A yeast-based genetic system for functional analysis of viral mRNA cappingenzymes

Virus-encoded mRNA capping enzymes are attractive targets for antiviral the rapy, but functional studies have been limited by the lack of genetically t ractable in vivo systems that focus exclusively on the RNA processing activ ities of the viral proteins. Were we have developed such a system by engine ering a viral capping enzyme-vaccinia virus D1(1-545)p, an RNA triphosphata se and RNA guanylyltransferase-to function in the budding yeast Saccharomyc es cerevisiae in lieu of the endogenous fungal triphosphatase (Cet1p) and g uanylyltransferase (Ceg1p), This was accomplished by fusion of D1(1-545)p t o the C-terminal guanylyltransferase domain of mammalian capping enzyme, Mc e1(211-597)p, which serves as a vehicle to target the viral capping enzyme to the RNA polymerase II elongation complex: An inactivating mutation (K294 A) of the mammalian guanylyltransferase active site in the fusion protein h ad no impact on genetic complementation of cet1 Delta ceg1 Delta cells, thu s proving that (i) the viral guanylyltransferase was active in vivo and (ii ) the mammalian domain can serve purely as a chaperone to direct other prot eins to the transcription complex. Alanine scanning had identified five ami no acids of vaccinia virus capping enzyme-Glu37, Glu39, Arg77, Glu192, and Glu194-that are essential for gamma phosphate cleavage in vitro. Here we sh ow that the introduction of mutation E37A, R77A, or E192A into the fusion p rotein abrogates RNA triphosphatase function in vivo. The essential residue s are located within three motifs that define a family of viral and fungal metal-dependent phosphohydrolases with a distinctive capacity to hydrolyze nucleoside triphosphates to nucleoside diphosphates in the presence of mang anese or cobalt, The acidic residues Glu37, Glu39, and Glu192 likely compri se the metal-binding site of vaccinia virus triphosphatase, insofar as thei r replacement by glutamine abolishes the RNA triphosphatase and ATPase acti vities.