In the past year, we advanced the goals of this project as follows. (1) To understand the function of the rotavirus RNA-dependent RNA polymerase (RDRP), we created a battery of recombinant baculoviruses expressing forms of the polymerase with mutations of residues potentially linked to recognition and replication of viral RNAs. Methods for the purification of the recombinant viral polymerases in their active forms were also refined in the past year. In vitro assays performed with the purified proteins will be used to assess the role of individual residues and structural motifs on polymerase activities. (2) To understand the structure and function of the rotavirus inner shell capsid protein VP2, we produced mutant forms of the protein using recombinant baculoviruses. Analysis of these mutant species allowed the identification of epitopes on the surface of the VP2 capsid accessible to monoclonal antibodies. In vitro replication assays carried out with the mutant species also allowed the mapping of domains on VP2 required for induction of the catalytic activity of the viral RNA polymerase. Additional mutants will be produced to further define the interactive domain of VP2 that triggers polymerase activity. (3) X-ray crystallographic analysis of NSP2 with its nucleotide substrates allowed the characterization of intermediate states in nucleotide hydrolysis by the protein. In addition, enzymatic analyses performed on NSP2 indicate that the protein has NDP kinase activity, a property possibly allowing NSP2 to maintain pools of nucleotides for RNA replication in viral inclusion factories. (4) Enzymatic analysis of NSP5 revealed that the protein has catalytic activity capable of hydrolyzing ATP. This ATPase activity may provide its infected-cell partner NSP2 with the gamma-phosphate that it needs to maintain pools of nucleotides for RNA replication in viral inclusion factories.