Abstract
Influenza A viruses (IAV) primarily target respiratory epithelial cells, but can also replicate in immune cells, including human dendritic cells (DCs). Super-resolution microscopy provides a novel method of visualizing viral trafficking by overcoming the resolution limit imposed by conventional light microscopy, without the laborious sample preparation of electron microscopy. Using three-color Stimulated Emission Depletion (STED) microscopy, we visualized input IAV nucleoprotein (NP), early and late endosomal compartments (EEA1 and LAMP1 respectively), and HLA-DR (DC membrane/cytosol) by immunofluorescence in human DCs. Surface bound IAV were internalized within 5 min of infection. The association of virus particles with early endosomes peaked at 5 min when 50% of NP+ signals were also EEA1+. Peak association with late endosomes occurred at 15 min when 60% of NP+ signals were LAMP1+. At 30 min of infection, the majority of NP signals were in the nucleus. Our findings illustrate that early IAV trafficking in human DCs proceeds via the classical endocytic pathway.
MeSH terms
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Dendritic Cells / metabolism
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Dendritic Cells / ultrastructure*
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Dendritic Cells / virology
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Endosomes / virology
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Epithelial Cells / ultrastructure
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Epithelial Cells / virology
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HLA-DR Antigens / isolation & purification
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Host-Pathogen Interactions*
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Humans
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Influenza A virus / genetics
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Influenza A virus / isolation & purification
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Influenza A virus / pathogenicity
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Influenza A virus / ultrastructure*
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Lysosomal Membrane Proteins / isolation & purification
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Microscopy
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Nucleocapsid Proteins
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RNA-Binding Proteins / genetics
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RNA-Binding Proteins / isolation & purification
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Vesicular Transport Proteins / isolation & purification
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Viral Core Proteins / genetics
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Viral Core Proteins / isolation & purification
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Virion / genetics
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Virion / pathogenicity
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Virion / ultrastructure*
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Virus Replication / genetics
Substances
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HLA-DR Antigens
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LAMP1 protein, human
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Lysosomal Membrane Proteins
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NP protein, Influenza A virus
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Nucleocapsid Proteins
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RNA-Binding Proteins
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Vesicular Transport Proteins
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Viral Core Proteins
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early endosome antigen 1
Grants and funding
This work was supported by grants to AS-S from the Swedish Research Council, the Swedish Heart-Lung Foundation, and Karolinska Institutet. Genentech, Inc. provided support in the form of salaries for CC and IM, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the author contributions’ section.