Silencing of rotavirus NSP4 or VP7 expression reduces alterations in Ca2+ homeostasis induced by infection of cultured cells.
Rotavirus infection of cells in culture induces major changes in Ca(2+) 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.
Zambrano
José Luis
JL
Center for Microbiology and Cell Biology, Venezuelan Institute for Scientific Research (IVIC), Caracas 1020-A, Venezuela.
Díaz
Yuleima
Y
Peña
Franshelle
F
Vizzi
Esmeralda
E
Ruiz
Marie-Christine
MC
Michelangeli
Fabián
F
Liprandi
Ferdinando
F
Ludert
Juan Ernesto
JE
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000956:Antigens, Viral / Q000235:genetics / Q000378:metabolism*; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D002132:Calcium Radioisotopes / Q000378:metabolism; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D019084:Fluorescent Antibody Technique, Indirect; D020868:Gene Silencing; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006706:Homeostasis; D034741:RNA, Small Interfering / Q000378:metabolism; D012401:Rotavirus / Q000502:physiology*; D014118:Toxins, Biological / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D002132:Calcium Radioisotopes; D036022:Capsid Proteins; D006023:Glycoproteins; C081369:NS28 protein, rotavirus; D034741:RNA, Small Interfering; D014118:Toxins, Biological; C050779:VP7 protein, Rotavirus; D017361:Viral Nonstructural Proteins; D002118:Calcium