monash_89873.pdf (8.12 MB)

Intracellular trafficking of adenovirus protein II (Hexon) and its significance with respect to design of non-viral Gene delivery system

Download (8.12 MB)
thesis
posted on 06.02.2017, 06:15 by Hsieh, Ming Je
A successful non-viral gene delivery system needs to accomplish its tasks by delivering the therapeutic gene to the nucleus of the targeted cell. To do so, the system needs to cross the cell membrane, travels towards the nuclear pore complex, and enters the nucleus. To enter the cytoplasm, the delivery system can be tagged with signal peptides that promote endocytosis, and the nuclear entry can also be achieved similarly via the help of the nuclear localization signal. It is the cytoplasmic trafficking that poses as a major obstacle for the efficient delivery of the cargo, thus, only with the assistance of an active cytoplasmic transport system, cargo(s) can then achieve efficient mobility in a crowded, dense cytoplasm. Viruses can navigate through the host’s cytoplasm with relative ease, which indicates the likelihood that viruses possess endogenous signals, presumably as native viral component(s) that can be recognized by the host’s cytoplasmic transport system. Using the human serotype 5 adenovirus as a model, this study investigated the capsid protein hexon as the putative signal-bearing component of the virus, with a view towards the adaptation of hexon or hexon fragments as the cytoplasmic trafficking component of a non-viral delivery system. In this study, the hexon ORF was fused with EmGFP, and transfected into several common laboratory cell lines to investigate the localization of the hexon fusion protein. Using a water-immersion objective, the transfected cells were analysed by laser-scanning confocal microscopy. Results indicated that hexon-EmGFP localized to the nuclear membrane of HeLa cells, but no clear localization pattern could be seen in Cos-7, 293, and A549 cells. The hexon ORF was truncated so as to investigate multiple fragments, and then fused with β-Gal to prevent passive diffusion of the reporter fusion proteins into the nucleus. Using the Gateway Pro Multisite system, a truncation study mapped the nuclear membrane localizing signal to hexon 1501-1575, with a corresponding amino acid sequence of 501YDYMNKRVVAPGLVDCYINLGARWS525, this represents an approximately 97% reduction in the size of the functional peptide from the hexon ORF, and importantly, this peptide is not situated within the immunogenicity region of the hexon ORF. In order to test the function of this peptide in an innate adenovirus, attempts were made to generate a reporter adenovirus by introducing an expression cassette encoding mCherry fused to core protein V into the adenovirus’s genome, making use of the pAdEasy system. The first attempt was made to introduce the pV-mCherry cassette, and a modified promoter, into an IRES-hrGFP carrying pShuttle vector. The second attempt was made by introducing the mCherry-pV cassette directly into the pAdEasy vector via homologous recombination, such that the fusion replaced the native protein V open reading frame. The two recombined adenovirus genomes showed fluorescent protein expression, but with low infectivity in the packaging cell line. Sizing analysis indicated the failure of packaging the modified viral core with the viral capsid components of the recombined adenovirus. Attempts were also made to purify hexon particles from the cytosol of virus-infected 293 cells by FPLC chromatography. While monoclonal anti-hexon antibody did detect the presence of hexon in its trimeric and monomeric formats from the initial virus-infected cell lysate, fractions from the chromatography assay showed bands that were 30% lower in terms of the detected mass. This indicates either the presence of anomalous migration on the SDS-PAGE gel, or the disintegration of the hexon monomer during fractionation process.

History

Campus location

Australia

Principal supervisor

Paul White

Additional supervisor 1

Colin Pouton, Helen Irving

Year of Award

2012

Department, School or Centre

Monash Institute of Pharmaceutical Sciences

Course

Doctor of Philosophy

Degree Type

DOCTORATE

Faculty

Faculty of Pharmacy and Pharmaceutical Sciences