ABSTRACT OF THE DISSERTATION
Engineering and Virology of the Cowpea Mosaic Virus and Flock House Virus,
Positive Sense Icosahedral RNA Viruses
by
Jamie Patrick Phelps
Doctor of Philosophy in Chemistry
University of California, San Diego, 2017
Professor John Johnson, Chair
Professor Tim Baker, Co-Chair
Small positive sense icosahedral RNA viruses include polio, rhinovirus, dengue, Norwalk virus, cowpea mosaic virus, and flock house virus. Cowpea mosaic virus (CPMV) and flock house virus (FHV) are excellent model viral systems for studying and engineering these viruses.
Engineering BSA conjugated to CPMV attenuated 99% of the virus through 10,000-fold serial dilutions and conjugating RNase safely inactivated the virus. RNase activity was inhibited 26-fold.
Engineering additional cysteine mutations to virus particles adds functional reactive potential. Reducing agents stabilize this reactive potential while protecting the proteins from unwanted aggregation. Mercaptopyridine based molecules are oxidizing agents that stabilize proteins by capping reactive thiols forming stable aqueous disulfide bonds that can be reduced as needed with traditional reducing agents. Stably bound mercaptopyridine or 2- mercaptopyridine-3-carboxylic acid was visualized with gel shifts in SDS-PAGE analysis.
The fixed positioning of metal nanoparticles on functional virus particles produced plasmonic nanoclusters with resonances at visible and infrared wavelengths. The metal cluster arrangement was controlled with placement of cysteine on the virus and VLPs. The fully assembled NCs gave rise to a 10-fold surface averaged enhancement of the resonance signal, which was consistent with calculated simulations.
The viral coat protein plays a role in almost every step of the life cycle of the virus. The stability of the capsid is crucial for protecting the genome from physical, chemical, and enzymatic damages. Magnetic tweezer forces were applied to the coat protein to understand the mechanical stability of the virus. Unfolding of individual coat proteins occurred over the entire force range while maintaining their attachment to the surrounding capsid proteins.
The emergence of new viruses into humans costs billions of dollars of medical relief. It is vital to understand the mechanisms of cross species viral transmission. The virology of cross species FHV infection in stressed mammalian cells was studied. A new approach of analyzing cross species infection unveiled a new organelle association structure, various forms of organelle malformation, multiple virus assemblies, and a more realistic understanding of the range of phenotypes in viral infection.
