INTRODUCTION

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Eukaryotic viruses have evolved diverse strategies to acquire a 5' cap structure for their mRNAs (4). RNA viruses that encode RNA-dependent RNA polymerases to synthesize their mRNAs either steal the caps from cellular transcripts, encode their own enzymes that cap and methylate the plus-strand transcripts, or circumvent the capping problem by including cis-acting elements in the plus strand that promote cap-independent translation. The mRNAs of most DNA viruses are synthesized by cellular RNA polymerase II (pol II) and are therefore capped by the cellular capping and methylating enzymes. However, vaccinia virus and other poxviruses, which replicate entirely in the cytoplasm, encode and encapsidate their own DNA-dependent RNA polymerase and mRNA capping apparatus (12, 16). African swine fever virus, which has a cytoplasmic replication phase, also encodes and encapsidates its own RNA polymerase and capping enzyme (39). Baculoviruses, which replicate in the nucleus, use pol II to transcribe early genes and then switch at late times to a virus-encoded transcription system that includes RNA polymerase and capping activities (14, 15, 26). Chlorella virus PBCV-1 encodes a capping enzyme (25) but appears not to encode its own RNA polymerase.

Cap formation by the enzymes of DNA viruses, double-stranded RNA viruses, and eukaryotic cells occurs via three sequential reactions: (i) the 5' triphosphate end of the nascent pre-mRNA is hydrolyzed to a diphosphate by RNA 5' triphosphatase, (ii) the diphosphate end is capped with GMP by GTP:RNA guanylyltransferase, and (iii) the GpppN cap is methylated by S-adenosylmethonine:RNA (guanine-N7) methyltransferase (48). This "conventional" pathway of cap synthesis was defined using soluble enzymes purified from vaccinia virus particles (12, 54). The mechanisms and structures of cellular and DNA virus capping enzymes have since been delineated through mutagenesis and crystallography (10, 11, 17, 21, 28, 30, 33, 37, 42, 56, 57, 60-62). Several single-stranded RNA viruses have evolved alternative cap synthetic pathways, which entail unconventional chemistry and are less well understood with respect to enzyme structure and mechanisms (1-3, 47).

The genetic and physical organizations of the known virus-encoded mRNA capping enzymes are significantly different from those of metazoan host cells (48). Hence, the viral cap-forming enzymes are potential targets for antiviral drugs that would interfere with capping of pathogen mRNAs but spare the host capping enzymes. For any given virus that provides its own caps, there are a number of questions that need to be addressed before capping can be validated as a target, such as (i) whether the viral gene encoding the capping enzyme is essential for virus replication and (ii) whether the capping activity of the viral gene product is essential for virus replication. These questions have not been answered fully, even where the biochemistry of viral cap formation is well understood. For example, vaccinia virus capping enzyme is a multifunctional protein with RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-7-) methyltransferase activities (45, 54). The enzyme is a heterodimer of 95- and 33-kDa subunits encoded by the vaccinia virus D1 and D12 genes, respectively. The vaccinia virus D1 and D12 genes are essential for virus replication, insofar as mutations that elicit temperature-sensitive virus growth phenotypes have been mapped to the two capping enzyme subunits (6, 18). However, the genetic landscape is complicated, because vaccinia virus capping enzyme plays a larger role in viral gene expression; it serves as a transcription termination factor during the synthesis of viral early mRNAs (29, 50) and as an initiation factor during the transcription of intermediate genes (55). Amazingly, the D1 and D12 temperature-sensitive mutant viruses display no gross defect in viral gene expression at the restrictive temperature but are instead defective in resolving concatemeric DNA replication intermediates into the hairpin telomeres of the mature viral genome (6, 18). This mysterious phenotype has no obvious connection to the known mRNA-processing or transcription functions of the D1 and D12 proteins. Similar problems arise in interpreting the essentiality of the baculovirus LEF-4 capping enzyme, which is an intrinsic subunit of baculovirus RNA polymerase, because it is not clear if the conditional phenotype of a lef-4 mutant virus is a consequence of failure to transcribe or failure to cap viral mRNAs (15, 26).

