Rotavirus NSP4 in extrareticular sites: support for its pathogenic role as an enterotoxin

Rotavirus 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.