The impact of interferon-stimulated genes in the pathobiology of Influenza A viruses

Gardiner, Emma Louise and Munir, Muhammad and Unterholzner, Leonie (2025) The impact of interferon-stimulated genes in the pathobiology of Influenza A viruses. PhD thesis, Lancaster University.

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Abstract

Innate immunity defines the first line of defence against viral infection and is mainly attributed to cytokines, chemokines, interferons, and interferon-stimulated and regulated genes. Amongst myriads of interferons-stimulated genes, interferon-inducible transmembrane (IFITM) proteins are responsive to virus-induced transcriptional activation and display dynamic roles in regulating viruses with diverse genetic backgrounds. IFITM proteins are genetically and functionally characterised in humans against several medically important viruses, including influenza; however, the nature and functional insights into the roles of IFITM proteins in chickens remain less characterised. In this thesis, a range of approaches were applied to investigate the roles of chicken IFITM proteins against avian influenza viruses, ranging from stable and transient expression, cellular distributions, in vivo investigation, and structural and mechanistic insights into different domains of IFITM proteins, which form the basis of the antiviral potential of IFITM proteins. Using transcriptomic datasets, it was identified that chicken IFITM genes are profoundly activated in chickens in response to influenza viral infections. Next, deploying transient expression system we mapped the cellular distribution of chIFITM1 and chIFITM3 in chicken fibroblasts (DF-1 cells) and concluded functionally important residues which retain IFITM proteins in the plasma membrane. Additionally, residues that define the antiviral roles of IFITM proteins (IFITM1 and IFITM3) against influenza viruses were mapped. Extensive mutation and deletion mapping catalogued the C-terminus amphipathic helix and hydrophobic domain residues, which defined the antiviral roles of IFITM1 and IFITM3 against influenza viruses. Next, efforts were made to establish a basis for structural modelling and functional characterisation of chicken IFITM1 and IFITM3. Finally, by applying an avian sarcoma-leucosis virus (RCAS)-based gene transfer system, transgenic chickens constitutively expressing IFITM3 showed resistance against highly pathogenic avian influenza virus (HPAIV) subtype H5N1. These findings establish solid foundations to characterise chIFITM1 and chIFITM3 in regulating influenza viruses both in vitro and in vivo.