Date of Award
Doctor of Philosophy (PhD)
Genes required for the lysosomal degradation pathway of autophagy play key roles in topologically distinct cellular processes with significant physiologic importance. One of the first-described of these ATG gene-dependent processes is the requirement for a subset of ATG genes in interferon-γ (IFNγ)-induced inhibition of norovirus and Toxoplasma gondii replication. In this dissertation we identified novel components that are required for or that negatively regulate this immune mechanism. Enzymes involved in the conjugation of UFM1 to target proteins including UFC1 and UBA5, negatively regulated IFNγ-induced inhibition of norovirus replication via effects of Ern1. We identified and confirmed that IFNγ-induced inhibition of norovirus replication required Wipi2b, Atg9a and Gabarapl2 while Becn1, Atg14 and Sqstm1 were not important. The phosphatidylinositol-3-phosphate and ATG16L1 binding domains of WIPI2B were required for IFNγ-induced inhibition of norovirus replication. WIPI2 was also required for IFNγ-induced inhibition of Toxoplasma gondii replication in HeLa cells. Moreover, we utilized an unbiased genome wide CRISPR-Cas9 screening approach to identify additional novel components of this immune mechanism. We identified 164 candidate genes regulating this immune process, Uba5 among them. We identified 51 candidate genes required for IFNγ-induced control of norovirus and significant enrichment of the Cul3 ubiquitin-ligase network. We confirmed Cul3 and the cullin-RING ligase substrate adaptor Klhl9 were required for IFNγ-induced control of norovirus. Cul3, Klhl9 and ubiquitin localized to norovirus replication complexes containing the viral protease-polymerase protein in infected cells. This work further delineates the mechanisms of a programmable form of cytokine-induced intracellular immunity that relies on an expanding cassette of essential ATG genes and additional novel genes pertaining to the ubiquitin pathway to restrict the growth of phylogenetically diverse pathogens.
Chair and Committee
David A. Hunstad Herbert Virgin
Megan Baldridge, Haina Shin, Christina Stallings,
Mcallaster, Michael, "Investigation of IFNγ-induced Control of Intracellular Pathogens" (2021). Arts & Sciences Electronic Theses and Dissertations. 2514.