![]() In rotavirus-infected Caco-2 cells, we showed that an important proportion of VP4 rapidly associates with lipid rafts and is early targeted to the apical membrane. The sphingolipids, which constitute the backbone of rafts, are only synthesized in the Golgi apparatus of mammalian cells ( 37). Rafts are membrane microdomains enriched in cholesterol and sphingolipids and are thought to play a key role in apical trafficking of epithelial cells ( 33). We have recently shown that rafts would be involved in this atypical pathway ( 32). In clear contrast, in Caco-2 cells, a well-polarized and differentiated intestinal cell line, rotavirus is released from the apical surface through a nonconventional pathway that bypasses the Golgi apparatus ( 15). Virus release from MA 104 cells is associated with the concomitant lysis of infected cells. However, triple-layered particles containing VP7 are assembled in cells in which VP4 synthesis was inhibited through a short interfering RNA (siRNA) approach ( 7). It has been proposed that VP4 forms hetero-oligomeric complexes with NSP4 and VP7 ( 19) and assembles with DLPs together with VP7 inside the ER. A juxtanuclear localization of VP4 similar to that of NSP4 and VP7 has also been described ( 12). VP4 is also present on the cell surface and along cytoskeletal structures early after infection ( 26 A. VP4 has been localized between the periphery of the viroplasm and outside the ER ( 29). The step at which VP4 is assembled is not clear. This maturation step involves calcium ( 30) and protein glycosylation ( 24, 28, 31, 34). ![]() Once this envelope is lost, the mature particles containing VP7 and VP4 appear ( 10). During this process, which is mediated by the interaction of DLPs with the ER transmembrane protein NSP4, the particles acquire a transient membrane envelope ( 1). DLPs are then translocated from these structures into the adjacent ER. VP4 is synthesized on free ribosomes, and it has been assumed that this protein is directly released within the cytosol.ĭuring the virus life cycle, mostly studied in MA 104 cells, double-layered particles (DLPs) are assembled in cytoplasmic inclusions called viroplasms. VP7 is an integral endoplasmic reticulum (ER) resident membrane protein ( 16). The glycoprotein VP7 constitutes, with the spike protein VP4, the outermost layer. The capsid of rotavirus is composed of three concentric layers of proteins surrounding a segmented double-stranded RNA genome (reviewed in reference 9). Rotaviruses, which are the leading cause of infantile diarrhea worldwide, are nonenveloped viruses belonging to the Reoviridae family. These results strongly support a primary role for raft membranes in rotavirus final assembly and the fact that VP4 assembly with the rest of the particle is an extrareticular event. VP4 does not colocalize with the ER marker protein disulfide-isomerase even when viral particles were blocked by TM in this compartment. Interestingly, TM does not prevent the targeting of VP4 to the cell surface nor its association with raft membranes, whereas the infectivity associated with the raft fractions strongly decreased. In the meantime, viral particles were blocked as enveloped particles in the ER. We show that, as expected, TM blocks viral protein glycosylation and also decreases virus infectivity. In this study, we used tunicamycin (TM), a drug known to block the first step of protein N glycosylation, as a tool to dissect rotavirus assembly. However, we recently described a strong association of VP4 with raft-type membrane microdomains, a result that makes the ER a highly questionable site for the final assembly of rotavirus, since rafts are thought to be absent from this compartment. VP7 and NSP4 are two glycosylated proteins. It has been assumed until now that VP4, the most external viral protein that forms the spikes of mature virions, associates with double-layer particles within the endoplasmic reticulum (ER) in conjunction with VP7 and with the help of a nonstructural protein, NSP4. Rotavirus assembly is a multistep process that requires the successive association of four major structural proteins in three concentric layers.
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