Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

Summary

We present a protocol describing a semi-quantitative method for measuring both, the attachment of influenza A virus to A549 cells, as well as the internalization of virus particles into the target cells by flow cytometry.

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.

Introduction

The entry of influenza A virus (IAV) is a multi-step process that starts with the binding of the virus to receptors on the plasma membrane of target cells ¹. The receptor for IAV is sialic acid which is present on a large variety of glycoproteins and glycolipids. The hemagglutinin (HA) protein of IAV which is present in the viral envelope binds to sialic acid and thereby mediates attachment of the viral particles to the plasma membrane of target cells ². The virus enters the cells via clathrin-mediated endocytosis but also alternative entry pathways, such as macropinocytosis, have been described ^3-6. The interaction between HA and sial....

Protocol

Note before start: Use laminar flow hood and appropriate biocontainment when working with live virus. Here, we describe growth conditions suitable to culture influenza virus strain A/WSN/33. Multiplicity of infection (MOI) and incubation times may vary depending on virus strain used.

1. Preparation of Biotinylated A/WSN/33 Virus

1. Seed Madin-Darby canine kindey (MDCK) cells into a T175 cm² flask using Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% penicillin-streptomycin (Pen/Strep). Culture cells at 37 °C until approximately 70% confluency is reached.
2. Before infection, rem....

Results

A cartoon describing the four different experimental conditions is shown in Figure 1. Results of a representative experiment are presented in Figures 2-5. In the “0 min” sample, biotinylated virus is cold-bound to target cells which can be visualized by STV-Cy3 staining (Figures 1 and 2). When a blocking step (in the “0 min + STV” sample) is applied, virus at the cell surface can no longer be detected by STV-Cy3 staining. As a result, th.......

Discussion

Our protocol describes an easy way of measuring virus attachment and internalization by flow cytometry. It allows the use of labeled wildtype virus which mimics more closely virus infections compared to the use of virus-like particles (VLP). While our protocol has been optimized to measure attachment and internalization of IAV it can easily be adapted for other viruses. In addition, as flow cytometry is used for readout, co-stainings can be easily added to the protocol e.g. to test expression levels following kn.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Life Technologies	41966-052
FBS	Life Technologies	10270-106
Penicillin-Streptomycin	Life Technologies	15140-163
PBS	Life Technologies	14190-169
BSA	VWR Calbiochem	126579
HEPES	Life Technologies	15630-100
D-Sucrose	Fluka	84100
TRIS	Biosolve BV	20092391
EDTA	Sigma-Aldrich	3680
EZ-LINK NHS-SS-BIOTIN kit	Fisher Scientific	W9971E
Bio Rad Protein Bio Assay	Bio Rad	500-0006
deepwell tubes (1.2 ml microtubes)	Milian	82 00 001
PFA	Lucerna chem  Electron microscopy sciences	15710
ultracentrifuge tubes	Hemotec HmbH	253070
Triton X-100	Fluka	93420
STV-Cy3	Life Technologies	43-4315
STV	Life Technologies	43-4302
sodium azide	Fluka	71290
bacterial neuraminidase/sialidase	Sigma-Aldrich	N6514-1UN