METHYLTRANSFERASE-SPECIFIC DOMAINS WITHIN VP39, A BIFUNCTIONAL PROTEIN THAT PARTICIPATES IN THE MODIFICATION OF BOTH MESSENGER-RNA 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 fu sion protein indicated the presence of a protease-sensitive C-terminal ''tail'' 36-43 amino acids in length that is non-essential for VP39 f unction. Fourteen new VP39 point mutants, containing either single or multiple-clustered amino acid substitutions, were expressed in Escheri chia coli. Of the eight that retained either one or both of the activi ties of VP39, seven were specifically methyltransferase-defective. Non e was specifically defective in adenylyltransferase stimulation. The n ature of the methyltransferase defects in 10 of the methyltransferase- specific defectives, identified both herein and in a previous study (S chnierle BS, Gershon PD, Moss B, 1994, J Biol Chem 269:20700-20706), w as investigated using two novel substrate-binding assays. Three of the mutants (and possibly a fourth), whose lesions were juxtaposed and ce ntrally located within VP39, exhibited anomalous S-adenosyl-L-methioni ne (AdoMet) binding behavior, identifying residues important for AdoMe t 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 par tially defective. The lesions within the latter three mutants were clo sely apposed, and located to ward the N-terminus of VP39. We have thus identified regions of VP39 important for interaction with its two sub strates for cap-dependent methyltransferase activity: AdoMet and cap 0 .