Nonstructural protein 4 (NSP4), encoded by rotavirus, exhibits various properties linked to viral pathogenesis, including enterotoxic activity. A recent study (O. V. Kavanagh et al., Vaccine 28:3106-3111, 2010) indicated that NSP4 also has adjuvant properties, suggesting a possible role in the innate immune response to rotavirus infection. We report here that NSP4 purified from the medium of rotavirus-infected Caco-2 cells triggers the secretion of proinflammatory cytokines from macrophage-like THP-1 cells and nitric oxide from murine RAW 264.7 cells. Secretion is accompanied by the stimulation of p38 and JNK mitogen-activated protein kinases (MAPKs) and nuclear factor NF-κB. NSP4 triggered the secretion of cytokines from murine macrophages derived from wild-type but not MyD88 ^−/− or Toll-like receptor 2 (TLR2 ^−/− ) mice and induced secretion of interleukin-8 (IL-8) from human embryonic kidney cells transfected with TLR2 but not TLR4. Our studies identify NSP4 as a pathogen-associated molecular pattern (PAMP) encoded by rotavirus and provide a mechanism for the production of proinflammatory cytokines associated with the clinical symptoms of infection in humans and animals.

Membrane trafficking from the endoplasmic reticulum (ER) to the Golgi complex is mediated by pleiomorphic carrier vesicles that are driven along microtubule tracks by the action of motor proteins. Here we describe how NSP4, a rotavirus membrane glycoprotein, binds to microtubules and blocks ER‐to‐Golgi trafficking in vivo. NSP4 accumulates in a post‐ER, microtubule‐associated membrane compartment and prevents targeting of vesicular stomatitis virus glycoprotein (VSV‐G) at a pre‐Golgi step. NSP4 also redistributes β‐COP and ERGIC53, markers of a vesicular compartment that dynamically cycles between the ER and Golgi, to structures aligned along linear tracks radiating throughout the cytoplasm. This block in membrane trafficking is released when microtubules are depolymerized with nocodazole, indicating that vesicles containing NSP4 are tethered to the microtubule cytoskeleton. Disruption of microtubule‐mediated membrane transport by a viral glycoprotein may represent a novel pathogenic mechanism and provides a new experimental tool for the dissection of early steps in exocytic transport.

Rotavirus infection induces profound alterations in the morphology and biochemistry of the host cell. Using two-dimensional (2D) gel electrophoresis combined with metabolic labeling, we have identified four proteins that are specifically upregulated in rotavirus-infected cells. Two of these have been identified as BiP (GRP78) and endoplasmin (GRP94), members of a family of glucose-regulated chaperone proteins that reside in the endoplasmic reticulum (ER) lumen, the site of rotavirus morphogenesis. The level of mRNA and the transcriptional activity of the BiP and endoplasmin genes are increased markedly in rotavirus-infected cells, and these genes are also induced when a single rotavirus protein, the nonstructural glycoprotein NSP4, is expressed in MA104 cells. However, NSP4 does not associate with either BiP or endoplasmin, implying that the mechanism of BiP and endoplasmin gene activation by NSP4 may differ from that triggered by viral membrane glycoproteins of other viruses. The interaction of BiP and endoplasmin with rotavirus structural polypeptides suggests that these chaperones are involved in the process of viral maturation in the ER lumen.

NSP4, a nonstructural glycoprotein encoded by rotavirus, is involved in the morphogenesis of virus particles in the endoplasmic reticulum of infected cells. NSP4 is also implicated in the pathophysiology of rotavirus-induced diarrhea by acting as an enterotoxin. To mediate enterotoxic effects in vivo, NSP4 must be secreted or released from rotavirus-infected cells in a soluble form; however, previous studies have indicated that NSP4 is a transmembrane glycoprotein localized within endomembrane compartments in infected cells. In this study, we examined the fate of NSP4 synthesized in Caco-2 cells infected with bovine rotavirus. Our studies reveal that NSP4 is actively secreted into the culture medium, preferentially from the infected-cell apical surface. The secretion of NSP4 is dramatically inhibited by brefeldin A and monensin, suggesting that a Golgi-dependent pathway is involved in release of the protein. In agreement with the proposed involvement of the Golgi apparatus during secretion, secreted NSP4 appears to undergo additional posttranslational modification compared to its cell-associated counterpart and is partially resistant to deglycosylation by endoglycosidase H. Our experiments identify a novel, soluble form of NSP4 secreted from virus-infected cells with the potential to carry out the enterotoxigenic role previously attributed to recombinant forms of the protein.

Expression of the rotavirus non-structural glycoprotein NSP4 inE.colileads to a decrease in optical density of the culture and release of [³H]uridine into the medium, effects attributable to the ability of NSP4 to perturb the bacterial membrane. To identify a domain of NSP4 responsible, different regions of the polypeptide were expressed inE.coli. Membrane destabilization is associated with a region of the protein located within residues 48–91, which includes a potential cationic amphipathic helix. A second region of NSP4 that contains a coiled-coil oligomerization domain and a sequence reported to function as a viral enterotoxin enhances the membrane-destabilizing activity of residues 48–91, but has no direct effect on the membrane stability. These studies suggest that the membrane-destabilizing and enterotoxic properties of NSP4 may be mediated by different regions of the polypeptide and suggest a possible basis for the cytotoxicity of NSP4 in mammalian cells.

Background: Nonstructural glycoprotein 4 (NSP4) encoded by rotavirus is the only viral protein currently believed to function as an enterotoxin. NSP4 is synthesized as an intracellular transmembrane glycoprotein and as such is essential for virus assembly. Infection of polarized Caco-2 cells with rotavirus also results in the secretion of glycosylated NSP4 apparently in a soluble form despite retention of its transmembrane domain. We have examined the structure, solubility and cell-binding properties of this secreted form of NSP4 to further understand the biochemical basis for its enterotoxic function. We show here that NSP4 is secreted as discrete detergent-sensitive oligomers in a complex with phospholipids and demonstrate that this secreted form of NSP4 can bind to glycosaminoglycans present on the surface of a range of different cell types. Methods: NSP4 was purified from the medium of infected cells after ultracentrifugation and ultrafiltration by successive lectin-affinity and ion exchange chromatography. Oligomerisation of NSP4 was examined by density gradient centrifugation and chemical crosslinking and the lipid content was assessed by analytical thin layer chromatography and flame ionization detection. Binding of NSP4 to various cell lines was measured using a flow cytometric-based assay. Results: Secreted NSP4 formed oligomers that contained phospholipid but dissociated to a dimeric species in the presence of non-ionic detergent. The purified glycoprotein binds to the surface of various non-infected cells of distinct lineage. Binding of NSP4 to HT-29, a cell line of intestinal origin, is saturable and independent of divalent cations. Complementary biochemical approaches reveal that NSP4 binds to sulfated glycosaminoglycans on the plasma membrane. Conclusion: Our study is the first to analyze an authentic (i.e. non-recombinant) form of NSP4 that is secreted from virus-infected cells. Despite retention of the transmembrane domain, secreted NSP4 remains soluble in an aqueous environment as an oligomeric lipoprotein that can bind to various cell types via an interaction with glycosaminoglycans. This broad cellular tropism exhibited by NSP4 may have implications for the pathophysiology of rotavirus disease.