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Measuring attachment and internalization of influenza a virus in A549 cells by flow cytometry


Pohl, M O; Stertz, S (2015). Measuring attachment and internalization of influenza a virus in A549 cells by flow cytometry. Journal of Visualized Experiments (Jove), (105):e53372.

Abstract

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed protocol for measuring relative changes in the amount of viral particles that attach to A549 cells, a human lung epithelial cell line, as well as in the amount of particles that are internalized into the cell. We use biotinylated virus which can be easily detected following staining with Cy3-labeled streptavidin (STV-Cy3). We describe the growth, purification and biotinylation of A/WSN/33, a widely used IAV laboratory strain. Cold-bound biotinylated IAV particles on A549 cells are stained with STV-Cy3 and measured using flow cytometry. To investigate uptake of viral particles, cold-bound virus is allowed to internalize at 37 °C. In order to differentiate between external and internalized viral particles, a blocking step is applied: Free binding spots on the biotin of attached virus on the cell surface are bound by unlabeled streptavidin (STV). Subsequent cell permeabilization and staining with STV-Cy3 then enables detection of internalized viral particles. We present a calculation to determine the relative amount of internalized virus. This assay is suitable to measure effects of drug-treatments or other manipulations on attachment or internalization of IAV.

Abstract

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed protocol for measuring relative changes in the amount of viral particles that attach to A549 cells, a human lung epithelial cell line, as well as in the amount of particles that are internalized into the cell. We use biotinylated virus which can be easily detected following staining with Cy3-labeled streptavidin (STV-Cy3). We describe the growth, purification and biotinylation of A/WSN/33, a widely used IAV laboratory strain. Cold-bound biotinylated IAV particles on A549 cells are stained with STV-Cy3 and measured using flow cytometry. To investigate uptake of viral particles, cold-bound virus is allowed to internalize at 37 °C. In order to differentiate between external and internalized viral particles, a blocking step is applied: Free binding spots on the biotin of attached virus on the cell surface are bound by unlabeled streptavidin (STV). Subsequent cell permeabilization and staining with STV-Cy3 then enables detection of internalized viral particles. We present a calculation to determine the relative amount of internalized virus. This assay is suitable to measure effects of drug-treatments or other manipulations on attachment or internalization of IAV.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Medical Virology
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Uncontrolled Keywords:Infection,flow cytometry, influenza A virus, attachment, internalization, virus entry, A549 cells, biotinylated virus
Language:English
Date:11 April 2015
Deposited On:15 Feb 2016 15:23
Last Modified:08 Dec 2017 17:24
Publisher:Journal of Visualized Experiments
ISSN:1940-087X
Funders:Swiss National Science Foundation (31003A_135278) to SSt, doctoral grant from the AXA Research Fund to MOP
Free access at:PubMed ID. An embargo period may apply.
Publisher DOI:https://doi.org/10.3791/53372
Official URL:http://www.jove.com/video/53372/measuring-attachment-internalization-influenza-virus-a549-cells-flow
PubMed ID:26575457

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