scholarly journals TMPRSS2 and TMPRSS4 Facilitate Trypsin-Independent Spread of Influenza Virus in Caco-2 Cells

2010 ◽  
Vol 84 (19) ◽  
pp. 10016-10025 ◽  
Author(s):  
Stephanie Bertram ◽  
Ilona Glowacka ◽  
Paulina Blazejewska ◽  
Elizabeth Soilleux ◽  
Paul Allen ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Serine Proteases ◽  
Alveolar Epithelium ◽  
Influenza Viruses ◽  
Target Cells ◽  
Human Respiratory Tract ◽  
Proteolytic Activation ◽  
Viral Spread ◽  
Endogenous Expression ◽  
Influenza Virus Hemagglutinin

ABSTRACT Proteolysis of influenza virus hemagglutinin by host cell proteases is essential for viral infectivity, but the proteases responsible are not well defined. Recently, we showed that engineered expression of the type II transmembrane serine proteases (TTSPs) TMPRSS2 and TMPRSS4 allows hemagglutinin (HA) cleavage. Here we analyzed whether TMPRSS2 and TMPRSS4 are expressed in influenza virus target cells and support viral spread in the absence of exogenously added protease (trypsin). We found that transient expression of TMPRSS2 and TMPRSS4 resulted in HA cleavage and trypsin-independent viral spread. Endogenous expression of TMPRSS2 and TMPRSS4 in cell lines correlated with the ability to support the spread of influenza virus in the absence of trypsin, indicating that these proteases might activate influenza virus in naturally permissive cells. Indeed, RNA interference (RNAi)-mediated knockdown of both TMPRSS2 and TMPRSS4 in Caco-2 cells, which released fully infectious virus without trypsin treatment, markedly reduced the spread of influenza virus, demonstrating that these proteases were responsible for efficient proteolytic activation of HA in this cell line. Finally, TMPRSS2 was found to be coexpressed with the major receptor determinant of human influenza viruses, 2,6-linked sialic acids, in human alveolar epithelium, indicating that viral target cells in the human respiratory tract express TMPRSS2. Collectively, our results point toward an important role for TMPRSS2 and possibly TMPRSS4 in influenza virus replication and highlight the former protease as a potential therapeutic target.

scholarly journals Proteolytic Activation of Influenza Viruses by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium

2006 ◽  
Vol 80 (19) ◽  
pp. 9896-9898 ◽  
Author(s):  
Eva Böttcher ◽  
Tatyana Matrosovich ◽  
Michaela Beyerle ◽  
Hans-Dieter Klenk ◽  
Wolfgang Garten ◽  
...  
Keyword(s):  
Serine Proteases ◽  
Mammalian Cells ◽  
Mdck Cells ◽  
Influenza Viruses ◽  
Human Respiratory Tract ◽  
Proteolytic Activation ◽  
Mammalian Expression ◽  
Human Airway Epithelium

ABSTRACT Host cell proteases that cleave the hemagglutinin (HA) of influenza viruses in the human respiratory tract are still not identified. Here we cloned two human type II transmembrane serine proteases with known airway localization, TMPRSS2 and HAT, into mammalian expression vector. Cotransfection of mammalian cells with plasmids encoding HA and either protease resulted in HA cleavage in situ. Transient expression of either protease in MDCK cells enabled multicycle replication of influenza viruses in these cells in the absence of exogenous trypsin. These data suggest that TMPRSS2 and HAT are candidates for proteolytic activation of influenza viruses in vivo.

scholarly journals Expression of Factor X in BHK-21 Cells Promotes Low Pathogenic Influenza Viruses Replication

2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
Author(s):  
Shahla Shahsavandi ◽  
Mohammad Majid Ebrahimi ◽  
Shahin Masoudi ◽  
Hasan Izadi
Keyword(s):  
Influenza Virus ◽  
Mdck Cells ◽  
High Titer ◽  
Influenza Viruses ◽  
Factor X ◽  
Proteolytic Activation ◽  
Influenza Virus Hemagglutinin ◽  
Pathogenic Avian Influenza Virus ◽  
Replication Procedure ◽  
Cell Expression

A cDNA clone for factor 10 (FX) isolated from chicken embryo inserted into the mammalian cell expression vector pCDNA3.1 was transfected into the baby hamster kidney (BHK-21) cell line. The generated BHK-21 cells with inducible expression of FX were used to investigate the efficacy of the serine transmembrane protease to proteolytic activation of influenza virus hemagglutinin (HA) with monobasic cleavage site. Data showed that the BHK-21/FX stably expressed FX after ten serial passages. The cells could proteolytically cleave the HA of low pathogenic avian influenza virus at multiplicity of infection 0.01. Growth kinetics of the virus on BHK-21/FX, BHK-21, and MDCK cells were evaluated by titrations of virus particles in each culture supernatant. Efficient multicycle viral replication was markedly detected in the cell at subsequent passages. Virus titration demonstrated that BHK-21/FX cell supported high-titer growth of the virus in which the viral titer is comparable to the virus grown in BHK-21 or MDCK cells with TPCK-trypsin. The results indicate potential application for the BHK-21/FX in influenza virus replication procedure and related studies.

scholarly journals Antibody to influenza virus matrix protein detects a common antigen on the surface of cells infected with type A influenza viruses.

