The molecular biology of rotaviruses X: Intercellular dissemination of rotavirus NSP4 requires glycosylation and is mediated by direct cell-cell contact through cytoplasmic extrusions

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, which, through site-directed mutagenesis of the N-linked glycosylation sites near the amino terminus of the protein, were shown to be dependent on its ability to become fully glycosylated. Real-time confocal microscopy was used to follow the appearance of similar cytoplasmic extrusions in virus-infected cells and revealed them to grow at a rate of ~2 microns/min to more than three cell diameters and to have a lifespan of 30-60 minutes. CellTracker dyes were used to label cell populations and facilitate the monitoring of the transfer of cytoplasm from virus-infected to surrounding uninfected cells through cytoplasmic extrusions that broke down to vesicles, seen both on the surface of and within uninfected cells in mixed cell populations. Staining of these tagged cell mixtures with monospecific antibody to NSP4 revealed the presence of the protein on uninfected cells, suggesting that the cytoplasmic extrusions formed by virus-infected cells facilitates the direct cell-cell spread of NSP4. This direct cell-cell transfer of infected cell material triggered by expression of glycosylated NSP4 in virus-infected cells may contribute to viral pathogenesis and facilitate host invasion by rotaviruses.