Matching Items (2)
Filtering by
- All Subjects: Proteins--Analysis.
- Creators: Roberson, Robert W.
Description
Coronaviruses are a medically significant group of viruses that cause respiratory and enteric infections in humans and a broad range of animals. Coronaviruses assemble at the internal membranes of the endoplasmic reticulum- Golgi intermediate compartment (ERGIC). While there is a basic understanding of how viruses assemble at these membranes, the full mechanistic details are not understood. The coronavirus envelope (E) protein is a small multifunctional viroporin protein that plays a role in virus assembly but its function is unknown. The two goals of this study were : 1. To identify and analyze the localization of MHV E and 2. To identify the functions of conserved residues in the tail of the E protein. This study closely examined the localization, dynamics and mobility of the mouse hepatitis virus (MHV) E protein to gain insight into its functions. The results from the first aim of this study showed that the MHV E protein localizes at the site of assembly in the ERGIC-Golgi region based on analysis by immunofluorescence and correlative electron microscopy. A novel tetra-cysteine tagged MHV E protein was used to study the dynamics of the protein in cells. A recombinant MHV E Lumio virus was used to study the trafficking and mobility of the E protein. Live cell imaging and surface biotinylation confirmed that the E protein does not traffic to the cell surface. Fluorescence recovery after photo-bleaching (FRAP) analyses revealed that the E protein is mobile at the site of localization. As a part of the second aim, conserved prolines and tyrosine in the tail of the protein were targeted by site directed mutagenesis and analyzed for functionality. While none of the residues were absolutely essential for localization or virus production, the mutations had varying degrees of effect on envelope formation, protein stability and virus release. Differential scanning calorimetry data suggests that the proline and tyrosine residues enhance interaction with lipids. A wild type (WT) peptide contained the conserved residues was also able to significantly reduce the hexagonal phase transition temperature of lipids, whereas a mutant peptide with alanine substitutions for the residues did not cause a temperature shift. This suggests that the peptide can induce a negative curvature in lipids. The E protein may be playing a role as a scaffold to allow membrane bending to initiate budding or possibly scission. This data, along with the localization data, suggests that the E protein plays a mechanistic role at the site of virus assembly possibly by remodeling the membrane thereby allowing virus budding and/or scission.
ContributorsVenkatagopalan, Pavithra (Author) / Hogue, Brenda G (Thesis advisor) / Jacobs, Bertram L (Committee member) / Roberson, Robert W. (Committee member) / Fromme, Petra (Committee member) / Arizona State University (Publisher)
Created2012
Evolutionary analysis of the CAP superfamily of proteins using amino acid sequences and splice sites
Description
Here I document the breadth of the CAP (Cysteine-RIch Secretory Proteins (CRISP), Antigen 5 (Ag5), and the Pathogenesis-Related 1 (PR)) protein superfamily and trace some of the major events in the evolution of this family with particular focus on vertebrate CRISP proteins. Specifically, I sought to study the origin of these CAP subfamilies using both amino acid sequence data and gene structure data, more precisely the positions of exon/intron borders within their genes. Counter to current scientific understanding, I find that the wide variety of CAP subfamilies present in mammals, where they were originally discovered and characterized, have distinct homologues in the invertebrate phyla contrary to the common assumption that these are vertebrate protein subfamilies. In addition, I document the fact that primitive eukaryotic CAP genes contained only one exon, likely inherited from prokaryotic SCP-domain containing genes which were, by nature, free of introns. As evolution progressed, an increasing number of introns were inserted into CAP genes, reaching 2 to 5 in the invertebrate world, and 5 to 15 in the vertebrate world. Lastly, phylogenetic relationships between these proteins appear to be traceable not only by amino acid sequence homology but also by preservation of exon number and exon borders within their genes.
ContributorsAbraham, Anup (Author) / Chandler, Douglas E. (Thesis advisor) / Buetow, Kenneth H. (Committee member) / Roberson, Robert W. (Committee member) / Arizona State University (Publisher)
Created2016