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|Title:||Proteomic characterizations of cGAS and the virus microenvironment to decipher innate immune regulation|
|Advisors:||Cristea, Ileana M|
|Contributors:||Molecular Biology Department|
|Keywords:||Herpes virus infection|
Innate immune signaling
Post-translational modification (PTM)
|Publisher:||Princeton, NJ : Princeton University|
|Abstract:||The ability of mammalian cells to detect invading pathogens and engage in intra- and inter-cellular host defense signaling cascades is at the core of determining the outcome of infection. Cellular intrinsic and innate immune responses rely on germline-encoded pattern-recognition receptors (PRR) that defend against pathogens by recognizing pathogen-associated molecular patterns (PAMP), thereby stimulating expression of pro-inflammatory cytokines and interferon-induced proteins. This study integrates molecular virology with proteomics to examine intra- and intercellular host responses to viral infection, including PRR signaling and viral countermeasures within the local infected microenvironment. Cyclic GMP-AMP synthase (cGAS) is a PRR that detects pathogenic DNA from DNA viruses including herpesviruses, and synthesizes cyclic GMP-AMP, which activates the STING-TBK1-IRF3 axis to induce cytokines and apoptosis. While this pathway is well-established, how cGAS is homeostatically maintained or regulated upon infection is less clear. We defined cGAS protein-protein interactions upon herpes simplex virus type 1 (HSV-1) infection using immunoaffinity purification coupled to mass spectrometry (IP-MS). We identified an interaction between cGAS and 2ʹ-5ʹ-oligoadenylate synthase-like protein OASL, whose OAS-like domain interacted with the cGAS Mab21 domain, while the ubiquitin-like domain inhibited cGAS-mediated interferon response. By IP-MS and targeted MS, I further identified several critical cGAS phosphorylations and acetylations in human primary fibroblasts, HEK293Ts, and macrophage-like THP-1s. Acetyl-mimic mutations at Lys384 and Lys414 inhibited cGAS-dependent apoptosis, while acetyl-mimic Lys198 increased cGAS-dependent interferon signaling. Moreover, Lys198 acetylation levels were decreased upon infections with HSV-1 or human cytomegalovirus (HCMV), indicating a viral mechanism to inhibit host immunity. As virus-infected cells secrete cytokines to prevent virus spread, viral proteins are secreted to dampen host responses. However, it remains largely unknown how cells either in close or distant proximity to the infected cell respond to infection. I adapted a cell-penetrating mCherry labelling approach paired with cell sorting and MS to isolate and characterize infected, neighboring, and distal cell populations within the HCMV microenvironment. In neighboring compared to distal cells, I discovered lower levels of interferon-inducible proteins, differential cell cycle progression, and higher susceptibility to superinfection with either HCMV or HSV-1. These findings provide insights into how infection reshapes the local microenvironment to facilitate virus spread.|
|Alternate format:||The Mudd Manuscript Library retains one bound copy of each dissertation. Search for these copies in the library's main catalog: catalog.princeton.edu|
|Type of Material:||Academic dissertations (Ph.D.)|
|Appears in Collections:||Molecular Biology|
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