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J. Virol. doi:10.1128/JVI.02751-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Endoplasmic reticulum chaperones are involved in the morphogenesis of rotavirus infectious particles

Departamento de Genética del Desarrollo y Fisiología Molecular. Instituto de Biotecnología. Universidad Nacional Autónoma de México. Cuernavaca, Morelos, 62210, Mexico

^* To whom correspondence should be addressed. Email: arias{at}ibt.unam.mx.

	Abstract

The final assembly of rotavirus particles takes place in the endoplasmic reticulum (ER). In this work we evaluated by RNA interference the relevance on rotavirus assembly and infectivity of grp78, PDI, grp94, calnexin, calreticulin, and ERp57, members of the two described ER folding systems. Silencing the expression of grp94 and Erp57 had no effect on rotavirus infectivity, while knocking-down the expression of any of the other four chaperons caused a reduction in the yield of infectious virus of about 50%. In grp78-silenced cells the maturation of the oligosaccharide chains of NSP4 was retarded; in cells with reduced levels of calnexin the oxidative folding of VP7 was impaired and the trimming of NSP4 was accelerated, while the formation of disulfide bonds of VP7 was also accelerated in calreticulin-silenced cells. The knock-down of PDI impaired the formation and/or rearrangement of the VP7 disulfide bonds. All these conditions also affected the correct assembly of virus particles, since when compared with virions from control cells, they showed an altered susceptibility to EGTA and heat treatments, a decreased specific infectivity, and a diminished reactivity with MAb M60 to VP7, which only recognizes this protein when its disulfide bonds have been correctly formed. In the case of grp78-silenced cells the virus produced bound less efficiently to MA104 cells. All these results suggest that these chaperones are involved in the quality control of rotavirus morphogenesis. The complexity of the steps of rotavirus assembly that occur in the ER provide a useful model to study the organization and operation of the complex network of chaperones involved in maintaining the quality control of this organelle.