Identification of the RNA-binding, dimerization, and eIF4GI-binding domains of rotavirus nonstructural protein NSP3

The rotavirus nonstructural protein NSP3 is a sequence-specific RNA binding protein that binds the nonpolyadenylated 3' end of the rotavirus mRNAs, NS P3 also interacts with the translation initiation factor eIF4GI and compete s with the poly(A) binding protein. Deletion mutations and point mutations of NSP3 from group A rotavirus (NSP3A), expressed in Escherichia coli, intr icate that the RNA binding domain lies between amino acids 4 and 149. Simil ar results were obtained with NSP3 from group C rotaviruses, Data also indi cate that a dimer of NSP3A binds one molecule of RNA and that dimerization is necessary for strong RNA binding. The dimerization domain of NSP3 was ma pped between amino acids 150 and 206 by using the yeast two-hybrid system, The eukaryotic initiation factor 4 GI subunit (eIF-4GI) binding domain of N SP3A has been mapped in the last 107 amino acids of its C terminus by using a pulldown assay and the yeast two-hybrid system. NSP3 is composed of two functional domains separated by a dimerization domain.