Rotaviruses, members of the Reoviridae, are the major cause of severe acute gastroenteritis in infants and young children. While their medical significance has stimulated extensive efforts towards the development of rotavirus vaccines, such attempts have yet to lead to the production of commercially available vaccines of widespread efficacy. The overall goal of this project is to gain information about the molecular biology of rotavirus genome replication and capsid assembly that can be used in generating effective rotavirus vaccines. An important aim of the project includes establishing a method for introducing genetic mutations into the rotavirus genome. Such a reverse genetic system would have a positive impact on rotavirus vaccine development by providing a mechanism for modifying the antigenicity and virulence of the rotaviruses and for exploring the potential usefulness of the rotaviruses as vectors for immunization against other enteric pathogens. During the last year, advances important in defining rotavirus molecular biology were made in three areas. (i) Rotavirus core particles consist of VP1, VP2 and VP3 and have replicase activity which supports the replication of the double-stranded (ds)RNA genome from viral messenger (m)RNA in a cell-free system. Using this system, we identified in viral mRNA a cis-acting signal that is essential for replication and two cis-acting signals which, while not essential for replication, serve to enhance the process. (ii) To determine which of the core proteins recognizes viral mRNA during replication, a gel mobility shift assay was developed to study the interaction between core proteins and viral RNA. The results showed that VP1 possesses specific affinity for the essential 3'-terminal cis-acting replication signal of the rotavirus mRNA. Despite its affinity for the cis-acting signal and its polymerase-like structural properties, VP1 was found to lack replicase activity in the absence of other core proteins. (iii) To determine which core proteins were required for RNA replication, recombinant(r) VP1 and VP2 were expressed and assayed individually and together for associated replicase activity. The results demonstrated that, while neither protein alone has activity, rVP1 when combined with core-like particles formed by rVP2 possesses replicase activity. Together these data have identified the proteins and regions of the viral mRNA that play crucial roles in the replication of the rotavirus genome.