Host envelope glycoprotein processing proteases are indispensable for entry into human cells by seasonal and highly pathogenic avian influenza viruses

Division of Enzyme Chemistry, Institute for Enzyme Research, The University of Tokushima, Kuramoto-cho 3-18-15, Tokushima 770-8503, Japan.
Journal of molecular and genetic medicine: an international journal of biomedical research 02/2008; 3(1):167-75.
Source: PubMed


Influenza A virus (IAV) is one of the most common infectious pathogens in humans and causes considerable morbidity and mortality. The recent spread of highly-pathogenic avian IAV H5N1 viruses has reinforced the importance of pandemic preparedness. In the pathogenesis of IAV infection, cellular proteases play critical roles in the process of viral entry into cells that subsequently leads to tissue damage in the infected organs. Since there are no processing protease for the viral membrane fusion glycoprotein hemagglutinin precursor (HA(0)) in IAV, entry of the virus into cells is determined primarily by the host cellular HA(0) processing proteases that proteolytically activate membrane fusion activity. HA(0) of seasonal human IAV has the consensus cleavage site motif Q(E)-T/X-R and is selectively processed by at least seven different trypsin-type processing proteases identified to-date in animal model experiments using mouse-adapted IAV or gene expression system in MDCK cells. As is the case for the highly pathogenic avian influenza (HPAI) A virus, endoproteolytic processing of the HA(0) occurs through ubiquitous cellular processing proteases, which selectively recognize the multi-basic consensus cleavage site motifs, such as R-X-K/R-R, and K-X-K/R-R. The cleavage enzymes for the R-X-K/R-R motif, but not K-X-K/R-R motif, have been reported to be furin and pro-protein convertase (PC)5/6 in the trans-Golgi network. Here we report new members of type II transmembrane serine proteases of the cell membrane, mosaic serine protease large form (MSPL) and its splice variant TMPRSS13, which recognize and cleave both R-X-K/R-R and K-X-K/R-R motifs without calcium. Furthermore, IAV infection significantly up-regulates a latent ectopic pancreatic trypsin, one of the potent HA processing proteases, and pro-matrix metalloprotease-9, in various organs. These proteases may synergistically damage the blood-brain barrier in the brain and basement membrane of blood vessels in various organs, resulting in severe edema and multiple organ failure. In this review, we discuss these proteases as new drug target molecules for IAV treatment acting by inhibition of IAV multiplication and prevention of multiple organ failure, other than anti-viral agents, viral neuraminidase inhibitors.