1977 ◽  
Vol 146 (3) ◽  
pp. 690-697 ◽  
Author(s):  
W E Biddison ◽  
P C Doherty ◽  
R G Webster
Keyword(s):  
Influenza Virus ◽  
Matrix Protein ◽  
Type A ◽  
Cross Reactivity ◽  
T Cell Response ◽  
Influenza Viruses ◽  
Cytotoxic T Cell ◽  
Target Cells ◽  
Common Antigen ◽  
Infected Cells

Antisera to the type-specific internal influenza virus matrix (M) protein of a type A influenza virus were produced in goats. In the presence of complement, anti-M serum was cytotoxic for target cells which were infected with a variety of serologically distinct type A influenza viruses, but did not react with type B influenza virus-infected cells. Absorption experiments indicated that anti-M serum detected a common antigen(s) on the surface of type A-infected cells. This serological cross-reactivity parallels the cross-reactivity observed for the cytotoxic T-cell response to type A viruses.

scholarly journals Cleavage of Influenza A Virus Hemagglutinin in Human Respiratory Epithelium Is Cell Associated and Sensitive to Exogenous Antiproteases

2002 ◽  
Vol 76 (17) ◽  
pp. 8682-8689 ◽  
Author(s):  
Oleg P. Zhirnov ◽  
Mine R. Ikizler ◽  
Peter F. Wright
Keyword(s):  
Molecular Weight ◽  
Influenza Virus ◽  
Serine Proteases ◽  
Influenza A ◽  
Extracellular Fluid ◽  
Respiratory Epithelium ◽  
Influenza Viruses ◽  
Secretory Cells ◽  
Low Molecular Weight ◽  
Virus Growth

ABSTRACT Proteolytic cleavage of the hemagglutinin (HA) of human influenza viruses A/Aichi/2/68 (H3N2) and A/WSN/34 (H1N1) from HA0 to HA1/HA2 was studied in primary human adenoid epithelial cells (HAEC). HAEC contain a mixture of ciliated and nonciliated secretory cells and mimic the epithelium membrane of the human respiratory tract. Pulse-chase labeling with [35S]methionine and Western blot analysis with anti-HA antibodies of cellular and virion polypeptides showed that HAEC cleaved newly synthesized HA0 to HA1/HA2 (“cleavage from within”) and significant amounts of cleaved HA accumulated within cells. It was also shown that HAEC was able to cleave HA0 of incoming virions (“cleavage from without”), whereas the HA0 of nonabsorbed virions free in extracellular fluid were not cleaved, supporting the conclusion that HA0 cleavage in HAEC is cell associated. Low-molecular-weight inhibitors of serine proteases, aprotinin and leupeptin, when added to influenza virus-infected HAEC suppressed HA0 cleavage and reduced the amount of cleaved HA1/HA2 both in cells and in progeny virions and thus diminished the infectivity of the virus. In contrast, the addition of fetal bovine serum, containing a number of high-molecular-weight antiproteases that compete for proteases in the extracellular environment, did not inhibit influenza virus growth in HAEC. These data suggest that in human respiratory epithelium the cleavage of influenza virus HA containing a single arginine in the proteolytic site (i) is a cell-associated process accomplished by serine-type protease(s) and (ii) is sensitive to low-molecular-weight exogenous inhibitors of serine proteases.

scholarly journals Entry of influenza A virus: host factors and antiviral targets

2014 ◽  
Vol 95 (2) ◽  
pp. 263-277 ◽  
Author(s):  
Thomas O. Edinger ◽  
Marie O. Pohl ◽  
Silke Stertz
Keyword(s):  
Influenza Virus ◽  
Cell Nucleus ◽  
Influenza A ◽  
Current Knowledge ◽  
Influenza Viruses ◽  
Host Factors ◽  
Target Cells ◽  
Endosomal Trafficking ◽  
Substantial Impact ◽  
Entry Inhibitors