Genetic, and ultimately pharmacologic, analysis of viral capping enzymes would be facilitated 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. Here we explore the possibility of using the budding yeast Saccharomyces cerevisiae as a genetic model for the study of viral capping enzymes. S. cerevisiae encodes a three-component capping system consisting of separate triphosphatase (Cet1p), guanylyltransferase (Ceg1p), and methyltransferase (Abd1p) gene products (22, 31, 43, 53). All three genes are essential for yeast cell growth. Mutational analyses of Cet1p, Ceg1p, and Abd1p have resulted in the delineation of minimal catalytic domains for each protein and the identification of catalytically important amino acid side chains that comprise the triphosphatase, guanylyltransferase, and methyltransferase active sites (21, 27, 30, 37, 56, 57). By correlating mutational effects on catalysis in vitro with effects on function in vivo, we have shown that the triphosphatase, guanylyltransferase, and methyltransferase activities are essential for yeast cell growth. The feasibility of using yeast growth as a readout of the function of capping enzymes from heterologous sources is underscored by the demonstration that the entire three-component yeast capping apparatus can be replaced in vivo by the two-component mammalian capping system, consisting of a bifunctional triphosphatase-guanylyltransferase protein (Mce1p) and a separate cap methyltransferase (Hcm1p) (41). This result is remarkable because the structure and catalytic mechanism of the mammalian RNA triphosphatase are completely different from those of the yeast RNA triphosphatase (48).

The salient question here is whether a viral capping enzyme can function in place of one or more of the yeast capping enzymes. To address this issue, we tested the ability of the N-terminal triphosphatase-guanylyltransferase domain of the vaccinia virus D1 polypeptide [vD1(1-545)p] to complement the growth of yeast cet1 or ceg1 cells. We report that the vaccinia virus enzyme is active in vivo, provided that it is targeted to the pol II transcription complex by fusion to a cellular protein that binds to pol II. Mutations of vD1(1-545)p that abrogate triphosphatase activity in vitro abolish complementation of cet1. Additional mutational analysis of vD1(1-545)p defines the triphosphatase active site and suggests a mechanism of metal-dependent catalysis common to DNA viral and fungal capping enzymes. Isogenic strains bearing vaccinia virus and human capping enzymes can be used to screen for cytotoxic compounds that specifically inhibit the poxvirus capping apparatus.

	MATERIALS AND METHODS

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Yeast expression plasmids for vaccinia virus capping enzyme. The vD1(1-545) gene encoding the N-terminal triphosphatase-guanylyltransferase domain of vaccinia virus capping enzyme (35, 61) was excised from pET-D1(1-545) with NdeI and HindIII and inserted between the NdeI and HindIII sites of the customized yeast expression vector pYX1 (CEN TRP1), a derivative of pYX132 (Novagen) that contains six tandem histidine codons and a unique NdeI site between the NcoI and BamHI sites. The resulting yeast plasmid, pYX1-vD1(1-545), encodes vaccinia virus D1(1-545)p fused in-frame with an amino-terminal 12-amino-acid leader peptide (MGSHHHHHHSGH). An AatII-NheI fragment containing the vD1(1-545) gene was excised from pYX1-vD1(1-545) and inserted into the yeast multicopy expression plasmid pYX232 (2µm TRP1) to generate p232-vD1(1-545). Expression of the vD1(1-545) gene is under the control of the yeast TPI1 promoter.

Yeast strains. Strain YBS20 (MATa trp1 his3 ura3 leu2 ade2 can1 cet1::LEU2 p360-CET1) is deleted at the chromosomal CET1 locus encoding yeast RNA triphosphatase. Growth of YBS20 depends on maintenance of plasmid p360-CET1 (CEN URA3 CET1). Strain YBS2 (MATa ura3 trp1 lys2 leu2 ceg1::hisG pGYCE-360) is deleted at the chromosomal CEG1 locus encoding yeast RNA guanylyltransferase. Growth of YBS2 depends on maintenance of plasmid pGYCE-360 (CEN URA3 CEG1). Strain YBS50 (MATa leu2 ade2 trp1 his3 ura3 can1 ceg1::hisG cet1::LEU2 p360-CET1/CEG1) is deleted at the chromosomal CET1 and CEG1 loci (41). Growth of YBS50 is contingent on the maintenance of plasmid p360-CET1/CEG1 (CEN URA3 CET1 CEG1).

T7-based plasmids for expression of vD1(1-545)p in bacteria. vD1(1-545) mutations E37D, E37Q, E39D, E39Q, E192D, E192Q, E194D, and E194Q were programmed by synthetic oligonucleotides, using the two-stage PCR-based overlap extension strategy. NdeI-BglII restriction fragments of the PCR-amplified mutated vD1(1-545) DNAs were inserted into the T7-based expression plasmid pET16b that had been digested with NdeI and BamHI. The resulting plasmids contained the mutated vD1(1-545) coding sequence fused in frame with a 63-bp 5' leader sequence that encodes 10 consecutive histidine residues. The presence of the desired mutations was confirmed in each case by sequencing the entire 1.7-kbp insert; the occurrence of PCR-generated mutations outside the targeted region was thereby excluded.

