Browsing by Subject "NSP4"
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Item Intracellular trafficking and plasma membrane microdomain distribution of the NSP4 enterotoxin during rotavirus infection in epithelial cells(2009-05-15) Storey, Stephen MichaelRotavirus (RV) nonstructural protein 4 (NSP4) is a multifunctional glycoprotein that induces secretory diarrhea in mouse pups in the absence of other viral proteins. The intracellular transport route(s) and functional mechanism(s) of NSP4 are poorly understood; however, the recent association of the enterotoxin with cellular caveolin-1 may provide a link between NSP4 transport and function. To determine if NSP4 traffics to a specific subset of lipid rafts at the plasma membrane (PM), we isolated caveolae from a PM-enriched fraction with a new method that yielded endoplasmic reticulum (ER)-free caveolae membranes with a unique membrane structure and composition. Comparison of these caveolae with other detergent- and non-detergent-extracted membranes revealed that each caveolae/raft fraction contained caveolae markers; however, only our PM caveolae fraction mimicked the membrane structure and sterol exchange dynamics of intact PM without ER or non-raft PM contaminants. When these PM caveolae were isolated from RV-infected cells, full-length, high-mannose glycosylated NSP4 was present. Confocal imaging showed association of NSP4 with caveolin-1 moving from perinuclear and cytoplasmic sites toward the PM as the infection progressed. Fluorescent imaging also indicated exposure of the NSP4 Cterminus at the exofacial PM surface without transport of the enterotoxin through the Golgi apparatus. Surface-specific biotinylation was used to confirm NSP4 exposure at the surface of infected MDCK cells and to determine that the exposed protein was fulllength and high-mannose glycosylated. This study presents an ER contaminant-free PM caveolae isolation methodology, identifies the presence of full-length, high-mannose glycosylated NSP4 in both PM caveolae and exposed at the cell surface, and confirms the Golgi-bypassing nature of NSP4 ER to PM transport in RV-infected MDCK cells.Item Mapping of the rotavirus nonstructural protein-4-caveolin-1 binding site to three hydrophobic residues within the extended, c-terminal amphipathic alpha helix(2009-05-15) Williams, Cecelia V.Rotavirus NSP4, the first described viral enterotoxin, localizes to the plasma membrane of infected cells, possibly through interaction with caveolin-1. A direct interaction between NSP4 and caveolin-1, the structural protein of caveolae, was shown by yeast two-hybrid, peptide binding, and FRET analyses. To dissect the precise NSP4 binding domain to caveolin-1, mutants were prepared by altering either the charged or hydrophobic face of the NSP4 C-terminal amphipathic alpha-helix and examined for binding to caveolin-1. Replacing six charged residues with alanine (FLNSP4Ala) disrupted the charged face, while the hydrophobic face was disrupted by replacing selected hydrophobic residues with charged amino acids (aa) (FLNSP4HydroMut). In yeast two-hybrid and peptide binding assays, FLNSP4Ala retained its binding capacity, whereas FLNSP4HydroMut failed to bind caveolin-1. Mutants were generated with an Nterminal truncated clone (NSP446-175), which removed the hydrophobic domains and aided in yeast-two hybrid assays. These mutants exhibited the same binding pattern as FLNSP4 confirming that the N-terminus of NSP4 lacks the caveolin-1 binding site and NSP446-175 is sufficient for binding. Seven additional mutants were prepared from NSP4HydroMut in which individually charged residues were reverted to the original hydrophobic aa or were replaced with alanine. Analyses of the interaction of these revertants with caveolin-1 localized the NSP4 binding domain to one critical hydrophobic aa (L116) and one or two additional aa (I113, L127, and/or L134) on the hydrophobic face. Those mutants that bound caveolin-1 bound both the N- and C-terminal caveolin-1 peptides, but lacked binding to a centrally located peptide. These data suggest conformational and hydrophobic constraints play a role in the NSP4-caveolin-1 association. The mutant NSP4 molecules also were evaluated for transport to the plasma membrane. Mammalian cells were transfected with FLNSP4, FLNSP41-175Ala, and NSP41-175HydroMut plasmid DNA, surface