Subcellular Barriers to Viral Infection and Gene Delivery

Jefferson, Matthew (2020) Subcellular Barriers to Viral Infection and Gene Delivery. Doctoral thesis, University of East Anglia.

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Abstract

Viruses are obligate intracellular parasites that survive by gaining entry into cells and suppressing subsequent antiviral responses. Pestiviruses encode a single polyprotein which is processed by proteases to produce 12 major viral proteins. The N-terminal protease, Npro cleaves itself from the N-terminus of the polyprotein and targets IRF3 for degradation to supress interferon signalling. This study shows that Npro also inhibits apoptosis by inhibiting the IRF3-dependent Bax pathway and uses mitochondria and peroxisomes as sites for IRF3 regulation. Mass spectrometry was used to identify proteins that interact with Npro. Of the 55 proteins identified most were ribosomal proteins and ribonucleoproteins including RHA, YBX1, DDX3, DDX5, eIF3, IGF2BP1, IEBP3, and PABP-1. These are components of the translation machinery, ribonucleoprotein particles (RNPs), and stress granules (SGs) which can halt translation and prevent translation of viral genomes.

Sindbis virus (SINV) infection induces eIF2α phosphorylation, which leads to SG assembly. SINV infection also stimulates autophagy. We showed that the autophagy protein ATG16L1 not only regulates eIF2α phosphorylation and the translation of viral and antiviral proteins, but also controls SG assembly. SINV capsid was recruited by ribonuclear proteins TIA1, YBX1, and VCP, to a single perinuclear body, which co-localised with protein-kinase R (PKR) and RIG-I. Loss of ATG16L1 inhibited SG assembly and the capsid remained in small foci containing YBX1, TIA1 with RIG-I. It is possible that ATG16L1 is required for SG assembly, and for attenuating the innate immune response.

A non-canonical autophagy pathway called LC3-associated phagocytosis (LAP) targets LC3 to phagosomes and endosome membranes during uptake of pathogens. LAP is dependent on the WD domain of ATG16L1 and this study has used a mouse lacking the WD domain (δWD) to investigate the roles played by LAP during influenza virus infection in vivo. The growth, fertility and tissue homeostasis of δWD mice were comparable with wild type mice, but δWD mice were extremely sensitive to IAV resulting in fulminant pneumonia, lung inflammation and high mortality rates. A conditional mouse model (δWDphag) where δWD was expressed solely in myeloid cells showed that protection against IAV infection of lung was independent of phagocytes. This establishes non-canonical autophagy pathways in non-phagocytes such as epithelial cells as a novel innate defence mechanism that can restrict IAV infection.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Medicine and Health Sciences > Norwich Medical School
Depositing User:	Chris White
Date Deposited:	10 Nov 2021 10:45
Last Modified:	10 Nov 2021 10:45
URI:	https://ueaeprints.uea.ac.uk/id/eprint/82044
DOI:

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