Caveats of Using Overexpression Approaches to Screen Cellular Host IFITM Proteins for Antiviral Activity
Ectopic protein overexpression in immortalised cell lines is a commonly used method to screen host factors for their antiviral activity against different viruses. However, the question remains as to what extent such artificial protein overexpression recapitulates endogenous protein function. Previou...
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Published in | Pathogens (Basel) Vol. 12; no. 4; p. 519 |
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Main Authors | , , , , , , |
Format | Journal Article |
Language | English |
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27.03.2023
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Abstract | Ectopic protein overexpression in immortalised cell lines is a commonly used method to screen host factors for their antiviral activity against different viruses. However, the question remains as to what extent such artificial protein overexpression recapitulates endogenous protein function. Previously, we used a doxycycline-inducible overexpression system, in conjunction with approaches to modulate the expression of endogenous protein, to demonstrate the antiviral activity of IFITM1, IFITM2, and IFITM3 against influenza A virus (IAV) but not parainfluenza virus-3 (PIV-3) in A549 cells. We now show that constitutive overexpression of the same IFITM constructs in A549 cells led to a significant restriction of PIV-3 infection by all three IFITM proteins. Variable IFITM mRNA and protein expression levels were detected in A549 cells with constitutive versus inducible overexpression of each IFITM. Our findings show that overexpression approaches can lead to levels of IFITM1, IFITM2, and IFITM3 that significantly exceed those achieved through interferon stimulation of endogenous protein. We propose that exceedingly high levels of overexpressed IFITMs may not accurately reflect the true function of endogenous protein, thus contributing to discrepancies when attributing the antiviral activity of individual IFITM proteins against different viruses. Our findings clearly highlight the caveats associated with overexpression approaches used to screen cellular host proteins for antiviral activity. |
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AbstractList | Ectopic protein overexpression in immortalised cell lines is a commonly used method to screen host factors for their antiviral activity against different viruses. However, the question remains as to what extent such artificial protein overexpression recapitulates endogenous protein function. Previously, we used a doxycycline-inducible overexpression system, in conjunction with approaches to modulate the expression of endogenous protein, to demonstrate the antiviral activity of IFITM1, IFITM2, and IFITM3 against influenza A virus (IAV) but not parainfluenza virus-3 (PIV-3) in A549 cells. We now show that constitutive overexpression of the same IFITM constructs in A549 cells led to a significant restriction of PIV-3 infection by all three IFITM proteins. Variable IFITM mRNA and protein expression levels were detected in A549 cells with constitutive versus inducible overexpression of each IFITM. Our findings show that overexpression approaches can lead to levels of IFITM1, IFITM2, and IFITM3 that significantly exceed those achieved through interferon stimulation of endogenous protein. We propose that exceedingly high levels of overexpressed IFITMs may not accurately reflect the true function of endogenous protein, thus contributing to discrepancies when attributing the antiviral activity of individual IFITM proteins against different viruses. Our findings clearly highlight the caveats associated with overexpression approaches used to screen cellular host proteins for antiviral activity. |
Audience | Academic |
Author | Meischel, Tina Villalón-Letelier, Fernando Smith, Jeffrey M Brooks, Andrew G Fritzlar, Svenja Londrigan, Sarah L Reading, Patrick C |
AuthorAffiliation | 2 WHO Collaborating Centre for Reference and Research on Influenza, Victorian Infectious Diseases Reference Laboratory, The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St., Melbourne, VIC 3000, Australia 1 Department of Microbiology and Immunology, University of Melbourne, The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St., Melbourne, VIC 3000, Australia |
AuthorAffiliation_xml | – name: 1 Department of Microbiology and Immunology, University of Melbourne, The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St., Melbourne, VIC 3000, Australia – name: 2 WHO Collaborating Centre for Reference and Research on Influenza, Victorian Infectious Diseases Reference Laboratory, The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St., Melbourne, VIC 3000, Australia |
Author_xml | – sequence: 1 givenname: Tina orcidid: 0000-0002-0911-6574 surname: Meischel fullname: Meischel, Tina organization: Department of Microbiology and Immunology, University of Melbourne, The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St., Melbourne, VIC 3000, Australia – sequence: 2 givenname: Svenja orcidid: 0000-0003-3407-1758 surname: Fritzlar fullname: Fritzlar, Svenja organization: Department of Microbiology and Immunology, University of Melbourne, The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St., Melbourne, VIC 3000, Australia – sequence: 3 givenname: Fernando surname: Villalón-Letelier fullname: Villalón-Letelier, Fernando organization: Department of Microbiology and Immunology, University of Melbourne, The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St., Melbourne, VIC 3000, Australia – sequence: 4 givenname: Jeffrey M surname: Smith fullname: Smith, Jeffrey M organization: Department of Microbiology and Immunology, University of Melbourne, The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St., Melbourne, VIC 3000, Australia – sequence: 5 givenname: Andrew G surname: Brooks fullname: Brooks, Andrew G organization: Department of Microbiology and Immunology, University of Melbourne, The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St., Melbourne, VIC 3000, Australia – sequence: 6 givenname: Patrick C orcidid: 0000-0002-8860-5308 surname: Reading fullname: Reading, Patrick C organization: WHO Collaborating Centre for Reference and Research on Influenza, Victorian Infectious Diseases Reference Laboratory, The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St., Melbourne, VIC 3000, Australia – sequence: 7 givenname: Sarah L orcidid: 0000-0003-0989-4971 surname: Londrigan fullname: Londrigan, Sarah L organization: Department of Microbiology and Immunology, University of Melbourne, The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St., Melbourne, VIC 3000, Australia |
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Keywords | host antiviral factors IFITM proteins innate immunity parainfluenza virus respiratory viruses influenza A virus |
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SubjectTerms | Antibodies Antiviral activity Antiviral agents Cell lines Cells Cloning Dengue fever Doxycycline Eggs Gene expression host antiviral factors IFITM proteins Infections Influenza A influenza A virus innate immunity Interferon Membrane proteins mRNA Parainfluenza parainfluenza virus Plasmids Protein expression Proteins respiratory viruses Software Testing Viral infections Viruses West Nile virus |
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Title | Caveats of Using Overexpression Approaches to Screen Cellular Host IFITM Proteins for Antiviral Activity |
URI | https://www.ncbi.nlm.nih.gov/pubmed/37111405 https://www.proquest.com/docview/2806594256 https://search.proquest.com/docview/2807919769 https://pubmed.ncbi.nlm.nih.gov/PMC10145288 https://doaj.org/article/e3fc0f0b7607426fa04e535f76ee790e |
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