Methyltransferase-specific domains within VP-39, a bifunctional protein that participates in the modification of both mRNA ends.

VP39 is a bifunctional vaccinia virus protein that acts as both a cap- dependent 2'-O-Methyltransferase and a poly(A) polymerase processivity factor. An analysis of C-terminal truncation mutants of a GST-VP39 fusion protein indicated the presence of a protease-sensitive C-terminal "tail" 36-43 amino acids in length that is non-essential for VP39 function. Fourteen new VP39 pointmutants, containing either single or multiple-clustered amino acid substitutions, were expressed in Escherichia coli. Of the eight that retained either one or both of the activities of VP39, seven were specifically methyltransferase-defective. None was specifically defective in adenylyltransferase stimulation. The nature of the methyltransferase defects in 10 of the methyltransferase-specific defectives, identified both herein and in a previous study (Schnierle BS, Gershon PD, Moss B, 1994, J biol Chem 269:20700-20706), was investigated using two novel substrate-binding assays. Three of the mutants (and possible a fourth), whose lesions were juxtaposed and centrally located within VP39, exhibited anomalous S-adenosyl-(L)-methionine (AdoMet) binding behavior, identifying residues important for AdoMet binding and possible also for catalysis. A surface plasmon resonance-based assay measured the interaction of VP39 with uncapped and 5'-cap 0-terminated oligo(A). A cap 0- dependent association-rate enhancement was observed for wild-type VP39 and 4 of the 10 mutant proteins. Two others were identified as defective in cap binding, and a third as partially defective. The lesions within the latter three mutants were closely apposed, and located toward the N-terminus of VP39. We have thus identified regions of VP39 important for interaction with its two substrates for cap-dependent methyltransferase activity: AdoMet and cap 0.