Methyltransferase-Specific Domains Within Vp39, 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 point mutants, 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 possibly 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 possibly 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 to ward 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.