Princeton University Library Catalog
- Hung, George [Browse]
- Senior thesis
- Flint, Jane [Browse]
- Princeton University. Department of Molecular Biology [Browse]
- Class year:
- 113 pages
- Restrictions note:
- Walk-in Access. This thesis can only be viewed on computer terminals at the Mudd Manuscript Library.
- Summary note:
- The efficacy of adenoviral vectors as a system for gene transfer has been hindered due to their propensity to induce strong innate immune responses within both host cells and human patients. A deeper understanding of these antiviral immune responses is necessary in order to optimize these vectors for clinical use and to reveal novel mechanisms of innate antiviral immunity. The human adenovirus (group C) serotype 5 (Ad5) E1B 55 kDa protein has been recently shown to repress the transcription of multiple innate immune response genes and to inhibit the antiviral effects of type I interferon (IFN) in primary human cells. Both of these functions are independent of the well-established role of E1B 55 kDa in the adenovirus-specific E3 ubiquitin ligase, but the mechanism(s) by which they occur remains unresolved. This study sought to elucidate the mechanism of E1B 55 kDa-mediated immune modulation through identification of an expanded interactome. Co-immunoprecipitation (co-IP) and subsequent mass spectrometric (MS) studies of E1B 55 kDa protein complexes were employed in order to provide an enlarged set of candidate E1B 55 kDa interacting partners. Co-IPs were performed on whole-cell lysates generated from human foreskin fibroblasts (HFFs) infected with wild-type AdE, the E1B 55 kDa null mutant dl-1, and the E1B 55 kDa substitution mutant S19. Resolution of eluted proteins by SDS-PAGE followed by high-resolution, high mass-accuracy nano-flow UPLC-mass spectrometry and database searching identified 845 proteins, 267 of which were absent from the population isolated from dl-1 infected HFFs.
This set of proteins represents the most comprehensive set of candidate E1B 55 kDa interacting partners compiled to date. Subsequent gene ontology (GO) enrichment analyses of these candidate partners revealed that the overrepresented GO categories were related to known functions of the E1B 55 kDa protein, including RNA transport, ubiquitindependent degradation, apoptosis, and the cellular response to stress. Among the candidate interacting partners was pp32, a conserved multifunctional cellular protein recently implicated in the regulation of IFN-stimulated genes. Reciprocal IP, immunofluorescence, and knockdown experiments were conducted in order to examine the precise involvement of pp32 in adenoviral infection. Reciprocal IP confirmed the interaction between the E1B 55 kDa and pp32 proteins, siRNA knockdown of pp32 resulted in an increase in viral DNA synthesis in IFN treated HFFs infected with wild-type adenovirus, and immunofluorescence analyses showed that pp32 colocalized with both E1B 55 kDa and DBP at viral replication centers. These findings collectively suggest that E1B 55 kDa binds to pp32 at viral replication centers, and that the repression of the IFN-mediated anti-viral response by E1B 55 kDa is at least partly mediated through pp32.