In vivo interactions among rotavirus nonstructural proteins

Abstract

The rotavirus genome encodes six nonstructural (NS) proteins, five of which (NSP1, NSP2, NSP3, NSP5, and NSP6) have been suggested to be involved in a variety of events, such as genome replication, regulation of gene expression, and gene assortment. These NS proteins have been found to be associated with replication complexes that are precursors of the viral core, however, little information is available about the intermolecular interactions that may exist among them. Using the yeast two-hybrid system, which allows the detection of protein-protein interactions in vivo, all possible combinations among the rotavirus NS proteins were tested, and several interactions were observed. NSP1 interacted with the other four proteins tested; NSP3 associated with itself; and NSP5 was found to form homodimers and to interact with NSP6. Co-immunoprecipitation of proteins from rotavirus-infected cells, using hyperimmune sera monospecific for the NS proteins, showed the same interactions for NSP1 as those observed in yeast. Immunofluorescence co-localization analysis of virus-infected epithelial cells revealed that the intracellular distribution of proteins that were seen to interact in yeast had patterns of distribution that would allow such intermolecular interactions to occur. These findings should contribute to the understanding of the role these proteins play in different aspects of the virus replication cycle.