Influenza virus is a major human pathogen that causes annual epidemics and occasional pandemics. Moreover, the virus causes outbreaks in poultry and other animals, such as pigs, requiring costly and laborious countermeasures. Therefore, influenza virus has a substantial impact on health and the global economy. Here, we review entry of this important pathogen into target cells, an essential process by which viral genomes are delivered from extracellular virions to sites of transcription/replication in the cell nucleus. We summarize current knowledge on the interaction of influenza viruses with their receptor, sialic acid, and highlight the ongoing search for additional receptors. We describe receptor-mediated endocytosis and the recently discovered macropinocytosis as alternative virus uptake pathways, and illustrate the subsequent endosomal trafficking of the virus with advanced live microscopy techniques. Release of virus from the endosome and import of the viral ribonucleoproteins into the host cell nucleus are also outlined. Although a focus has been on viral protein function during entry, recent studies have revealed exciting information on cellular factors required for influenza virus entry. We highlight these, and discuss established entry inhibitors targeting viral and host factors, as well as the latest prospects for designing novel ‘anti-entry’ compounds. New entry inhibitors are of particular importance for current efforts to develop the next generation of anti-influenza drugs – entry is the first essential step of virus replication and is an ideal target to block infection efficiently.

scholarly journals A Prevalent Focused Human Antibody Response to the Influenza Virus Hemagglutinin Head Interface

mBio ◽  
2021 ◽  
Author(s):  
Kevin R. McCarthy ◽  
Jiwon Lee ◽  
Akiko Watanabe ◽  
Masayuki Kuraoka ◽  
Lindsey R. Robinson-McCarthy ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Herd Immunity ◽  
Influenza Viruses ◽  
Influenza Vaccines ◽  
Human Antibody ◽  
Human Influenza ◽  
Human Antibodies ◽  
Influenza Virus Hemagglutinin ◽  
Selection For ◽  
Rapid Appearance

The rapid appearance of mutations in circulating human influenza viruses and selection for escape from herd immunity require prediction of likely variants for an annual updating of influenza vaccines. The identification of human antibodies that recognize conserved surfaces on the influenza virus hemagglutinin (HA) has prompted efforts to design immunogens that might selectively elicit such antibodies.

scholarly journals Host DNA released by NETosis in neutrophils exposed to seasonal H1N1 and highly pathogenic H5N1 influenza viruses

2020 ◽  
Author(s):  
Louisa L.Y. Chan ◽  
John M. Nicholls ◽  
J.S. Malik Peiris ◽  
Yu Lung Lau ◽  
Michael C.W. Chan ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Virus Infection ◽  
H1n1 Virus ◽  
Influenza Virus Infection ◽  
Alveolar Epithelium ◽  
Influenza Viruses ◽  
H5n1 Influenza Virus ◽  
H5n1 Influenza ◽  
Influenza H1n1

Abstract Background Neutrophil (Nϕ) is of the most abundant number in human immune system. During acute influenza virus infection, Nϕs are already active in the early phase of inflammation-a time in which clinical biopsy or autopsy material is not readily available. However, the role of Nϕ in virus infection is not well understood. Here, we studied the role of Nϕ in host defense during influenza A virus infection, specifically assessing if it contributes to the differential pathogenesis in H5N1 disease. Methods Nϕs were freshly isolated from healthy volunteers and subjected to direct influenza H1N1 and H5N1 virus infection in vitro . The ability of the naïve Nϕs to infiltrate from the basolateral to the apical phase of the influenza virus infected alveolar epithelium was assessed. The viral replication, innate immune responses and Neutrophil extracellular trap (NET) formation of Nϕs upon influenza virus infection were evaluated. Results Our results demonstrated that influenza virus infected alveolar epithelium allowed more Nϕs transmigration. Significantly more Nϕs migrated across the H5N1 influenza virus infected the epithelium than the counterpart infected by the seasonal influenza H1N1 virus infected. Nϕs were equally susceptible to H5N1 and H1N1 virus infection with similar viral gene transcription. Productive replication was observed in H5N1 infected Nϕs. Both H5N1 and H1N1 infected Nϕs induced cytokines and chemokines including TNF-α, IFN-β, CXCL10, MIP-1α and IL-8. This inferred a more intense inflammatory response posed by H5N1 than H1N1 virus. Strikingly, NADPH oxidase-independent NET formation was observed in H1N1 infected Nϕs at 6 hpi while no NET formation was observed upon H5N1 infection. Conclusion Our data is the first to demonstrate that NET formation is abrogated in H5N1 influenza virus infection. Its contribution to the differential severity of H5N1 disease requires further investigation.

scholarly journals Dissection of Epitope-Specific Mechanisms of Neutralization of Influenza Virus by Intact IgG and Fab Fragments

