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

The N-terminal 60 kDa (amino acids 1 to 545) of the D1 subunit of vacc inia virus mRNA capping enzyme is an autonomous bifunctional domain wi th triphosphatase and guanylyltransferase activities. We previously de scribed two alanine cluster mutations, R77 to A (R77A)-K79A and E192A- E194A, which selectively inactivated the triphosphatase component. Her e, we characterize the activities of 11 single alanine mutants-E37A, E 39A, Q60A, E61A, T67A, T69A, K75A, R77A, K79A, E192A, and E194A-and a quadruple mutant in which four residues (R77, K79, E192, and E194) wer e replaced by alanine. We report that Glu-37, Glu-39, Arg-77, Glu-192, and Glu-194 are essential for gamma-phosphate cleavage. The five esse ntial residues are conserved in the capping enzymes of Shope fibroma v irus, molluscum contagiosum virus, and African swine fever virus. Prob ing the structure of D1(1-545) by limited V8 proteolysis suggested a b ipartite subdomain structure. The essential residue Glu-192 is the pri ncipal site of V8 cleavage. Secondary cleavage by V8 occurs at the ess ential residue Glu-39. The triphosphatase-defective quadruple mutant t ransferred GMP to the triphosphate end of poly(A) to form a tetraphosp hate cap structure, GppppA. We report that GppppA-capped RNA is a poor substrate for cap methylation by the vaccinia virus and Saccharomyces cerevisiae RNA (guanine-7) methyltransferases. The transcription term ination factor activity of the D1-D12 capping enzyme heterodimer was n ot affected by mutations that abrogated ATPase activity. Thus, the cap ping enzyme is not responsible for the requirement for ATP hydrolysis during transcription termination.