Caveats of Using Overexpression Approaches to Screen Cellular Host IFITM Proteins for Antiviral Activity

Author:

Meischel Tina1ORCID,Fritzlar Svenja1ORCID,Villalón-Letelier Fernando1,Smith Jeffrey M.1,Brooks Andrew G.1,Reading Patrick C.12ORCID,Londrigan Sarah L.1ORCID

Affiliation:

1. Department of Microbiology and Immunology, University of Melbourne, The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St., Melbourne, VIC 3000, Australia

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

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.

Funder

National Health and Medical Research Council (NHMRC) of Australia

Publisher

MDPI AG

Subject

Infectious Diseases,Microbiology (medical),General Immunology and Microbiology,Molecular Biology,Immunology and Allergy

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