biotinylated, and examined by IFA or Western blot for NSP4 expression. Epifluorescence revealed FLNSP4 and FLNSP4Ala were exposed on the cell surface in the absence of other viral proteins, whereas NSP4HydroMut remained intracellular. Further, NSP4-transfected cells displayed an intracellular association of with caveolin-1 or the caveolin-1 chaperone complex proteins. These data indicate NSP4 interacts with caveolin-1 in the absence of other viral proteins.Item Rotavirus NSP4 in extrareticular sites: support for its pathogenic role as an enterotoxin(2009-05-15) Gibbons, Thomas FieldRotavirus non structural protein 4 (NSP4) was initially characterized as an endoplasmic reticulum intracellular receptor. Continued studies of NSP4 revealed additional functions performed by or dependent on NSP4, some of which required trafficking from the ER to other areas of the cell. Chiefly, purified NSP4 exogenously added to the PM has been shown to mobilize intracellular calcium by a phospholipase C/inositol trisphosphate signaling pathway, yet the details whereby NSP4 are able to exert enterotoxic actions are still unknown. Our initial hypothesis included the protein caveolin 1, which subsequently was proven to bind NSP4 and prompted continued investigation as to whether or not NSP4 utilized caveolin 1 for extrareticular transport and or function. Caveolin 1 is the defining protein of caveolae, a region of the plasma membrane rich in multiple molecules that function in signal transduction, including possible receptor mediated activation of the phospholipase C/inositol triphosphate pathway. To determine if NSP4 trafficked to caveolae, a novel isolation procedure was developed and utilized to show NSP4 in PM caveolae. Expanding on the caveolae/NSP4 finding, temporal and spatial analyses of NSP4 in relation to progeny virus were conducted. NSP4?s appearance at the exofacial surface of the PM was carried out utilizing surface biotinylation, exofacial staining of live cells, and confocal imaging of the PM with fluorescent resonance energy transfer studies. During these studies soluble NSP4, which was isolated from RV infected cells, was also shown to interact with the PM of multiple cell lines. These studies provided confirmation of the NSP4-caveolin interaction in the presence and absence of other viral proteins. Our studies indicate the presence of full length NSP4 glycans at caveolae and the exofacial PM and are in agreement with studies indicating NSP4 traffics independent of the Golgi network. To further explore the NSP4/caveolin 1 interaction, we conducted a comparative analysis of NSP4 in relation to two separate pools of proteins. The first pool included proteins collocated with the classical secretory pathway proteins, including caveolin 1, which traffick through the Golgi. The second pool included proteins collocated with a subset of caveolin 1, which traffick independently of the Golgi.Item The rotavirus nonstructural protein 4 (NSP4) interacts with both the N- and C- termini of caveolin-1(Texas A&M University, 2006-08-16) Mir, Kiran DRotavirus (RV) is an etiologic agent of viral gastroenteritis in children and infants worldwide, accounting for an estimated 500,000 deaths annually. NSP4, the first described viral enterotoxin, contributes to RV pathogenesis by mobilizing intracellular calcium through multiple mechanisms that promote abnormal ion transport and subsequent secretory diarrhea. NSP4 and the enterotoxic peptide 114-135 preferentially interact with model membranes mimicking caveolae in lipid composition and radius of curvature. Our laboratory has recently reported the colocalization and coimmunoprecipitation of NSP4 with caveolin-1, the structural protein of caveolae. Moreover, the caveolin-1 binding domain of NSP4 has been localized to the enterotoxic peptide. We now report that caveolin-1 binds NSP4 via the N- and C-termini and one terminus is sufficient for binding. A panel of caveolin-1 deletion mutants was expressed in a yeast two-hybrid assay against an NSP4 bait. Caveolin-1 mutants retaining at least one terminus were capable of binding the NSP4 bait. An in vitro binding assay confirmed the two-hybrid results and localized the NSP4 binding domains to caveolin-1 residues 2-22 and 161-178. These data support the hypothesis that caveolin-1 mediates NSP4 signaling and/or intracellular trafficking.