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Authors: Lum, Krystal Kar-Yan
Advisors: Cristea, Ileana M
Contributors: Molecular Biology Department
Keywords: cGAS
Innate immunity
Mass spectrometry
Subjects: Molecular biology
Issue Date: 2019
Publisher: Princeton, NJ : Princeton University
Abstract: The mammalian immune system is equipped with an impressive array of intrinsic and innate components that serve to clear pathogen infections by recognizing pathogen-associated molecular patterns. Among this group of cellular defense factors are receptors that bind directly to pathogen-derived DNA, including viral DNA genomes. This binding event engages shared and distinct immune and cell-homeostatic signaling cascades, which culminate in the generation of pro-inflammatory cytokines, suppression of pathogen replication and spread, and regulation of cell death pathways. Of these DNA sensors, the interferon-inducible protein 16 (IFI16) is the first to be characterized as functioning within the nucleus. IFI16 has since emerged as a prominent sensor against nuclear-replicating DNA viruses, including herpes simplex virus-1 (HSV-1). Despite its identified contributions to inducing antiviral cytokines and suppressing virus gene expression, the mechanisms through which IFI16 initiates these functions remain to be fully understood. More recently, the cyclic GMP-AMP synthase (cGAS) was identified as a prominent cytoplasmic DNA sensor, including upon HSV-1 infection. However, how cGAS is homeostatically maintained or regulated upon its activation remains unclear. This study explores the regulation of the nuclear DNA sensor IFI16 and the cytoplasmic DNA sensor cGAS, at the levels of protein-intrinsic properties and intramolecular protein interactions. To accomplish this, we used an integrative approach that combined proteomics with virology, biochemistry, microscopy, and classical molecular biology. Altogether, we uncovered intrinsic properties that govern IFI16 oligomerization and interactions with antiviral proteins in primary human fibroblasts. Our findings suggest that IFI16 oligomerization serves as a signal amplification platform to in-part activate cytokine expression and to recruit transcriptional regulatory proteins that suppress HSV-1 replication. To explore cGAS regulation, we defined cytoplasmic cGAS protein interactions upon HSV-1 infection. We placed these interactions in the context of temporal proteome changes induced by infection. We established that cGAS associates with the 2’-5’-oligoadenylate synthase-like protein OASL in resting cells and upon various contexts of nuclear and cytoplasmic DNA stimulation. We observed that OASL inhibited cGAS-mediated interferon responses. This finding provides an explanation for how cGAS may be inactively maintained in cellular homeostasis, with OASL functioning as a negative feedback loop for cytokine induction.
Alternate format: The Mudd Manuscript Library retains one bound copy of each dissertation. Search for these copies in the library's main catalog:
Type of Material: Academic dissertations (Ph.D.)
Language: en
Appears in Collections:Molecular Biology

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