NSP4, encoded by rotavirus genome segment 10 has been shown to be a transmembrane, endoplasmic reticulum (ER) specific N-linked glycoprotein. Consistent with its localization to the ER membrane, NSP4 was first shown to have a role in the morphogenesis of the infectious virion. The protein has also been reported to have cytotoxic activity when applied extracellularly to cells. Consequently it has been earmarked as an enterotoxin being secreted from virus-infected cells to cause early cellular pathology in the gut. The effect of expressing the NSP4 protein of group A rotaviruses in cells has been studied. It led to the rapid appearance of long cytoplasmic extrusions. Site-directed mutagenesis was used to block N-linked glycosylation at both of the known glycosylation sites near the amino terminus of NSP4. This revealed that the NSP4 induced formation of the cytoplasmic extrusions was dependent on the protein’s ability to become fully glycosylated. The cytoplasmic extrusions seen in cells expressing glycosylated NSP4 were also evident in virus-infected cells. Using real-time confocal microscopy a dynamic elongation of the cytoplasmic extrusions with a growth speed of 2 μm/min was observed in virus-infected cells. The cytoplasmic extrusions were found to contain β-tubulin and F-actin. Inhibiting their polymerization prevented the formation of the extrusions from virus-infected cells. Functional studies using Cell Tracker dyes showed that the cytoplasmic extrusions could disseminate vesicles from virus-infected cells onto the plasma membrane surface of uninfected cells. The vesicles were then found in the interior of the uninfected cells. Mono-specific antibody to NSP4 revealed the presence of the protein in the vesicles suggesting that the cytoplasmic extrusions facilitated the direct cell-cell spread of NSP4. The effect of NSP4 expression on the microtubular network of cells was analysed. It was found that NSP4 de-polymerized the microtubular network from the centre of cells and promoted the assembly of microtubules at the periphery of the cells in a glycosylation independent manner. Similar de-polymerization and re-assembly of the microtubules was observed in the virus-infected cells. Interestingly in the presence of nocodazole, tubular structures containing tubulin and viral proteins excluding NSP4 were found in virus-infected cells. A YFP-PCA assay was established to screen for cellular partners of NSP4. The functionality and the sensitivity of the assay were examined, but only two false positive colonies were isolated in the first screening. In conclusion, the function of glycosylated and unglycosylated NSP4 was examined with the former possessing the ability to promote the formation of the cytoplasmic extrusions from cells and both being capable of disrupting the microtubular network indicating that two forms of NSP4 play different roles in NSP4 function. The cytoplasmic extrusions seen in our studies may be relevant to rotavirus infection and pathogenesis.
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