Expression and purification of recombinant vD1(1-545)p. The recombinant His-tagged vaccinia virus proteins were purified by nickel-agarose chromatography of soluble extracts of 1-liter cultures of Escherichia coli BL21(DE3) bearing wild-type or mutated pET-His-D1(1-545) plasmids as described previously (61). The 200 mM imidazole eluate fractions of each preparation were dialyzed and purified further by phosphocellulose chromatography (61). The polypeptide compositions of the column fractions were monitored by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Protein concentrations were determined using the Bio-Rad dye reagent with bovine serum albumin as a standard.

	RESULTS

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Engineering vaccinia virus capping enzyme to function in yeast. vD1(1-545)p is an autonomous trypsin-resistant domain with RNA triphosphatase and RNA guanylyltransferase activities equivalent to those of the native vaccinia virus capping enzyme (35, 36, 44, 61). The triphosphatase and guanylyltransferase active sites are distinct, but the structural elements that comprise the active sites are partially interdigitated within the primary structure, such that vD1(1-545)p cannot be separated into catalytically active subdomains. To express vaccinia virus triphosphatase-guanylyltransferase in yeast, we cloned the vD1(1-545) gene into a yeast 2µm plasmid and placed it under the transcriptional control of the strong constitutive TPI1 promoter. The capacity of vD1(1-545)p to replace the yeast RNA triphosphatase Cet1p was tested by plasmid shuffle in yeast cet1 cells that contain CET1 on a URA3 plasmid. The cet1 strain is unable to form colonies on medium containing 5-fluoro-orotic acid (5-FOA), a drug that selects against the URA3 plasmid, unless it is transformed with a second plasmid bearing CET1 or a functional homologue from another source. For example, transformation with a TRP1 plasmid bearing the MCE1 gene, which encodes the 597-amino-acid mammalian triphosphatase-guanylyltransferase, allowed growth of cet1 cells on 5-FOA, whereas a plasmid expressing Mce1(211-597)p, the C-terminal guanylyltransferase domain of mammalian capping enzyme, did not (Fig. 1A). The key finding was that expression of vD1(1-545)p did not complement the cet1 mutation (Fig. 1A).

View larger version (49K): [in this window] [in a new window]   FIG. 1.   Complementation of yeast cet1 and ceg1 mutations by expression of a vD1(1-545)-Mce1(211-597)p chimeric capping enzyme. Yeast strains YBS20 (cet1), YBS2 (ceg1), and YBS50 (cet1 ceg1) were transformed with 2µm TRP1 plasmids containing either vD1(1-545) or vD1(1-545)-MCE1(211-597). Control transformations were performed with CEN TRP1 plasmids containing MCE1 or MCE1(211-597). Single Trp1+ transformants were patched to agar plates lacking tryptophan and then streaked on agar medium containing 5-FOA (0.75 mg/ml). The plates were photographed after incubation for 4 days at 30°C.

cet1 ceg1

Vaccinia virus guanylyltransferase is active in yeast. vD1(1-549)-MCE1(211-597) cells contain a single source of RNA triphosphatase activity derived from the vaccinia virus component of the fusion protein, but they contain two potential guanylyltransferase activities: one from the N-terminal vaccinia virus domain and one from the C-terminal mammalian domain. In order to determine whether the vaccinia virus guanylyltransferase could sustain cell growth when it is the only guanylyltransferase present, we introduced an inactivating missense mutation into the mouse guanylyltransferase domain. RNA guanylyltransferases are structurally and mechanistically conserved among fungi, mammals, and DNA viruses. The signature features of the guanylyltransferases are a two-step ping-pong reaction mechanism of nucleotidyl transfer through a covalent enzyme-(lysyl-N)-GMP intermediate (49). In the first step, a lysine nucleophile on the enzyme attacks the  phosphorus of GTP to form the covalent intermediate and expel pyrophosphate. In the second step, a nonbridging  phosphate oxygen of diphosphate-terminated RNA attacks the covalently bound guanylate to form the GpppN cap. A set of six conserved peptide motifs comprises the active site (17, 48, 56). The lysine nucleophile that becomes covalently attached to GMP is located within motif I (KxDG). The active-site lysine of Mce1p is Lys294. Substitution of alanine for Lys294 abrogates the guanylyltransferase activity and precludes complementation of ceg1 cells by mammalian capping enzyme (24, 59, 62).

