IN-VIVO INTERACTIONS AMONG ROTAVIRUS NONSTRUCTURAL PROTEINS

The rotavirus genome encodes six nonstructural (NS) proteins, five of which (NSP1, NSP2, NSP3, NSP5, and NSP6) have been suggested to be inv olved in a variety of events, such as genome replication, regulation o f gene expression, and gene assortment. These NS proteins have been fo und to be associated with replication complexes that are precursors of the viral core, however, little information is available about the in termolecular interactions that may exist among them. Using the yeast t wo-hybrid system, which allows the detection of protein-protein intera ctions in vivo, all possible combinations among the rotavirus NS prote ins were tested, and several interactions were observed. NSP1 interact ed with the other four proteins tested; NSP3 associated with itself; a nd NSP5 was found to form homodimers and to interact with NSP6. Co-imm unoprecipitation of proteins from rotavirus-infected cells, using hype rimmune sera monospecific for the NS proteins, showed the same interac tions for NSP1 as those observed in yeast. Immunofluorescence co-local ization analysis of virus-infected epithelial cells revealed that the intracellular distribution of proteins that were seen to interact in y east had patterns of distribution that would allow such intermolecular interactions to occur. These findings should contribute to the unders tanding of the role these proteins play in different aspects of the vi rus replication cycle.