Protein interaction studies on the rotavirus non-structural protein NSP1
Rotavirus encodes six structural and six non-structural proteins. In contrast to the structural proteins, the functional roles of the non-structural proteins are not well defined beyond a realisation that they must have a role in the viral replication cycle. A fuller understanding of the replication cycle must therefore rest on determining the specific roles played by the non-structural proteins. Non-structural protein NSP1 shows high levels of sequence divergence. A generally well conserved cysteine-rich region at the amino-terminus may form a zinc finger structure. It has been shown to possess non-specific RNA-binding activity, and has been found associated with the smallest of three replication intermediates (RIs) found in infected cells, together with the viral proteins VP1, VP3 and NSP3. VP2 and VP6 are added sequentially to the pre-core RI to form the core RI and single-shelled RI respectively. The function of NSP1 in the replication cycle and the importance of its presence in early replication complexes has not been determined. The intermolecular interactions that occur between the components of the RIs have not been defined. Protein-protein interactions between NSP1 and VP1, VP2, VP3, and NSP3, from the UKtc strain of bovine rotavirus, were investigated using a variety of approaches, the first of which was the yeast two-hybrid system. In this assay a self-interaction of NSP1 was not detected. Protein-protein interactions between NSPl and VPl, VP2, VP3, and NSP3, were also not detected. Both the full-length protein and a truncated NSPl, consisting of only the amino terminal third of the protein, were tested. A direct self-interaction of NSP3 was shown and quantified. Radio-immunoprecipitation analysis of in vitro translated viral proteins using specific anti-NSP1 serum was also employed. However, it failed to detect direct protein-protein interactions between NSP1 and VPI, VP2, and VP3. Immunoprecipitation of UKtc rotavirus-infected celllysates with anti-NSP1 serum showed the co-precipitation of viral proteins VPl, VP2, VP3NP4, VP6 and NSP3, with NSP1. It was proposed that NSP1 formed a previously unrecognised complex with these proteins. Immunoprecipitation of nuclease-treated infected cell lysates showed a reduction in the co-precipitation of VP2, VP3NP4 and NSP3 with NSP1. No reduction in the co-precipitation of VP6 was seen. The association of the complex proteins may be mediated by RNA binding. Immunoprecipitation with an anti-VP6 monoclonal antibody reciprocally precipitated small amounts of NSP1, VP2, VP3/VP4, and NSP3, with VP6.