Activation of calcium/calmodulin-dependent kinase II following bovine rotavirus enterotoxin NSP4 expression

Objective(s): The rotavirus nonstructural protein 4 (NSP4) is responsible for the increase in cytoplasmic calcium concentration through a phospholipase C-dependent and phospholipase C-independent pathways in infected cells. It is shown that increasing of intracellular calcium concentration in rotavirus infected cells is associated with the activation of some members of protein kinases family such as calcium/calmodulin-dependent kinase II, which plays a crucial role in replication and pathogenesis of the virus. The aim of this study was to expression bovine rotavirus NSP4 gene in HEK293 cell and evaluation of its biological effect related to activation of calcium/calmodulin-dependent kinase II in cell culture. Materials and Methods: MA104 cells was used as a sensitive cell for propagation of virus and defined as a positive control. The NSP4 gene was amplified and inserted into an expression vector, and introduced as a recombinant plasmid into HEK293T cells. Western blot analysis was performed as a confirmation test for both expression of NSP4 protein and activation of calcium/calmodulin-dependent kinase II. Results:Expression of NSP4 and activated form of calcium/calmodulin-dependent kinase II were demonstrated by western blotting. Conclusion: It was shown that the expression of biologically active full- length NSP4 protein in HEK293T cells may be associated with some biological properties such as calcium calmodulin kinase II activation, which was indicator of rotaviruses replication and pathogenesis