An Enzymatic Assay for Detection of Viral Entry

Donna M. Tscherne1, Adolfo García‐Sastre2

1 Department of Microbiology, Mount Sinai School of Medicine, New York, New York, 2 Global Health and Emerging Pathogens Institute, Mount Sinai School of Medicine, New York, New York
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 26.12
DOI:  10.1002/0471143030.cb2612s51
Online Posting Date:  June, 2011
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Abstract

This unit describes a viral entry assay where a beta‐lactamase reporter protein fused to the matrix protein of either influenza (BlaM1) or ebola virus (BlaVP40) is packaged as a structural component into virus‐like particles (VLPs). The Bla reporter is released upon fusion with target cells and can be detected in live cells by flow cytometry, microscopy, or a fluorometric plate reader for utility in high‐throughput screening approaches. The transfer of Bla to a target cell by BlaM1 or BlaVP40 VLPs requires the presence of influenza hemagglutinin (HA) and neuraminidase (NA) or EboV glycoprotein (GP), respectively. This straightforward assay has broad application for studying the entry steps of enveloped viruses, especially those that require high levels of biosafety containment. Curr. Protoc. Cell Biol. 51:26.12.1‐26.12.10. © 2011 by John Wiley & Sons, Inc.

Keywords: virus entry; influenza virus; ebola virus; Marburg virus; flow cytometry; microscopy

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • 293T cells
  • Cell culture medium (e.g., DMEM containing 10% FBS)
  • Phosphate‐buffered saline (PBS; Invitrogen, cat. no. 20012‐050)
  • OptiMEM I Reduced Serum Medium (Invitrogen)
  • Lipofectamine 2000 transfection reagent (Invitrogen)
  • Plasmids encoding BlaM1 or BlaVP40 under control of a eukaryotic promoter
  • Plasmids encoding the glycoprotein of interest (e.g., influenza HA and NA, EboV GP, MarV GP, VSV‐G) under control of a eukaryotic promoter
  • TPCK‐treated trypsin (Sigma): for stock solution, dissolve in water to a final concentration of 1 mg/ml
  • Soybean trypsin inhibitor (Sigma): for stock solution, dissolve in PBS to a final concentration of 10 mg/ml
  • Chicken red blood cells: 0.5% suspension in PBS
  • Target cells [(e.g., Madin Darby canine kidney cells (MDCK), A549 cells] cultured in 24‐well dishes or coverslip suitable for microscopy
  • Trypsin/EDTA
  • CCF2‐AM reagent and loading kit (see recipes)
  • Bovine serum albumin (BSA)
  • 6‐well plates
  • 37°C incubator
  • Centrifuge
  • V‐bottom 96‐well plates (Fisher Scientific)
  • Flow cytometer equipped with a violet laser (e.g., LSRIII; Becton Dickinson) or inverted confocal microscope (e.g., Leica SP5 DMI; Leica Microsystems)
NOTE: The BlaM1 and BlaVP40 constructs should lack the first 24 amino acids of the full‐length Bla protein in order to remove a bacterial secretion signal. The construct should express the modified Bla, followed by a short, glycine‐rich linker sequence, and M1 or VP40.
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Figures

Videos

Literature Cited

Literature Cited
   Manicassamy, B. and Rong, L. 2009. Expression of ebola virus glycoprotein on the target cells enhances viral entry. J. Virol. 6:75.
   Tscherne, D.M., Manicassamy, B., and García‐Sastre, A. 2009. An enzymatic virus‐like particle assay for sensitive detection of virus entry. J. Virol. Methods 163:336‐343.
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