2017 ◽  
Vol 92 (6) ◽  
Author(s):  
James A. Williams ◽  
Long Gui ◽  
Nancy Hom ◽  
Alexander Mileant ◽  
Kelly K. Lee
Keyword(s):  
Influenza Virus ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Target Cells ◽  
Virus Infectivity ◽  
Fab Fragment ◽  
Fab Fragments ◽  
Influenza Virus Hemagglutinin ◽  
Fusion Glycoprotein ◽  
The Impact

ABSTRACTThe neutralizing antibody (nAb) response against the influenza virus hemagglutinin (HA) fusion glycoprotein is important for preventing viral infection, but we lack a comprehensive understanding of the mechanisms by which these antibodies act. Here we investigated the effect of nAb binding and the role of IgG bivalency in the inhibition of HA function for nAbs targeting distinct HA epitopes. HC19 targets the receptor binding pocket at the distal end of HA, while FI6v3 binds primarily to the HA2 fusion subunit toward the base of the stalk. Surprisingly, HC19 inhibited the ability of HA to induce lipid mixing by preventing the structural rearrangement of HA under fusion-activating conditions. These results suggest that nAbs such as HC19 not only act by blocking receptor binding but also inhibit key late-stage HA conformational changes required for fusion. Intact HC19 IgG was also shown to cross-link separate virus particles, burying large proportions of HA within aggregates where they are blocked from interacting with target membranes; Fabs yielded no such aggregation and displayed weaker neutralization than IgG, emphasizing the impact of bivalency on the ability to neutralize virus. In contrast, the stem-targeting nAb FI6v3 did not aggregate particles. The Fab fragment was significantly less effective than IgG in preventing both membrane disruption and fusion. We infer that interspike cross-linking within a given particle by FI6v3 IgG may be critical to its potent neutralization, as no significant neutralization occurred with Fabs. These results demonstrate that IgG bivalency enhances HA inhibition through functionally important modes not evident in pared-down Fab-soluble HA structures.IMPORTANCEThe influenza virus hemagglutinin (HA) fusion glycoprotein mediates entry into target cells and is the primary antigenic target of neutralizing antibodies (nAbs). Our current structural understanding of mechanisms of antibody (Ab)-mediated neutralization largely relies on the high-resolution characterization of antigen binding (Fab) fragments in complex with soluble, isolated antigen constructs by cryo-electron microscopy (EM) single-particle reconstruction or X-ray crystallography. Interactions between full-length IgG and whole virions have not been well characterized, and a gap remains in our understanding of how intact Abs neutralize virus and prevent infection. Using structural and biophysical approaches, we observed that Ab-mediated inhibition of HA function and neutralization of virus infectivity occur by multiple coexisting mechanisms, are largely dependent on the specific epitope that is targeted, and are highly dependent on the bivalent nature of IgG molecules.

A Novel Influenza A Virus Activating Enzyme from Porcine Lung: Purification and Characterization

2003 ◽  
Vol 384 (2) ◽  
pp. 219-227 ◽  
Author(s):  
M. Sato ◽  
S. Yoshida ◽  
K. Iida ◽  
T. Tomozawa ◽  
H. Kido ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Gel Filtration ◽  
Influenza Viruses ◽  
Proteolytic Activation ◽  
Peptide Substrates ◽  
Sds Page ◽  
Natural Hosts ◽  
Processing Activity ◽  
Porcine Lungs

AbstractProteolytic activation of hemagglutinin, an envelope glycoprotein of the influenza virus, by host proteases is essential for infection and proliferation of the virus. However, there is no well-defined, inherent source of host proteases in man or swine, both of which are natural hosts for human influenza viruses. We have recently isolated a 32 kDa protein in a high salt extract from porcine lungs, which possess the hemagglutinin processing activity. In this study, we attempted to purify another hemagglutinin processing enzyme from porcine lung. The purified enzyme, named tryptase TC30, exhibited a molecular mass of about 30 kDa by SDS-PAGE and 28.5 kDa by gel filtration chromatography, suggesting that it is a monomer. Tryptase TC30 cleaved peptide substrates with Arg at the P1 position, and preferentially substrates with the Ser-Ile-Gln-Ser-Arg sequence corresponding to the HA cleavage site sequence of the A/PR/8/34 influenza virus. Among various inhibitors tested, trypsintype serine protease inhibitors, such as aprotinin, antipain, benzamidine and leupeptin, efficiently inhibited the proteolytic activity of the enzyme. The N-terminal 40 amino acid sequence of tryptase TC30 exhibits more than 60% homology to mast cell tryptases from mice MCP-6 and human tryptase-α and -β. These data indicate that tryptase TC30, the 30 kDa enzyme from porcine lung, is a novel hemagglutinin-cleaving enzyme.

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