cet1 ceg1

View larger version (24K): [in this window] [in a new window]   FIG. 2.   Mutational analysis of the vD1(1-545)-MCE1(211-597) fusion defines residues in vaccinia virus capping enzyme required for cap formation in vivo in yeast. The polypeptides encoded by the "wild-type" (WT) fusion gene vD1(1-545)-MCE1(211-597) and mutant alleles vD1(E37A), vD1(R77K), vD1(E192A), and MCE1(K294A) are depicted as horizontal bars with the N termini at the left and the C termini at the right. The positions of residues mutated in the vaccinia virus RNA triphosphatase subdomain (E37, R77, and E192) and the mouse guanylyltransferase domain (K294) are indicated. Complementation of the yeast cet1, ceg1, and cet1 ceg1 strains by the fusion genes on 2µm TRP1 plasmids was tested by plasmid shuffle as described for Fig. 1. Trp1+ isolates were streaked on agar medium containing 5-FOA. The plates were photographed after incubation for 4 days at 30°C.

In vivo effects of mutations in the putative active site of vaccinia virus RNA triphosphatase. Two distinct classes of eukaryotic RNA triphosphatases have been described. The RNA triphosphatases of mammals and other metazoan species belong to a superfamily of phosphatases that act via formation and hydrolysis of a covalent enzyme-(cysteinyl-S-)-phosphate intermediate (24, 52, 58). The metazoan RNA triphosphatase reaction requires no metal cofactor. In fact, metazoan RNA triphosphatases are inhibited by divalent cations. In contrast, the RNA triphosphatases of DNA viruses (poxviruses and baculoviruses), the budding yeast S. cerevisiae, and the pathogenic fungus Candida albicans are strictly dependent on a divalent cation cofactor (14, 21, 26, 38, 51). The viral-fungal triphosphatase family is defined by three conserved collinear motifs (A, B, and C) that include clusters of acidic and basic amino acids that are essential for catalytic activity (21, 37) (Fig. 3). Five essential residues within these motifs were initially uncovered by alanine scanning mutagenesis of the vaccinia virus RNA triphosphatase (60, 61). Single alanine substitutions for residues Glu37, Glu39, Arg77, Glu192, and Glu194 of vD1(1-545)p reduced triphosphatase activity by two to three orders of magnitude without affecting the guanylyltransferase activity of the mutant proteins (60).

View larger version (52K): [in this window] [in a new window]   FIG. 3.   Purification of wild-type and mutated versions of vaccinia virus D1(1-545)p. Conserved motifs A, B, and C of the RNA triphosphatases of vaccinia virus (vD1), Shope fibroma virus (SFV), molluscum contagiosum virus (MCV), Melanoplus sanguinipes entomopoxvirus (EPV), Yaba monkey tumor virus (YMV), African swine fever virus (ASF), Autographa californica baculovirus (Lef4), S. cerevisiae (Cth1 and Cet1), and C. albicans (CaCet1) are aligned in the top panel. Vaccinia virus D1 residues that are conserved in the other proteins are shaded. The five amino acids in vD1(1-545)p that were found by alanine scanning to be essential for triphosphatase activity are indicated by asterisks. Protein purification is shown in the bottom panel. Aliquots (0.5 µg) of the phosphocellulose preparations of recombinant wild-type (WT) vD1(1-545) and the indicated mutant proteins were electrophoresed through a 10% polyacrylamide gel containing 0.1% SDS. Polypeptides were visualized by staining with Coomassie Blue dye. The positions and sizes (in kilodaltons) of marker proteins are indicated on the left.

Conservative mutations of the active site of vaccinia virus RNA triphosphatase. We hypothesized previously that the glutamates in vaccinia virus motifs A and C facilitate catalysis by coordinating the essential divalent cation(s) (60, 61). The recently reported crystal structure of the S. cerevisiae RNA triphosphatase revealed that the glutamates in motif A and motif C do indeed comprise the metal-binding site of Cet1p (28). Mutational analysis showed that the glutamates are essential for Cet1p triphosphatase activity, and other studies indicate that their equivalents in motifs A and C of the C. albicans RNA triphosphatase CaCet1p (38) and baculovirus LEF4 (26) are also essential for catalysis.

