Yeast-Based Genetic System for Functional Analysis of Poxvirus mRNA Cap Methyltransferase

The m7GpppN cap structure of eukaryotic mRNAs is formed cotranscriptionally by the sequential action of three enzymes: an RNA triphosphatase that removes the gamma phosphate of the nascent transcript to yield a 5′ diphosphate end, a GTP:RNA guanylyltransferase that adds a GMP residue to the diphosphate end in a 5′-to-5′ orientation, and an RNA (guanine-N7) methyltransferase that uses S-adenosylmethionine to methylate the N7 position of the cap guanine (34, 35). The capping enzyme of vaccinia virus is a heterodimer of vD1 (844 amino acids [aa]) and vD12 (287 aa) polypeptides that catalyzes all three capping reactions (24, 38). The triphosphatase, guanylyltransferase, and methyltransferase active sites are arrayed in modular fashion within the vD1 subunit. The amino-terminal 60-kDa segment of vD1 (from aa 1 to 545) is an autonomous triphosphatase-guanylyltransferase domain in which the triphosphatase active site is located in the proximal half and the guanylyltransferase active site is in the distal half (8, 9, 12, 26, 27, 28, 37, 41, 42) The methyltransferase domain consists of the carboxyl-terminal portion of the vD1 subunit (vD1-C) heterodimerized with the vD12 subunit (7, 15, 16, 17, 20, 21, 29). The methyltransferase active site is located within vD1-C, which has a weak intrinsic methyltransferase activity that is stimulated 30- to 50-fold by the vD12 subunit, which is itself catalytically inert. Thus, the native vaccinia virus cap methyltransferase consists of a catalytic subunit (vD1-C) and a regulatory subunit (vD12). The physical organization of the poxvirus capping apparatus is quite distinct from that of cellular capping enzymes (34, 35). For example, the triphosphatase, guanylyltransferase, and methyltransferase components of the yeast capping apparatus reside on three different polypeptides encoded by three genes (CET1, CEG1, and ABD1, respectively). The metazoan capping enzyme consists of two components, a bifunctional triphosphatase-guanylyltransferase polypeptide and a separately encoded methyltransferase polypeptide. The cap methyltransferases of fungi and metazoa are monomeric enzymes. In the budding yeast Saccharomyces cerevisiae, the cap methylating enzyme Abd1 is essential for cell viability (22, 23, 40). Cap methyltransferases from other unicellular eukarya (Ccm1, Pcm1, and Ecm1 of Candida albicans, Schizosaccharomyces pombe, and Encephalitozoon cuniculi, respectively) can function in lieu of Abd1 to sustain yeast cell growth (14, 30). The human cap methyltransferase Hcm1 can also replace Abd1 in vivo, attesting to the conservation of structure and function between cap methylating enzymes from yeast to humans (30). Cellular cap methyltransferases display local amino acid sequence similarity to the vaccinia virus methyltransferase catalytic subunit but not to the stimulatory subunit (40). The structural differences between the poxvirus and human capping enzymes recommend cap formation as a target for antipoxviral drug discovery. The stimulatory subunit of the viral methyltransferase is an especially attractive molecular target, because there is no discernible homolog of vD12 in the known proteomes of any metazoan species. Although structure-function analyses of vaccinia virus cap methyltransferase have been carried out with purified vD1-C and D12 proteins expressed in bacteria, there has been no genetic analysis of the role of cap N7 methylation in poxvirus replication in vivo. Mutations that elicit temperature-sensitive virus growth phenotypes have been mapped to the two capping enzyme subunits (6, 13), but the picture is complicated because the capping enzyme plays multiple additional roles in viral gene expression. Capping enzyme serves as a transcription termination factor during the synthesis of viral early mRNAs (19, 36) and as an initiation factor during the transcription of intermediate genes (39). Surprisingly, the vD1 and vD12 temperature-sensitive mutant viruses display no gross defect in viral gene expression at the restrictive temperature but instead are defective in resolving concatameric DNA replication intermediates into the hairpin telomeres of the mature viral genome (6, 13). The telomere phenotype has no obvious connection to the known mRNA processing functions of the vD1 and vD12 proteins. Genetic and pharmacologic studies of poxvirus capping enzymes would be expedited by the development of in vivo assays in which the functional readout is clearly and exclusively dependent on the capacity of the viral gene product to catalyze cap synthesis. Previously, Ho et al. validated the concept of using the budding yeast S. cerevisiae as a genetic model for the study of the RNA triphosphatase and guanylyltransferase components of the poxvirus capping enzyme (18). Ho et al. showed that expression of vD1(1-545) in S. cerevisiae complemented the growth of mutant strains lacking the endogenous RNA triphosphatase or guanylyltransferase enzymes provided that vD1(1-545) was fused to a cellular protein capable of targeting it to the RNA polymerase II (Pol II) transcription complex. Here we tested the feasibility of using yeast as a genetic system to study the function of the poxvirus cap methyltransferase. We report that the vaccinia virus methyltransferase functions in lieu of the yeast Abd1 when the vD1-C and vD12 proteins are coexpressed in vivo. Genetic complementation by the viral methyltransferase did not require fusion to a cellular targeting protein. We exploit the yeast-based genetic system to identify constituents of the poxvirus catalytic and regulatory subunits that are important for cap methylation in vivo and in vitro.