Studies on the assembly of rotavirus viroplasmas

The processes that regulate Rotavirus replication are not fully understood and the lack of a reverse genetic approach represent an obstacle for the investigations in Rotavirus biology. Viroplasms are cytoplasmic structures that form soon after infection, and constitute the site of virus replication. Structural proteins like the viral RNA-dependent RNA -polymerase VP1, the capping enzyme VP3, the scaffolding protein VP2,and the middle layer VP6 localize in viroplasms; in addition, also the non-structural proteins NSP5 and NSP2 have been demonstrated to be essential components for viroplasm formation. Following the characterization of the interaction between NSP5 and VP1, we characterized the relationships between NSP5 and the structural protein VP2. In this work, interaction of NSP5 with VP2 was investigated by coexpression of the two proteins in uninfected cells, which resulted in a strong hyperphosphorylation of NSP5 and in the formation of viroplasm like structures (VLS). The behaviour of NSP5 in the presence of VP2 is very similar to that induced by NSP2 and already described (1), (60). Therefore, a comparison between the phosphorylation degree of NSP5 and VLS formation induced either by VP2 or by NSP2 was conducted. In both cases VLS formation was shown to assemble independently of the phosphorylation degree of NSP5, and to recruit the viroplasm-resident proteins VP1. However, VP6 (the protein forming the middle layer of the virion) was shown to be recruited only into VLS induced by VP2 (VLS(VP2i)), while it remains organized in tubular structures when VLSinduced by NSP2 (VLS(NSP2i)) were formed. Attempts to coimmunoprecipitate NSP5 and VP2 failed both from infected and co-transfected cells. However, promising preliminary results were obtained with a recently isolated monoclonal Ab specific for NSP5. Altogether, these data showed that two different viral proteins induced the same kind of modifications in NSP5, suggesting that these modifications may have a fundamental role for virus replication. Moreover, these data suggest that NSP5 plays a key role in architectural assembly of viroplasms and in recruitment of the other viroplasmic proteins.