During maturation, rotavirus double-layer particles (DLPs) bud into the endoplasmic reticulum (ER) by using the virally encoded nonstructural protein NSP4 and acquire an ER-derived lipid membrane that is eventually lost and replaced by two outer capsid proteins, VP7 and VP4 ( 14). In vivo, these viruses replicate primarily in the intestinal villus tip epithelial cells of the small bowel, and in vitro, the viral replication cycle has been primarily studied by using a variety of epithelial cell lines of renal or intestinal origin ( 15).The mechanism underlying rotavirus assembly and release is not totally understood. These viruses are nonenveloped viruses composed of three concentric layers of protein and 11 segments of double-stranded RNA (dsRNA) ( 14).
Rotaviruses are the single most important cause of severe diarrhea in infants and young children around the world ( 35). Finally, we analyzed the progression of rotavirus proteins in the exocytic pathway and found that VP4 and virion-assembled VP7 colocalized with ERGIC-53, suggesting that rotavirus particles transit through the intermediate compartment between the ER and the Golgi complex. We also present evidence to support the hypothesis that assembly of VP4 into mature virions takes place in the late stages of transit through the ER. We showed that two populations of VP4 exist, one small population that is independently targeted to rafts and a second large pool of VP4 whose association with rafts is mediated by particle formation in the ER. We found that inhibition of rotavirus migration into lipid rafts, by either siRNAs or tunicamycin, also specifically blocked the targeting of VP4 to rafts, suggesting that the association of VP4 with rafts is mostly mediated by the formation of viral particles in the endoplasmic reticulum (ER). Silencing of VP4 and NSP4 reduced the association of rotavirus particles with rafts in contrast, inhibition of VP7 synthesis slightly affected the migration of virions into rafts. In this study, we silenced the expression of VP4, VP7, and NSP4 by using small interfering RNAs (siRNAs) and evaluated the effect of shutting down the expression of these proteins on rotavirus-raft interactions. Furthermore, increasing evidence suggests that lipid rafts actively participate in the exit of rotavirus from the infected cell. Studies of rotavirus morphogenesis, transport, and release have shown that although these viruses are released from the apical surface of polarized intestinal cells before cellular lysis, they do not follow the classic exocytic pathway.
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