently, increased KLK1 secretion appeared to be associated with the recruitment of inflammatory cells in human airways regardless of the origin of inflammation. Thus, our findings point to the involvement of KLK5 in the proteolytic activation and spread of seasonal influenza viruses in humans.IMPORTANCE Influenza A viruses (IAVs) cause acute infection of the respiratory tract that affects millions of people during seasonal outbreaks every year. Cleavage of the hemagglutinin precursor by host proteases is a critical step in the life cycle of these viruses. Consequently, host proteases that activate HA can be considered promising targets for the development of new antivirals. However, the specific proteases that activate seasonal influenza viruses, especially H3N2 viruses, in the human respiratory tract have remain undefined despite many years of work. Here we demonstrate that the secreted, extracellular protease KLK5 (kallikrein-related peptidase 5) is efficient in promoting the infectivity of H3N2 IAV in vitro and in vivo. Furthermore, we found that its secretion was selectively enhanced in the human lower respiratory tract during a seasonal outbreak dominated by an H3N2 virus. Collectively, our data support the clinical relevance of this protease in human influenza pathogenesis.

“…Using mouse models, three research groups recently demonstrated that a type II transmembrane serine protease, TMPRSS2, expressed in the airway epithelium, is critically important for HA cleavage in vivo (1)(2)(3). Mice lacking TMPRSS2 expression (TMPRSS2 knockout [tmprss2 KO ] mice) are highly tolerant of challenge infection by low-pathogenic (LP) IAVs possessing a monobasic cleavage site in HA.…”

mentioning

confidence: 99%

“…The viral hemagglutinin (HA) glycoprotein, which is responsible for receptor binding and subsequent membrane fusion, is synthesized as the inactive precursor, HA 0 , and cleaved by a host cell protease(s) into HA 1 and HA 2 subunits. The cleavage is essential for HA to mediate membrane fusion.…”

mentioning

confidence: 99%

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A Mutant H3N2 Influenza Virus Uses an Alternative Activation Mechanism in TMPRSS2 Knockout Mice by Loss of an Oligosaccharide in the Hemagglutinin Stalk Region

Sakai

Sekizuka

Ami

et al. 2015

Self Cite

e The host protease TMPRSS2 plays an essential role in proteolytic activation of the influenza A virus (IAV) hemagglutinin (HA) protein possessing a monobasic cleavage site. However, after passages in TMPRSS2 knockout mice, an H3N2 subtype IAV began to undergo cleavage activation of HA, showing high virulence in the mice due to the loss of an oligosaccharide at position 8 in the HA stalk region. Thus, the H3N2 IAV acquired cleavability by an alternative HA activation mechanism/protease(s). Influenza A virus (IAV), a member of the Orthomyxoviridae family, affects and kills many humans worldwide. The viral hemagglutinin (HA) glycoprotein, which is responsible for receptor binding and subsequent membrane fusion, is synthesized as the inactive precursor, HA 0 , and cleaved by a host cell protease(s) into HA 1 and HA 2 subunits. The cleavage is essential for HA to mediate membrane fusion.Using mouse models, three research groups recently demonstrated that a type II transmembrane serine protease, TMPRSS2, expressed in the airway epithelium, is critically important for HA cleavage in vivo (1-3). Mice lacking TMPRSS2 expression (TMPRSS2 knockout [tmprss2 KO ] mice) are highly tolerant of challenge infection by low-pathogenic (LP) IAVs possessing a monobasic cleavage site in HA. Our group (1) and Hatesuer et al.(2) showed that TMPRSS2 is essential for proteolytic activation of both H1N1 and H3N2 subtype IAVs. However, Tarnow et al. (3) reported that TMPRSS2 is not critical for activation of H3N2 IAV. Since the three groups used different H3N2 IAV strains, the discrepancy likely arose from possible variations in HA among the H3N2 IAV strains. The present study reveals a molecular determinant that modulates the TMPRSS2 dependency of H3N2 IAV in mouse lungs.A mouse-adapted human H3N2 IAV strain, MA-A/Guizhou-X (H3 WT ) (1), was used as a reference wild-type (WT) virus. The virus was a reassortant H3N2 virus of A/Guizhou/54/89 (H3N2), a Chinese human isolate from 1989, and A/Puerto Rico/8/34 (H1N1) (4). The H3WT virus was passaged 10 times in tmprss2 KO mice, and a tmprss2 KO mouse-adapted MA-A/Guizhou-X virus ( H3 p10 ) was obtained.The parental H3 WT and H3 p10 viruses were inoculated intranasally into tmprss2 KO mice (n ϭ 5). After challenge infection with 3.0 ϫ 10 3 PFU of H3 WT , tmprss2 KO mice showed no body weight loss, while all TMPRSS2 ϩ/ϩ wild-type mice (tmprss2 WT ) showed severe body weight loss (Fig. 1A). Conversely, both tmprss2 KO and tmprss2 WT mice infected with 1.7 ϫ 10 2 PFU of H3 p10 showed severe body weight loss and required euthanasia on day 6 postinfection (p.i.) (Fig. 1B). To compare the susceptibility between the two mouse strains in detail, both mouse strains were also infected with 1.7 ϫ 10, 1.7, and 0.17 PFU of H3 p10 ( Fig. 1C to E). The H3 p10 infections exhibited pathogenicity in both tmprss2 KO and tmprss2 WT mice, although the pathological changes were slightly less severe in tmprss2 KO mice than in tmprss2 WT mice. The 50% lethal doses of H3 p10 for tmprss2 WT and tmprss2 KO mice w...