In the past year, we have advanced the objectives of this project along several lines. (1) To develop a reverse genetics system, we have determined the complete genome sequences of several lab-adapted strains of rotavirus. Subsequently, we have cloned all the genome segments of some strains into specialized T7-transcription vectors successfully used in creating reverse genetics systems for other viruses, including reovirus, a segmented dsRNA virus similar to rotavirus. We are currently attempting to use these vectors along with appropriate recombinant vaccinia viruses that express T7 RNA polymerase to generate recombinant rotaviruses. (2) To identify determinants in rotavirus affecting its virulence and pathogenesis, we have continued studies on one of its nonstructural proteins, NSP1. This protein was found earlier to subvert the innate immune system by interfering with the ability of the host to express interferon. During this past year, we have learned much about the mechanism by which NSP1 performs this function. Our experiments show that NSP1 interacts with and induces the degradation of a key family of cellular transcription factors in the infected cell required for interferon expression, and thus interferes with the activation of antiviral pathways. (3) To understand more about the diversity of rotavirus genes and its potential impact on the pathogenesis, evolution and epidemiology of these viruses, we sequenced the complete genomes of 10 distinct strains of human rotaviruses, representative of the 10 G-serotypes of viruses known to infect man. These data indicate that except for the immunodominant outer capsid proteins (VP4 and VP7) of rotavirus, the other nine viral proteins can be separated by-in-large into one of three phylogenically-distinct genogroups. Moreover, our results indicate that a hallmark feature of the major G-serotypes of rotaviruses circulating in nature is that they express preferred constellations of rotavirus proteins. Such constellations suggest the co-evolution of rotavirus genes falling within the same genogroup, raising the possibility that proteins within the same genogroup work best with one another. Thus, although rotavirus can undergo reassortment, such reassortants may not be viable in nature, because they create mismatched constellations of protein that fail to work efficiently with each other in support of virus replication.