On the expression, function and regulation of the murine Ifit family of antiviral RNA-binding proteins.

IFIT proteins are highly expressed as part of the cell-intrinsic immune response following viral infection. In humans, IFIT1 inhibits translation at the initiation stage by binding directly to the 5' terminus of foreign RNA, precluding the recruitment of the cap-binding translation initiation factor complex eIF4F. IFIT1 is highly specific for ‘non-self’ cap0 RNA, which lacks methylation on the first and second cap-proximal nucleotides, but at high concentrations may also restrict ‘self’ cap1 translation. Knock-out mouse models have been extensively used to study IFIT antiviral activity and, more recently, vaccines based on the antiviral activity of IFIT1 have been trialled for efficacy in mice. However, it is becoming clear that there are differences in murine and human IFIT function and regulation, which impacts the interpretation of these models. Mice lack a true orthologue of IFIT1 and instead the closely related Ifit1b has been duplicated twice, yielding three paralogues: Ifit1, Ifit1b and Ifit1c. Murine Ifit1, like human IFIT1, can bind to cap0 RNA and inhibit its translation, but lacks cap1 binding activity. Ifit1b and Ifit1c are closely related to Ifit1 and share many of the residues critical for RNA-binding, but their precise functions are unknown. In this thesis, the expression of the entire murine Ifit family was examined in different mouse cell lines, validating expression of Ifit1b and Ifit1c following interferon stimulation. The murine Ifit family was then recombinantly expressed and purified, allowing biochemical characterisation. It was discovered that Ifit1b, a previously uncharacterised protein, preferentially inhibited the translation of cap1 mRNA, while cap0 and cap2 mRNAs were inhibited to a much lesser extent. Specific cap1 binding allows Ifit1b to inhibit a proportion of cellular translation and block translation of murine hepatitis virus, a cap1 coronavirus. However, both Ifit1 and Ifit1b were incapable of inhibiting translation from a more structured Zika virus reporter mRNA. The same reporter was effectively inhibited by human IFIT1, highlighting a key difference between the activities of human and murine IFIT proteins. IFIT proteins are known to form both homo- and hetero-oligomers, but the functional significance of these interactions is unclear. Here, interaction between human IFIT1 and IFIT3 was shown to increase the stability of both proteins and stimulate translation inhibition by IFIT1. IFIT1 and IFIT3 interacted via a C-terminal YxxxL motif, and disruption of this motif in either protein abolished the cofactor activity of IFIT3, both in vitro and in cells. In mice, Ifit3 is truncated and lacks the YxxxL motif, thus precluding interaction with murine Ifit1. However, this motif is maintained in Ifit1, Ifit1b and Ifit1c, which were found to interact with one-another in vitro. Interaction between murine Ifit proteins increased their stability and Ifit1c enhanced translation inhibition by Ifit1 and Ifit1b in vitro, thereby acting analogously to human IFIT3. Together this work provides a better understanding of the function and regulation of murine Ifit proteins, to aid interpretation and improvement of mouse models in studying the role of IFIT proteins in the antiviral response.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2020

Author dc:creator

• Mears, Harriet Victoria

Advisor dc:contributor.advisor

• Sweeney, Trevor

• Innate immunity
• Interferon
• Antiviral response

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/ba6b9052-f95e-4018-a0b6-b80524d99c49/download
• https://creativecommons.org/licenses/by-nc-nd/4.0/

Language dc:language

en

Author Identifier

0000-0003-0300-0885

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/300608