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Rotavirus infection of cells in culture induces major changes in Ca2؉ homeostasis. These changes include increases in plasma membrane Ca 2؉ permeability, cytosolic Ca 2؉ concentration, and total cell Ca 2؉ content and a reduction in the amount of Ca 2؉ released from intracellular pools sensitive to agonists. Various lines of evidence suggest that the nonstructural glycoprotein NSP4 and possibly the major outer capsid glycoprotein VP7 are responsible for these effects. In order to evaluate the functional roles of NSP4 and other rotavirus proteins in the changes in Ca 2؉ homeostasis observed in infected cells, the expressions of NSP4, VP7, and VP4 were silenced using the short interfering RNA (siRNA) technique. The transfection of specific siRNAs resulted in a strong and specific reduction of the expression of NSP4, VP7, and VP4 and decreased the yield of new viral progeny by more than 90%. Using fura-2 loaded cells, we observed that knocking down the expression of NSP4 totally prevented the increase in Ca 2؉ permeability of the plasma membrane and cytosolic Ca 2؉ concentration measured in infected cells. A reduction in the levels of VP7 expression partially reduced the effect of infection on plasma membrane Ca 2؉ permeability and Ca 2؉ pools released by agonist (ATP). In addition, the increase of total Ca 2؉ content (as measured by 45 Ca 2؉ uptake) observed in infected cells was reduced to the levels in mock-infected cells when NSP4 and VP7 were silenced. Finally, when the expression of VP4 was silenced, none of the disturbances of Ca 2؉ homeostasis caused by rotaviruses in infected cells were affected. These data altogether indicate that NSP4 is the main protein responsible for the changes in Ca 2؉ homeostasis observed in rotavirus-infected cultured cells. Nevertheless, VP7 may contribute to these effects. Viral-associated diarrhea remains one of the most common causes of morbidity and mortality among infants and young children. Worldwide estimations indicate that rotaviruses are the leading viral agent associated with severe diarrhea in children younger than 5 years old (20). In addition, rotavirus infections are also a main cause of diarrhea in calves, piglets, and the young of other animals of economic importance (20). Thus, further knowledge of the virus-cell interactions and the events leading to pathogenesis are necessary to improve or develop new strategies that may prevent or reduce the health and economic impact caused by rotavirus infections.Rotaviruses are members of the Reoviridae family. The rotavirus virion is icosahedral, nonenveloped, and composed of three concentric layers of proteins and a genome of 11 segments of double-stranded RNA. Each genomic segment, with the exception of segment 11, encodes one viral protein for a total of six structural (VP1 to VP7) and six nonstructural proteins (NSP1 to NSP6). The inner layer of the virion is formed by VP2 and also contains the RNA-dependent RNA polymerase VP1 and the guanylyltransferase/methylase VP3. The middle capsid is composed of the major virion...

Rotavirus infection of cultured cells induces a progressive increase in plasma membrane permeability to Ca2+. The viral product responsible for this effect is not known. We have used tunicamycin and brefeldin A to prevent glycosylation and membrane traffic and study the involvement of viral glycoproteins, NSP4 and/or VP7, in rotavirus-infected HT29 and MA104 cells. In infected cells, we observed an increase of plasma membrane Ca2+ permeability and a progressive depletion of agonist-releasable ER pools measured with fura 2 and an enhancement of total Ca2+ content measured as 45Ca2+ uptake. Tunicamycin inhibited the increase in membrane Ca2+ permeability, induced a depletion of agonist-releasable and 45Ca2+-sequestered pools. Brefeldin A inhibited the increase of Ca2+ permeability and the increase in 45Ca2+ uptake induced by infection. We propose that the glycosylated viral product NSP4 (and/or VP7) travels to the plasma membrane to form a Ca2+ channel and hence elevate Ca2+ permeability.

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