The amino-terminal 42-kDa region of the 144-kDa mammalian reovirus lambda2 protein is a guanylyltransferase. It catalyzes the transfer of GMP from GTP to the 5' end of 5'-diphosphorylated mRNA via a phosphoamide with Lys-190. This amino acid is located at the base of a deep cleft. Based on sequence comparisons, the Kx[V/L/I]S motif is present in all known and proposed guanylyltransferases of the family Reoviridae. The requirement for this conserved sequence and other regions of the enzyme was analyzed by site-directed mutagenesis. Based on the enzymatic activity of the mutants, Lys-190 and Asp-191 are the only amino acids of the (KDLS)-K-190 sequence that are necessary for enzymatic activity. Since Asp-191 has its side chain oriented away from the cleft, most likely it plays an indirect role in forming a functional guanylyltransferase. (C) 2002 Elsevier Science (USA).