MUTATIONAL ANALYSIS OF THE RNA TRIPHOSPHATASE COMPONENT OF VACCINIA VIRUS MESSENGER-RNA CAPPING ENZYME

Vaccinia virus mRNA capping enzyme is a multifunctional protein with R NA triphosphatase, RNA guanylyltransferase, and RNA (guanine-7-) methy ltransferase activities. The enzyme is a heterodimer of 95- and 33-kDa subunits encoded by the vaccinia virus D1 and D12 genes, respectively . The N-terminal 60-kDa of the D1 subunit (from residues 1 to 545) is an autonomous domain which catalyzes the triphosphatase and guanylyltr ansferase reactions. Mutations in the D1 subunit that specifically ina ctivate the guanylyltransferase without affecting the triphosphatase c omponent have been described (P. Cong and S. Shuman, Mol. Cell. Biol. 15:6222-6231, 1995). In the present study, we identified two alanine-c luster mutations of D1(1-545), R77A-K79A and E192A-E194A, that selecti vely inactivated the triphosphatase, but not the guanylyltransferase. Concordant mutational inactivation of RNA triphosphatase and nucleosid e triphosphatase functions (to similar to 1% of wild-type specific act ivity) suggests that both gamma-phosphate cleavage reactions occur at a single active site. The R77A-K79A and E192A-E194A mutant enzymes wer e less active than wild-type D1(1-545) in the capping of triphosphate- terminated poly(A) but could be complemented in vitro by D1(1-545)-K26 0A, which is inert in nucleotidyl transfer but active in gamma-phospha te cleavage. Whereas wild-type D1(1-545) formed only the standard Gppp A cap, the R77A-K79A and E192A-E194A enzymes synthesized an additional dinucleotide, GppppA. This finding illuminates a novel property of th e vaccinia virus capping enzyme, the use of triphosphate RNA ends as a n acceptor for nucleotidyl transfer when gamma-phosphate cleavage is r ate limiting.