A direct role for NSP4 in the acute watery diarrhea caused by rotavirus infection was first proposed over a decade ago following the observation that a peptide derived from the cytoplasmic domain of the protein caused diarrhea in 3 day old mice when injected intra-illeally or intra peritoneally . These studies led to the identification of NSP4 as a functional enterotoxin, the first, and currently only such virus-encoded protein known to exhibit this property. Subsequent in vitro experiments confirmed that NSP4 could potentiate secretion of Cl- ions and water from isolated mouse crypts through PLC-dependant elevation of cytosolic Ca2+ [3, 15]. Notably, these physiological studies utilized synthetic peptides or recombinant forms of NSP4 purified from detergent-solubilized insect cells and, preceded direct evidence that NSP4 was secreted from rotavirus infected cells, a prerequisite likely to enable the protein to interact with the plasma membrane of intestinal epithelial or other physiologically relevant cells.
Rotavirus infection of non-differentiated cells is cytolytic with cell viability rapidly decreasing after as little as 8 h post infection. In the monkey kidney line MA104, a peptide corresponding to residues 112-175 of NSP4 was released into the medium prior to cell lysis and recombinant forms of this peptide were capable of causing diarrhea in mice when purified from insect cells . In contrast, infection of differentiated Caco-2 cells, a polarized cell line derived from colonic epithelia, support non-lytic infection in which rotavirus is actively secreted from the apical surface with cell viability only slightly decreased after 72 h . We reported apical secretion of NSP4 from rotavirus infected Caco-2 cells as an intact species without proteolytic cleavage . Secretion of the unprocessed fully glycosylated form of NSP4 was surprising given the presence of a transmembrane domain but has been confirmed recently by Gibbons et al., . The results presented here indicate that NSP4 is secreted as an oligomeric lipoprotein in complex with phospholipid. The affinity of NSP4 for negatively charged phospholipids and cholesterol has been reported previously . We propose that recruitment of lipids during oligomerisation of NSP4 subunits in the ER membrane or a post ER membrane membraneous compartment facilitates its extrusion from the bilayer and release from infected cells as a small lipoprotein particles.
Purification of NSP4 to homogeneity from the media of rotavirus-infected cells confirmed our previous demonstration that the glycoprotein is not released in membrane vesicles like exosomes that contain additional proteins. Biophysical studies reveal that the secreted NSP4 is homogeneous in size. Previous studies have demonstrated a tetrameric structure for soluble forms of truncated regions of the cytoplasmic domain of NSP4 following recombinant expression or chemical synthesis [19–21]. The experiments reported here are, to our knowledge, the first that address the biophysical form of the full-length protein produced in rotavirus-infected cells. Although we have tentatively assigned a hexameric or octameric structure to the oligomer on the basis of apparent MW and crosslinking data, these data may also reflect an elongated polypeptide conformation and/or the ability to form higher order oligomers through weak hydrophobic interactions and thus we do not exclude that full length NSP4 is tetrameric.
To function as an enterotoxin, NSP4 should bind to a range of target cells and activate signaling pathways via receptors in the plasma membrane. A study in rotavirus-infected mice revealed that NSP4 was located predominantly on the basement membrane of villous epithelia that were not directly infected and identified fibronectin as a putative receptor . More recently, Seo et al., demonstrated that the metal ion-dependent adhesion site (MIDAS) motif present on integrins α1β1 and α2β1 can function as a receptor for NSP4 on cultured cells . All previous studies have employed NSP4 produced in either recombinant bacteria or insect cell lines [23–25]. Therefore, the present study was carried out to establish whether the native, full-length form of NSP4 secreted from polarized mammalian cells infected with rotavirus, is able to bind to non-infected mammalian cells and whether putative receptors can be identified.
The range of cells to which NSP4 binds includes cells of epithelial, fibroblast and hematopoetic origin, though considerable variation in the amount of NSP4 binding was observed between different cell types. The degree of GAG sulfation is a critical determinant of NSP4 binding revealed by the effect of chlorate treatment and the fact that HP, HS and CSB, the most highly sulfated GAGs used in our experiments, exhibited the greatest ability to inhibit binding of NSP4 to cells. These results could indicate that NSP4 (pI = 8.4), binds primarily via electrostatic interactions and that spacing of sulfate groups rather distinct sugar residues is a critical determinant for binding. Our experiments do not define a precise GAG structure targeted by NSP4 but the effect of heparanase treatment strongly suggests that heparan sulphate is the major GAG species required for binding to HT-29 cells. The specificity of NSP4-GAG interactions could be further explored using engineered cell lines that lack specific proteoglycan forms or overexpress different sulfotransferase enzymes that are required to generate highly sulfated GAG structures.
Many viral and host molecules interact with GAGs on the surface of cells. Viruses including herpes simplex-1 (HSV-1), human papillomavirus, dengue virus, and human immunodeficiency virus engage HS on the surface of cells during the initial stages of infection [26–29]. For example, HSV-1 initiates infection by attaching to HS on the cell surface via its surface glycoprotein gB and/or gC. A third viral glycoprotein gD can then interact with a specific form of HS known as 3-O-sulfated heparan sulphate to trigger membrane fusion and viral entry . Our results are also consistent with the behavior of a nonstructural glycoprotein encoded by dengue viruses. NS1 secreted from cells infected with dengue virus can utilize HS and CSE to attach to the surface of various cells types and this interaction may be a factor in the vascular leakage associated with secondary dengue virus infection . It is unlikely that the ability of NSP4 to activate intracellular signaling in intestinal and potentially other cells types is directly mediated by an interaction with GAGs. Rather GAGs may serve to recruit and tether NSP4 to the surface of cells enabling its interact with additional specific receptors to activate signaling pathways . While the primary focus of NSP4 has been on its role as an enterotoxin and its pathophysiological effects on intestinal cells, our studies now reveal that the protein may have a broader cellular tropism and thus exert a wider range of molecular effects in the host of relevance to rotavirus disease.