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Title: Characterizing the Relationship Between Sumoylation and the E1B 55-Kilodalton Protein During Adenoviral Infection
Authors: Trinh, Peter
Advisors: Flint, Jane
Department: Molecular Biology
Class Year: 2014
Abstract: Understanding the mechanisms that underlie type I interferon (IFN)-mediated cellular immune responses to adenoviruses is of paramount importance for the development of safe and effective Ad-based vectors. Species C human adenovirus type 5 (Ad5) E1B 55-kilodalton (kDa) is a multifunctional protein that blocks IFN-induced inhibition of viral replication and transcriptionally represses expression of IFN-stimulated genes (ISGs). Both functions are accomplished independently of the E1B 55-kDa/E4- Orf6 E3 ubiquitin ligase complex. E1B 55-kDa also has been characterized as a SUMO1- modified protein and a SUMO1 E3 ligase. To elucidate the importance of SUMO1- modification of E1B 55-kDa to successful inhibition of the IFN-mediated antiviral response in normal human cells, viral genome replication was assessed via qPCR in normal human cells infected with wildtype and sumoylation-mutant Ad5 viruses. Quantitation of viral DNA concentrations revealed that sumoylation at residue K104 is not crucial for E1B 55-kDa to protect viral DNA replication against the IFN-induced response. In order to investigate the function of E1B 55-kDa as a SUMO1 E3 ligase and also gain insight into the global role of SUMO1-modification, cellular and viral proteomes from wildtype- and E1B 55-kDa null mutant-infected cells were quantitatively assessed using stable isotope labeling via amino acids in cell culture, SUMO1 immunoprecipitation, and liquid chromatography mass spectrometry. We report the successful identification of 4283 putative SUMO1-modified proteins along with a subset of SUMO1 conjugates with links to the cellular innate immune response. Of particular interest is the protein LGP2, an RNA helicase that is reportedly a negative regulator of IFN signaling. Exploitation of the putative SUMO1 conjugate LGP2 may be a novel mechanism by which E1B 55-kDa protects viral replication against the IFN-mediated immune response.
Extent: 114 pages
Type of Material: Princeton University Senior Theses
Language: en_US
Appears in Collections:Molecular Biology, 1954-2016

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