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Available from: Junji Chida
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    • "Hemagglutinin (HA) sequence alignments of influenza A virus A/Brisbane/59/2007 (H1N1), A/California/04/2009 (H1N1), A/Brisbane/10/2007 (H3N2), A/Anhui/1/2005 (H5N1) and A/Anhui/1/2013 (H7N9) were analyzed. The HA cleavage site sequence of H7N9 is P-K-G-R, which is consistent with low pathogenic avian influenza H7 [17]. Western blot analysis also showed that, in contrast to H5N1, the H7N9 HA0 protein cannot be effectively cleaved into functional HA1 and HA2 subunits by intracellular proteases (Fig. 1A). "
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    ABSTRACT: The recently identified human infections with a novel avian influenza H7N9 virus in China raise important questions regarding possible risk to humans. However, the entry properties and tropism of this H7N9 virus were poorly understood. Moreover, neuraminidase inhibitor resistant H7N9 isolates were recently observed in two patients and correlated with poor clinical outcomes. In this study, we aimed to elucidate the entry properties of H7N9 virus, design and evaluate inhibitors for H7N9 virus entry. We optimized and developed an H7N9-pseudotyped particle system (H7N9pp) that could be neutralized by anti-H7 antibodies and closely mimicked the entry process of the H7N9 virus. Avian, human and mouse-derived cultured cells showed high, moderate and low permissiveness to H7N9pp, respectively. Based on influenza virus membrane fusion mechanisms, a potent anti-H7N9 peptide (P155-185-chol) corresponding to the C-terminal ectodomain of the H7N9 hemagglutinin protein was successfully identified. P155-185-chol demonstrated H7N9pp-specific inhibition of infection with IC50 of 0.19 µM. Importantly, P155-185-chol showed significant suppression of A/Anhui/1/2013 H7N9 live virus propagation in MDCK cells and additive effects with NA inhibitors Oseltamivir and Zanamivir. These findings expand our knowledge of the entry properties of the novel H7N9 viruses, and they highlight the potential for developing a new class of inhibitors targeting viral entry for use in the next pandemic.
    Full-text · Article · Sep 2014 · PLoS ONE
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    • "The function of HA is to recognize host sialic acid residue as an entry receptor [4,5], and to fuse viral envelope with vesicle's membrane [5,6] after the linker peptide between subdomain HA1 and HA2 of HA is cleaved by host trypsin-like proteases. Virulent H5 and H7 hemagglutinins [7] have a polybasic cleavage site that is exposed and cleavable by furin or other proprotein convertases [8,9] which enables the virus to infect multiple organs and leads to multisystem failure [7]. A second factor correlating to the high pathogenicity of H5N1 influenza virus is the PB2 subunit in polymerase complex [10,11]. "
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    ABSTRACT: Avian influenza H5N1 virus is highly pathogenic partially because its H5 hemagglutinin contains a polybasic cleavage site that can be processed by proteases in multiple organs. Monoclonal antibodies (mAb) specific to the synthetic peptide of hemagglutinin polybasic cleavage site of H5N1 virus were raised and tested for their neutralizing potential. Purified mAb showed suppression of H5N1 pseudovirus infection on Madin-Darby Canine Kidney (MDCK) cells but the efficacy was less than 50%. Since those mAb are specific to the intact uncut polybasic cleavage site of hemagglutinin, their efficacy depends on the extent of hemagglutinin cleavage on the viral surface. Proteolytic analysis suggests the low efficacy associated with those mAb may be due to proteolytic cleavage already present on the majority of hemagglutinin prior to the infection of virus.
    Full-text · Article · Apr 2012 · Journal of Biomedical Science
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    • "Most of the TTSPs identified so far recognize a single R at position P1, but the newly isolated MSPL/TMPRSS13 has unique substrate specificities of the preferential recognition of a paired basic residue at the cleavage site [41] [43] [44]. Thus, MSPL/TMPRSS13, which are ubiquitously expressed in the plasma membranes, particularly highly expressed in the lungs, brain and vascular epithelial cells of human and chicken [42] [43], can activate various fusogenic viral envelope glycoproteins of HPAI viral strains. To clarify the roles of MSPL and TMPRSS13 in the proteolytic activation of HA of HPAI viruses, we searched for cells that express MSPL/TMPRSS13 at below detection levels and found human endothelial cell line ECV304 as the corresponding cell line. "
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    ABSTRACT: Influenza A virus (IAV) is one of the most common infectious pathogens in humans. Since the IVA genome does not have the processing protease for the viral hemagglutinin (HA) envelope glycoprotein precursors, entry of this virus into cells and infectious organ tropism of IAV are primarily determined by host cellular trypsin-type HA processing proteases. Several secretion-type HA processing proteases for seasonal IAV in the airway, and ubiquitously expressed furin and pro-protein convertases for highly pathogenic avian influenza (HPAI) virus, have been reported. Recently, other HA-processing proteases for seasonal IAV and HPAI have been identified in the membrane fraction. These proteases proteolytically activate viral multiplication at the time of viral entry and budding. In addition to the role of host cellular proteases in IAV pathogenicity, IAV infection results in marked upregulation of cellular trypsins and matrix metalloproteinase-9 in various organs and cells, particularly endothelial cells, through induced pro-inflammatory cytokines. These host cellular factors interact with each other as the influenza virus-cytokine-protease cycle, which is the major mechanism that induces vascular hyperpermeability and multiorgan failure in severe influenza. This mini-review discusses the roles of cellular proteases in the pathogenesis of IAV and highlights the molecular mechanisms of upregulation of trypsins as effective targets for the control of IAV infection. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.
    Full-text · Article · Jul 2011 · Biochimica et Biophysica Acta
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