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The Influenza A Virus M2 Protein -Complementation System Offers a Set of Tools for the Undergraduate Virology Laboratory

    Authors: Michael L. Grantham1,*, Matthew F. McCown2, Andrew Pekosz3
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    Affiliations: 1: Department of Biology, Missouri Western State University, Saint Joseph, MO 64507; 2: Monsanto Company, Chesterfield, MO 63017; 3: W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205
    AUTHOR AND ARTICLE INFORMATION AUTHOR AND ARTICLE INFORMATION
    • Received 04 August 2018 Accepted 04 December 2018 Published 26 April 2019
    • ©2019 Author(s). Published by the American Society for Microbiology
    • [open-access] This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial-NoDerivatives 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/ and https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode), which grants the public the nonexclusive right to copy, distribute, or display the published work.

    • Supplemental materials available at http://asmscience.org/jmbe
    • *Corresponding author. Mailing address: Department of Biology, Missouri Western State University, 4525 Downs Drive, Saint Joseph, MO 64507. Phone: 816-271-5603. E-mail: [email protected].
    Source: J. Microbiol. Biol. Educ. April 2019 vol. 20 no. 1 doi:10.1128/jmbe.v20i1.1667
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    Abstract:

    An authentic, hands-on experience in the laboratory is an important part of any undergraduate biology course. However, there are a limited number of mammalian virus systems that students can work with safely in an undergraduate teaching laboratory. For many systems, the risk to the students is too high. The influenza A virus M2 protein -complementation system bridges this gap. This system consists of a virus with mutations that prevent the expression of the essential M2 protein; therefore this virus can only replicate in a cell line that provides M2 in . Here, we describe the use of this system to carry out hemagglutination, real-time reverse transcriptase PCR, 50% tissue culture infectious dose, and plaque assays in an undergraduate lab setting.

References & Citations

1. McCown MF, Pekosz A 2006 Distinct domains of the influenza A virus M2 protein cytoplasmic tail mediate binding to the M1 protein and facilitate infectious virus production J Virol 80 8178 10.1128/JVI.00627-06 16873274 1563831 http://dx.doi.org/10.1128/JVI.00627-06
2. Takeda M, Pekosz A, Shuck K, Pinto LH, Lamb RA 2002 Influenza A virus M2 ion channel activity is essential for efficient replication in tissue culture J Virol 76 1391 1399 10.1128/JVI.76.3.1391-1399.2002 11773413 135863 http://dx.doi.org/10.1128/JVI.76.3.1391-1399.2002
3. Iwatsuki-Horimoto K, Horimoto T, Noda T, Kiso M, Maeda J, Watanabe S, Muramoto Y, Fujii K, Kawaoka Y 2006 The cytoplasmic tail of the influenza A virus M2 protein plays a role in viral assembly J Virol 80 5233 5240 10.1128/JVI.00049-06 16699003 1472145 http://dx.doi.org/10.1128/JVI.00049-06
4. McCown MF, Pekosz A 2006 Distinct domains of the influenza A virus M2 protein cytoplasmic tail mediate binding to the M1 protein and facilitate infectious virus production J Virol 80 8178 8189 10.1128/JVI.00627-06 16873274 1563831 http://dx.doi.org/10.1128/JVI.00627-06
5. McCown MF, Pekosz A 2005 The influenza A virus M2 cytoplasmic tail is required for infectious virus production and efficient genome packaging J Virol 79 3595 10.1128/JVI.79.6.3595-3605.2005 15731254 1075690 http://dx.doi.org/10.1128/JVI.79.6.3595-3605.2005
6. Grantham ML, Stewart SM, Lalime EN, Pekosz A 2010 Tyrosines in the influenza A virus M2 protein cytoplasmic tail are critical for production of infectious virus particles J Virol 84 8765 8776 10.1128/JVI.00853-10 20573832 2918991 http://dx.doi.org/10.1128/JVI.00853-10
7. Grantham ML, Wu WH, Lalime EN, Lorenzo ME, Klein SL, Pekosz A 2009 Palmitoylation of the influenza A virus M2 protein is not required for virus replication in vitro but contributes to virus virulence J Virol 83 8655 8661 10.1128/JVI.01129-09 19553312 2738213 http://dx.doi.org/10.1128/JVI.01129-09
8. Stewart SM, Pekosz A 2011 Mutations in the membrane-proximal region of the influenza A virus M2 protein cytoplasmic tail have modest effects on virus replication J Virol 85 12179 12187 10.1128/JVI.05970-11 21917980 3209349 http://dx.doi.org/10.1128/JVI.05970-11
9. Liu H, Grantham ML, Pekosz A 2018 Mutations in the influenza A virus M1 protein enhance virus budding to complement lethal mutations in the M2 cytoplasmic tail J Virol 92 e00858 17
10. World Health Organization 2009 CDC protocol of realtime RTPCR for influenza A (H1N1) WHO Collaborating Centre for influenza at CDC Atlanta, GA
11. Reed LJ, Muench H 1938 A simple method of estimating fifty per cent endpoints Am J Epidemiol 27 493 497 10.1093/oxfordjournals.aje.a118408 http://dx.doi.org/10.1093/oxfordjournals.aje.a118408
12. Ito T, Suzuki Y, Mitnaul L, Vines A, Kida H, Kawaoka Y 1997 Receptor specificity of influenza A viruses correlates with the agglutination of erythrocytes from different animal species Virology 227 2 493 499 10.1006/viro.1996.8323 9018149 http://dx.doi.org/10.1006/viro.1996.8323

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2019-04-26
2019-08-25

Abstract:

An authentic, hands-on experience in the laboratory is an important part of any undergraduate biology course. However, there are a limited number of mammalian virus systems that students can work with safely in an undergraduate teaching laboratory. For many systems, the risk to the students is too high. The influenza A virus M2 protein -complementation system bridges this gap. This system consists of a virus with mutations that prevent the expression of the essential M2 protein; therefore this virus can only replicate in a cell line that provides M2 in . Here, we describe the use of this system to carry out hemagglutination, real-time reverse transcriptase PCR, 50% tissue culture infectious dose, and plaque assays in an undergraduate lab setting.

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FIGURE 1

A) A hemagglutination assay was carried out using either a virus stock (rUdorn-M2Stop) or phosphate-buffered saline (negative control). Positive wells are those that show either a circle or button of cells in the bottom of the well. B) Students carried out real-time RT-PCR assays using either a virus stock (dashed lines) or a DNA standard plasmid (solid lines). C) A TCID was carried out using rUdorn-M2Stop. Positive wells are those that show no staining or non-uniform staining. D) A plaque assay was carried out using rUdorn-M2Stop. The well shown is from the 10 dilution of this particular stock of virus, and plaques can be seen as unstained areas in the well.

Source: J. Microbiol. Biol. Educ. April 2019 vol. 20 no. 1 doi:10.1128/jmbe.v20i1.1667
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