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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| Format: | Article |
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MDPI AG
2023-03-01
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| Series: | Pathogens |
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| Online Access: | https://www.mdpi.com/2076-0817/12/4/519 |
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| author | Tina Meischel Svenja Fritzlar Fernando Villalón-Letelier Jeffrey M. Smith Andrew G. Brooks Patrick C. Reading Sarah L. Londrigan |
| author_facet | Tina Meischel Svenja Fritzlar Fernando Villalón-Letelier Jeffrey M. Smith Andrew G. Brooks Patrick C. Reading Sarah L. Londrigan |
| author_sort | Tina Meischel |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-11T04:39:49Z |
| format | Article |
| id | doaj.art-e3fc0f0b7607426fa04e535f76ee790e |
| institution | Directory Open Access Journal |
| issn | 2076-0817 |
| language | English |
| last_indexed | 2024-03-11T04:39:49Z |
| publishDate | 2023-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Pathogens |
| spelling | doaj.art-e3fc0f0b7607426fa04e535f76ee790e2023-11-17T20:47:53ZengMDPI AGPathogens2076-08172023-03-0112451910.3390/pathogens12040519Caveats of Using Overexpression Approaches to Screen Cellular Host IFITM Proteins for Antiviral ActivityTina Meischel0Svenja Fritzlar1Fernando Villalón-Letelier2Jeffrey M. Smith3Andrew G. Brooks4Patrick C. Reading5Sarah L. Londrigan6Department of Microbiology and Immunology, University of Melbourne, The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St., Melbourne, VIC 3000, AustraliaDepartment of Microbiology and Immunology, University of Melbourne, The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St., Melbourne, VIC 3000, AustraliaDepartment of Microbiology and Immunology, University of Melbourne, The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St., Melbourne, VIC 3000, AustraliaDepartment of Microbiology and Immunology, University of Melbourne, The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St., Melbourne, VIC 3000, AustraliaDepartment of Microbiology and Immunology, University of Melbourne, The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St., Melbourne, VIC 3000, AustraliaDepartment of Microbiology and Immunology, University of Melbourne, The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St., Melbourne, VIC 3000, AustraliaDepartment of Microbiology and Immunology, University of Melbourne, The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St., Melbourne, VIC 3000, AustraliaEctopic 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.https://www.mdpi.com/2076-0817/12/4/519respiratory virusesinnate immunityIFITM proteinshost antiviral factorsinfluenza A virusparainfluenza virus |
| spellingShingle | Tina Meischel Svenja Fritzlar Fernando Villalón-Letelier Jeffrey M. Smith Andrew G. Brooks Patrick C. Reading Sarah L. Londrigan Caveats of Using Overexpression Approaches to Screen Cellular Host IFITM Proteins for Antiviral Activity Pathogens respiratory viruses innate immunity IFITM proteins host antiviral factors influenza A virus parainfluenza virus |
| title | Caveats of Using Overexpression Approaches to Screen Cellular Host IFITM Proteins for Antiviral Activity |
| title_full | Caveats of Using Overexpression Approaches to Screen Cellular Host IFITM Proteins for Antiviral Activity |
| title_fullStr | Caveats of Using Overexpression Approaches to Screen Cellular Host IFITM Proteins for Antiviral Activity |
| title_full_unstemmed | Caveats of Using Overexpression Approaches to Screen Cellular Host IFITM Proteins for Antiviral Activity |
| title_short | Caveats of Using Overexpression Approaches to Screen Cellular Host IFITM Proteins for Antiviral Activity |
| title_sort | caveats of using overexpression approaches to screen cellular host ifitm proteins for antiviral activity |
| topic | respiratory viruses innate immunity IFITM proteins host antiviral factors influenza A virus parainfluenza virus |
| url | https://www.mdpi.com/2076-0817/12/4/519 |
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