Biotechnology Commons^™

The Comparison Of Effects Of Synthetic And Natural Arachidin-3 On Rotavirus Infected Cells, Rebekah Napier-Jameson

Electronic Theses and Dissertations

Rotavirus (RV) causes severe, life-threatening diarrhea, in infants, young children and immunocompromised adults. There are several effective vaccines for young children, however they are strain specific and are not protective against many RV strains in developing countries. Therefore, it is important to investigate anti-RV therapeutic agents. Our laboratory has shown arachidin-1 (A1) and arachadin-3 (A3) significantly inhibit RV replication in two cell lines, however the molecular mechanism(s) of action are not known. A synthetic molecule of A3 (sA3) has been produced, but its’ antiviral effects have not been examined. Our hypothesis is that sA3 produces the same effects on RV-infected …

Mutational Analysis Of The Rotavirus Nsp4 Enterotoxic Domain That Binds To Caveolin-1, Judith M. Ball, Megan E. Schroeder, Cecelia V. Williams, Friedhelm Schroeder, Rebecca D. Parr

Faculty Publications

Background: Rotavirus (RV) nonstructural protein 4 (NSP4) is the first described viral enterotoxin, which induces early secretory diarrhea in neonatal rodents. Our previous data show a direct interaction between RV NSP4 and the structural protein of caveolae, caveolin-1 (cav-1), in yeast and mammalian cells. The binding site of cav-1 mapped to the NSP4 amphipathic helix, and led us to examine which helical face was responsible for the interaction.

Methods: A panel of NSP4 mutants were prepared and tested for binding to cav-1 by yeast two hybrid and direct binding assays. The charged residues of the NSP4 amphipathic helix were changed …

Rotavirus Nsp4: Cell Type-Dependent Transport Kinetics To The Exofacial Plasma Membrane And Release From Intact Infected Cells, Thomas F. Gibbons, Stephen M. Storey, Cecelia V. Williams, Avery Mcintosh, Deanne M. Mitchel, Rebecca D. Parr, Friedhelm Schroeder, Judith M. Ball

Faculty Publications

Background

Rotavirus NSP4 localizes to multiple intracellular sites and is multifunctional, contributing to RV morphogenesis, replication and pathogenesis. One function of NSP4 is the induction of early secretory diarrhea by binding surface receptors to initiate signaling events. The aims of this study were to determine the transport kinetics of NSP4 to the exofacial plasma membrane (PM), the subsequent release from intact infected cells, and rebinding to naïve and/or neighboring cells in two cell types.

Methods

Transport kinetics was evaluated using surface-specific biotinylation/streptavidin pull-downs and exofacial exposure of NSP4 was confirmed by antibody binding to intact cells, and fluorescent resonant energy …

Full-Length, Glycosylated Nsp4 Is Localized To Plasma Membrane Caveolae By A Novel Raft Isolation Technique, Stephen M. Storey, Thomas F. Gibbons, Cecelia V. Williams, Rebecca D. Parr, Friedhelm Schroeder, Judith M. Ball

Faculty Publications

Rotavirus NSP4, initially characterized as an endoplasmic reticulum intracellular receptor, is a multifunctional viral enterotoxin that induces diarrhea in murine pups. There have been recent reports of the secretion of a cleaved NSP4 fragment (residues 112 to 175) and of the association of NSP4 with LC3-positive autophagosomes, raft membranes, and microtubules. To determine if NSP4 traffics to a specific subset of rafts at the plasma membrane, we isolated caveolae from plasma membrane-enriched material that yielded caveola membranes free of endoplasmic reticulum and nonraft plasma membrane markers. Analyses of the newly isolated caveolae from rotavirus-infected MDCK cells revealed full-length, high-mannose glycosylated …

The Rotavirus Enterotoxin Nsp4 Directly Interacts With The Caveolar Structural Protein Caveolin-1, Rebecca D. Parr, Stephen M. Storey, Deanne M. Mitchell, Avery Mcintosh, Minglong Zhou, Kiran D. Mir, Judith M. Ball

Faculty Publications

Rotavirus nonstructural protein 4 (NSP4) is known to function as an intracellular receptor at the endoplasmic reticulum (ER) critical to viral morphogenesis and is the first characterized viral enterotoxin. Exogenously added NSP4 induces diarrhea in rodent pups and stimulates secretory chloride currents across intestinal segments as measured in Ussing chambers. Circular dichroism studies further reveal that intact NSP4 and the enterotoxic peptide (NSP4_114-135) that is located within the extended, C-terminal amphipathic helix preferentially interact with caveola-like model membranes. We now show colocalization of NSP4 and caveolin-1 in NSP4-transfected and rotavirus-infected mammalian cells in reticular structures surrounding the nucleus …