Analyzing Influenza Virus Internalization Using a Streptavidin Blocking Assay

Abstract

Source: Pohl, M. O. & Stertz, S. Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry. J. Vis. Exp. (2015)

This video demonstrates the use of a streptavidin blocking assay to distinguish surface-bound from internalized influenza virus in human lung epithelial cells. Comparing fluorescence signals shows that a higher intensity after incubation indicates successful viral internalization.

Protocol

1. Attachment/Internalization Assay

NOTE: Make sure to prepare all reagents before starting the assay. For the three control groups presented in the results section (mock-infected cells, neuraminidase pre-treated cells and sodium azide treated cells), small alterations to the protocol are required.

1. Prepare 16 deepwell tubes each containing 200,000 A549 cells. Next to the experimental set consisting of 4 tubes, there are 3 control conditions, each requiring 4 tubes: mock-infected cells, neuraminidase (NA)-pretreated cells and sodium azide-treated cells. Each group consists of 4 tubes labelled as ‘0 min’, ‘0 min + streptavidin (STV)’, ‘30 min’, ‘30 min + STV’.
2. Spin tubes at 4 °C for 3 min at 450 x g. Remove supernatant and resuspend pellet in 200 µl phosphate-buffered saline (PBS).
3. Spin tubes at 4 °C at 450 x g. Remove supernatant and resuspend pellet in 200 µl Dulbecco's modified Eagle medium (DMEM) containing 0.3% bovine serum albumin (BSA), 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 1% penicillin-streptomycin (incubation medium). Incubate for 30 min at 37 °C.
   NOTE: For the neuraminidase control: Use bacterial neuraminidase (e.g. sialidase from Vibrio cholera) at a concentration of 200 mU/ml diluted in incubation medium to resuspend the cells.
4. Spin tubes at 4 °C for 3 min at 450 x g. Remove supernatant and resuspend pellet in 200 µl PBS. Repeat this step one more time, then cool tubes down on ice for 10 min.
5. Spin tubes at 4 °C for 3 min at 450 x g. Remove supernatant and resuspend pellet in 100 µl biotinylated virus (use the virus concentration determined in section 2 diluted in infection-PBS). Incubate 1 hr on ice.
6. Spin tubes at 4 °C for 3 min at 450 x g. Remove supernatant and resuspend pellet in 200 µl PBS. Repeat this step one more time.
   NOTE: For the mock-infected control: Use infection-PBS without virus. For the sodium azide control: Use virus diluted in infection-PBS supplemented with 0.1 % sodium azide.
7. Processing of the samples after the infection on ice:
   1. For the ‘0 min’ samples, follow step 2.5 and store samples at 4 °C until the other samples are fixed.
   2. For the ‘0 min + STV’ samples, spin tubes at 4 °C for 3 min at 450 x g. Remove supernatant and resuspend pellets in 50 µl STV (use the concentration determined in section 3) diluted in PBS containing 2% BSA and 0.1% sodium azide (to prevent internalization while handling the sample). Incubate 30 min on ice.
   3. Spin tubes at 4 °C for 3 min at 450 x g. Remove supernatant and resuspend pellet in 200 µl PBS. Repeat this step one more time.
   4. For fixation, spin tubes at 4 °C for 3 min at 450 x g. Remove supernatant and resuspend pellet in 100 µl of 3.7% paraformaldehyde (PFA). Incubate for 10 min at RT. Then, repeat the wash step described in 1.6) and store samples at 4 °C.
   5. For the ’30 min’ samples, spin tubes at 4 °C for 3 min at 450 x g. Remove supernatant and resuspend pellet in 200 µl PBS containing 2% BSA. Incubate 30 min at 37 °C. Follow steps 1.7.3-1.7.4 and store samples at 4 °C.
      NOTE: For the sodium azide control: Use PBS supplemented with 2 % BSA and 0.1 % sodium azide for the 30 min incubation at 37 °C.
   6. For the ’30 min + STV’ samples spin tubes at 4 °C for 3 min at 450 x g. Remove supernatant and resuspend pellet in 200 µl PBS containing 2% BSA. Incubate 30 min at 37 °C. Then, spin tubes at 4 °C for 3 min at 450 x g. Remove supernatant and resuspend pellets in 50 µl STV (use the concentration determined in section 3) diluted in PBS containing 2% BSA and 0.1% sodium azide (to prevent internalization while handling the sample). Incubate 30 min on ice. Follow steps 1.7.3-1.7.4 and store samples at 4 °C.
      NOTE: For the sodium azide control: Use PBS supplemented with 2 % BSA and 0.1 % sodium azide for the 30 min incubation at 37 °C.
8. For permeabilization, spin tubes at 4 °C for 3 min at 450 x g. Remove supernatant and resuspend pellet in 100 µl PBS containing 0.5% Triton X-100. Incubate for 6 min at RT. Then, repeat the wash step described in 1.6).
9. Spin tubes at 4 °C for 3 min at 450 x g. Remove supernatant and resuspend pellet in 50 µl 2% BSA diluted in PBS containing STV-Cy3. (Please note, the appropriate concentration of STV-Cy3 should be determined before starting the experiment. Start with a concentration of 1:200 and test 2-fold dilutions up to 1:2,400. In our hands, a dilution of 1:1,000 proved to be optimal.)
10. Incubate 1 hr in the dark at RT. Then, repeat the washing step described in 1.6 but resuspend the pellet in 200 µl 2% BSA diluted in PBS.
11. Analyze samples by flow cytometry. Determine the percentage of Cy3-positive cells in each sample.

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
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