Changes in human airway cells transcriptome during epithelial wound repair

Human parainfluenza virus type 1 (hPIV1) and 3 (hPIV3) cause seasonal epidemics, but little is known about their interaction with human airway cells. In this study, we determined cytopathology, replication, and progeny virion release from human airway cells during long-term infection in vitro. Both viruses readily established persistent infection without causing significant cytopathic effects. However, assembly and release of hPIV1 rapidly declined in sharp contrast to hPIV3 due to impaired viral ribonucleocapsid (vRNP) trafficking and virus assembly. Transcriptomic analysis revealed that both viruses induced similar levels of type I and III IFNs. However, hPIV1 induced specific ISGs stronger than hPIV3, such as MX2, which bound to hPIV1 vRNPs in infected cells. In addition, hPIV1 but not hPIV3 suppressed genes involved in lipid biogenesis and hPIV1 infection resulted in ubiquitination and degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, a rate limiting enzyme in cholesterol biosynthesis. Consequently, formation of cholesterol-rich lipid rafts was impaired in hPIV1 infected cells. These results indicate that hPIV1 is capable of regulating cholesterol biogenesis, which likely together with ISGs contributes to establishment of a quiescent infection.

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TMPRSS2 Is the Major Activating Protease of Influenza A Virus in Primary Human Airway Cells and Influenza B Virus in Human Type II Pneumocytes

Journal of Virology ◽

10.1128/jvi.00649-19 ◽

2019 ◽

Vol 93 (21) ◽

Cited By ~ 29

Author(s):

Hannah Limburg ◽

Anne Harbig ◽

Dorothea Bestle ◽

David A. Stein ◽

Hong M. Moulton ◽

...

Keyword(s):

Influenza A ◽

Influenza B Virus ◽

Influenza B ◽

Virus Infectivity ◽

Type Ii ◽

Proteolytic Activation ◽

Human Airway ◽

Type Ii Pneumocytes ◽

Airway Cells

ABSTRACT Cleavage of influenza virus hemagglutinin (HA) by host cell proteases is essential for virus infectivity and spread. We previously demonstrated in vitro that the transmembrane protease TMPRSS2 cleaves influenza A virus (IAV) and influenza B virus (IBV) HA possessing a monobasic cleavage site. Subsequent studies revealed that TMPRSS2 is crucial for the activation and pathogenesis of H1N1pdm and H7N9 IAV in mice. In contrast, activation of H3N2 IAV and IBV was found to be independent of TMPRSS2 expression and supported by an as-yet-undetermined protease(s). Here, we investigated the role of TMPRSS2 in proteolytic activation of IAV and IBV in three human airway cell culture systems: primary human bronchial epithelial cells (HBEC), primary type II alveolar epithelial cells (AECII), and Calu-3 cells. Knockdown of TMPRSS2 expression was performed using a previously described antisense peptide-conjugated phosphorodiamidate morpholino oligomer, T-ex5, that interferes with splicing of TMPRSS2 pre-mRNA, resulting in the expression of enzymatically inactive TMPRSS2. T-ex5 treatment produced efficient knockdown of active TMPRSS2 in all three airway cell culture models and prevented proteolytic activation and multiplication of H7N9 IAV in Calu-3 cells and H1N1pdm, H7N9, and H3N2 IAV in HBEC and AECII. T-ex5 treatment also inhibited the activation and spread of IBV in AECII but did not affect IBV activation in HBEC and Calu-3 cells. This study identifies TMPRSS2 as the major HA-activating protease of IAV in human airway cells and IBV in type II pneumocytes and as a potential target for the development of novel drugs to treat influenza infections. IMPORTANCE Influenza A viruses (IAV) and influenza B viruses (IBV) cause significant morbidity and mortality during seasonal outbreaks. Cleavage of the viral surface glycoprotein hemagglutinin (HA) by host proteases is a prerequisite for membrane fusion and essential for virus infectivity. Inhibition of relevant proteases provides a promising therapeutic approach that may avoid the development of drug resistance. HA of most influenza viruses is cleaved at a monobasic cleavage site, and a number of proteases have been shown to cleave HA in vitro. This study demonstrates that the transmembrane protease TMPRSS2 is the major HA-activating protease of IAV in primary human bronchial cells and of both IAV and IBV in primary human type II pneumocytes. It further reveals that human and murine airway cells can differ in their HA-cleaving protease repertoires. Our data will help drive the development of potent and selective protease inhibitors as novel drugs for influenza treatment.

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A Novel Co-Culture System Of Human Airway Cells With Cardiomyoblasts

10.1164/ajrccm-conference.2011.183.1_meetingabstracts.a3666 ◽

2011 ◽

Author(s):

Tsuyoshi Tanabe ◽

Soichiro Kanoh ◽

William Moskowitz ◽

Bruce K. Rubin

Keyword(s):

Culture System ◽

Human Airway ◽

Airway Cells

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Characterization of H5N1 Influenza Virus Quasispecies with Adaptive Hemagglutinin Mutations from Single-Virus Infections of Human Airway Cells

Journal of Virology ◽

10.1128/jvi.02004-17 ◽

2018 ◽

Vol 92 (11) ◽

pp. e02004-17 ◽

Cited By ~ 9

Author(s):

Yohei Watanabe ◽

Yasuha Arai ◽

Norihito Kawashita ◽

Madiha S. Ibrahim ◽

Emad M. Elgendy ◽

...

Keyword(s):

Genetic Diversity ◽

Influenza Virus ◽

Virus Infection ◽

Clinical Samples ◽

Human Airway ◽

H5n1 Influenza ◽

Starting Point ◽

Virus Adaptation ◽

Quasispecies Diversity ◽

Airway Cells

ABSTRACTTransmission of avian influenza (AI) viruses to mammals involves phylogenetic bottlenecks that select small numbers of variants for transmission to new host species. However, little is known about the AI virus quasispecies diversity that produces variants for virus adaptation to humans. Here, we analyzed the hemagglutinin (HA) genetic diversity produced during AI H5N1 single-virus infection of primary human airway cells and characterized the phenotypes of these variants. During single-virus infection, HA variants emerged with increased fitness to infect human cells. These variants generally had decreased HA thermostability, an indicator of decreased transmissibility, that appeared to compensate for their increase in α2,6-linked sialic acid (α2,6 Sia) binding specificity and/or in the membrane fusion pH threshold, each of which is an advantageous mutational change for viral infection of human airway epithelia. An HA variant with increased HA thermostability also emerged but could not outcompete variants with less HA thermostability. These results provided data on HA quasispecies diversity in human airway cells.IMPORTANCEThe diversity of the influenza virus quasispecies that emerges from a single infection is the starting point for viral adaptation to new hosts. A few studies have investigated AI virus quasispecies diversity during human adaptation using clinical samples. However, those studies could be appreciably affected by individual variability and multifactorial respiratory factors, which complicate identification of quasispecies diversity produced by selective pressure for increased adaptation to infect human airway cells. Here, we found that detectable HA genetic diversity was produced by H5N1 single-virus infection of human airway cells. Most of the HA variants had increased fitness to infect human airway cells but incurred a fitness cost of less HA stability. To our knowledge, this is the first report to characterize the adaptive changes of AI virus quasispecies produced by infection of human airway cells. These results provide a better perspective on AI virus adaptation to infect humans.

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