Rotaviruses cause severe, life-threatening diarrheal disease in young children resulting in over a million deaths worldwide. In 1996, we identified the first viral enterotoxin, rotavirus NSP4, and introduced a new mechanism of rotavirus-induced diarrhea. NSP4-induced diarrhea is mediated by a phophoinositide signal transduction pathway that results in inositol triphosphate production, increased intracellular calcium, and chloride secretion. Yet, discrete lipid interactions and intracellular targeting of NSP4 in mammalian cells are unknown. Nor have structural studies been completed with defined model membranes. Our goals to define the intracellular transport and discrete cholesterol- and caveolin-1-interacting domains of NSP4 will be accomplished by combining innovative biophysical measurements, laser imaging, fluorescent spectroscopy and resonance energy transfer studies, with classical genetic and biochemical techniques. Our hypothesis is the enterotoxin-containing, cytoplasmic domain of NSP4 (cNSP4) is cleaved from the ER, transported to the cell surface in association with caveolin-1 and/or caveolar vesicles, and targeted to plasma membrane caveolae to interact with the signaling machinery of the cell. Our preliminary data show NSP4 and its active peptide, NSP4114-135, preferentially bind highly curved, anionic, cholesterol-rich membrane vesicles that mimic the plasma membrane microdomain, caveolae. Moreover, a cytoplasmic, C-terminal region of NSP4 is released from the ER when expressed in mammalian cells. We have shown cNSP4 colocalizes with caveolin-1, verifying that NSP4 and caveolin-1 are sorted to the same intracellular location. We now propose an in depth study of the mechanism of NSP4 transport in intestinal cells.
The specific aims are to: 1. Characterize the intracellular location of the cleaved NSP4 fragment (cNSP4) and cNSP4-caveolin-1 interaction(s) in mammalian cells. 2. Determine the role of caveolin-1/caveolae in the intracellular transport of cNSP4. 3. Delineate the domains of NSP4 that influence cNSP4 transport in mammalian cells. This investigation will contribute new insights into our understanding of the newly discovered plasma membrane microdomains (such as caveolae); broaden our knowledge of intracellular protein-membrane/lipid interactions; contribute to our understanding of enterotoxin function; and disclose basic intracellular processes whereby othcr toxins may interact with the cell. Further, this study may reveal new intracellular protein transport pathways.
| Yakshe, Krystle A; Franklin, Zachary D; Ball, Judith M (2015) Rotaviruses: Extraction and Isolation of RNA, Reassortant Strains, and NSP4 Protein. Curr Protoc Microbiol 37:15C.6.1-44 |
| Ball, Judith M; Schroeder, Megan E; Williams, Cecelia V et al. (2013) Mutational analysis of the rotavirus NSP4 enterotoxic domain that binds to caveolin-1. Virol J 10:336 |
| Gibbons, Thomas F; Storey, Stephen M; Williams, Cecelia V et al. (2011) Rotavirus NSP4: Cell type-dependent transport kinetics to the exofacial plasma membrane and release from intact infected cells. Virol J 8:278 |
| Martin, Gregory G; Hostetler, Heather A; McIntosh, Avery L et al. (2008) Structure and function of the sterol carrier protein-2 N-terminal presequence. Biochemistry 47:5915-34 |
| Mir, Kiran D; Parr, Rebecca D; Schroeder, Friedhelm et al. (2007) Rotavirus NSP4 interacts with both the amino- and carboxyl-termini of caveolin-1. Virus Res 126:106-15 |
| Parr, Rebecca D; Martin, Gregory G; Hostetler, Heather A et al. (2007) A new N-terminal recognition domain in caveolin-1 interacts with sterol carrier protein-2 (SCP-2). Biochemistry 46:8301-14 |
| Storey, Stephen M; Gibbons, Thomas F; Williams, Cecelia V et al. (2007) Full-length, glycosylated NSP4 is localized to plasma membrane caveolae by a novel raft isolation technique. J Virol 81:5472-83 |
| Parr, Rebecca D; Storey, Stephen M; Mitchell, Deanne M et al. (2006) The rotavirus enterotoxin NSP4 directly interacts with the caveolar structural protein caveolin-1. J Virol 80:2842-54 |
| Ball, Judith M; Mitchell, Deanne M; Gibbons, Thomas F et al. (2005) Rotavirus NSP4: a multifunctional viral enterotoxin. Viral Immunol 18:27-40 |
| Zhou, Minglong; Parr, Rebecca D; Petrescu, Anca D et al. (2004) Sterol carrier protein-2 directly interacts with caveolin-1 in vitro and in vivo. Biochemistry 43:7288-306 |
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