A genome wide screening approach for investigating the interplay between host defence and parainfluenza viruses

  • Chloe Eleanor Jones

Student thesis: Doctoral Thesis (PhD)

Abstract

Viruses are obligate intracellular pathogens, so virus-host interactions are crucial to their infectivity. A successful infection is a trade-off between the virus’s utilisation of host dependency factors that permit virus replication, and its ability to overcome the host innate immune response. Broadening our understanding of what permits or restricts replication may provide us with novel targets for antiviral therapies.

Human parainfluenza virus type 3 (hPIV3) is a leading cause of pneumonia and hospitalisation of children under 5 years worldwide. Using a genome wide CRISPR knockout screen, targeting approximately 18,000 individual genes, we identified host factors that facilitate hPIV3 replication, including solute carrier family 35 member A1 (SLC35A1), a sialic acid transporter, alongside antiviral factors. Infection of independent gene knockout cell lines validated a role of three genes important for the antiviral response: Merlin (NF2), Hydroxysteroid 17-Beta Dehydrogenase 12 (HSD1712), and Zinc Finger CCCH-Type Containing, Antiviral 1 (also known as zinc finger antiviral protein, ZAP). Using flow cytometry and RT-qPCR, we showed that IFN-treated knockout cells were permissive to hPIV3 infection when compared to control cells. We also showed, by RT-qPCR, that all three hits regulated IFN induction. Importantly, NF2 also regulated IFN signalling; a novel phenotype not previously described. Using plaque assays, we demonstrated that all three hits restricted the replication of other paramyxo- and pneumoviruses, revealing their broad antiviral activity.

However, the IFN response is a vast response with significant redundancy and where several interferon stimulated genes (ISGs), that individually have low activity, culminate to restrict infection. Current screening methods rely on strong phenotypes, leading to low power in elucidating antiviral restriction factors. To overcome this, we carried out proof-of-principle studies, using an established ISG15-deficient cell line and model Paramyxovirus, PIV5, to enhance signal-to-noise and expand the quantitative working window required for the discovery of low acting ISGs. Building on this, future work will provide a broad overview of the host antiviral response that could inform the development of new therapeutics.
Date of Award28 Nov 2023
Original languageEnglish
Awarding Institution
  • University of St Andrews
SupervisorDavid John Hughes (Supervisor)

Keywords

  • Innate immunity
  • CRISPR/Cas9
  • Genome-wide screens
  • Antiviral immunity
  • Interferon
  • Paramyxovirus

Access Status

  • Full text embargoed until
  • 1 November 2024

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