Effects of conservative mutations on metal-dependent ATPase activity. The metal-dependent viral and fungal RNA triphosphatases also have an intrinsic capacity to hydrolyze nucleoside triphosphates to nucleoside diphosphates and inorganic phosphate in the presence of a divalent cation cofactoreither manganese or cobalt in the case of the yeast and baculovirus enzymes and either manganese, cobalt, or magnesium in the case of vaccinia virus capping enzyme (14, 21, 26, 37, 38, 45, 51). The nucleoside triphosphatase activities of wild-type and mutant vD1(1-545)p proteins were assayed by the release of ³²P_i from [-³²P]ATP (1 mM) during a 30-min reaction in the presence of 10 mM magnesium chloride (the optimal magnesium concentration for ATP hydrolysis [data not shown]). The relative specific activities of the vD1(1-545)p motif A and C mutants (normalized to the wild-type activity) are shown in Table 1. The motif A mutational effects on magnesium-dependent ATPase generally mirrored the effects on RNA triphosphatase, insofar as ATPase activity was abolished by introducing glutamine in place of Glu37 and Glu39. However, the extent of restoration of ATPase activity when aspartic acid was introduced at positions 37 and 39 was about an order of magnitude less than it was for RNA triphosphatase (Table 1). Thus, ATP hydrolysis was more stringent in its dependence on glutamates in motif A. In motif C, a glutamine at position 192 eliminated ATPase activity, which was restored to greater-than-wild-type levels when an aspartic acid was present at this position. This was the same mutational effect seen for RNA triphosphatase. At position Glu194, the level of residual activity with glutamine was much lower for ATPase (9%) than for RNA triphosphatase (81%). Similarly, the E194D change, although it restored activity compared to E194Q, had a relatively greater impact on ATPase than on RNA triphosphatase.

View larger version (11K): [in this window] [in a new window]   FIG. 4.   Effect of pH on metal-dependent ATP hydrolysis by vaccinia virus D1(1-545)p. Reaction mixtures (10 µl) containing 50 mM buffer (either BTP [pH 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0] or Tris [Tris-acetate at pH 5.5, 6.0, 6.5, or 7.0 or Tris-HCl at pH 7.5, 8.0, 8.5, 9.0, or 9.5]), 5 ng of vD1(1-545)p, 1 mM [-32P]ATP, and either 10 mm MgCl2 (A), 1 mM MnCl2 (B), or 2 mM CoCl2 (C) were incubated for 30 min at 37°C. The reaction products were analyzed by polyethyleneimine-cellulose thin-layer chromatography (61). ATPase activity is plotted as a function of pH.

	DISCUSSION

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Viral RNA capping enzymes are attractive targets for antiviral drugs because the properties of the viral enzymes are often different from those of the host cell enzymes. This is especially so for the metal-dependent RNA triphosphatases of DNA viruses and double-stranded RNA viruses (5, 14, 26, 35, 60), which are mechanistically and structurally unrelated to the metal-independent cellular RNA triphosphatase. The fact that metazoan species encode no obvious homologues of the viral RNA triphosphatases suggests that an inhibitor of viral RNA triphosphatase should have selectivity for the viral pathogen and minimal effect on the host organism. The yeast-based genetic system we describe here provides a valuable tool for basic and applied studies of viral capping enzymes. We show that vaccinia virus RNA triphosphatase and vaccinia virus guanylyltransferase can function in lieu of the essential yeast enzymes Cet1p and Ceg1p, provided that the vaccinia virus protein is fused to the mammalian capping enzyme, which targets the viral protein to the pol II elongation complex. Hence, the capping functions of the viral enzyme can be studied in vivo independent of ancillary roles of the viral protein in the context of virus replication. We have used this system to establish a concordance between mutational effects on vaccinia virus RNA triphosphatase activity in vitro and in vivo.

We have illuminated structure-activity relationships for four essential amino acids of vaccinia virus RNA triphosphatase. Three essential glutamates, Glu37 and Glu39 in motif A and Glu192 in motif C, cannot be replaced by glutamine, suggesting that these side chains comprise the metal-binding site of vaccinia virus triphosphatase. These results are consistent with insights gained from the crystal structure of yeast RNA triphosphatase Cet1p complexed with the divalent cation manganese. Cet1p adopts a novel enzyme fold whereby an antiparallel eight-strand  barrel forms a hydrophilic "triphosphate tunnel" that is topologically closed (28). Multiple acidic side chains point into the tunnel cavity, including the essential glutamates of motifs A and C. The interior of the tunnel contains a single sulfate ion coordinated by basic side chains projecting into the tunnel. Insofar as sulfate is a structural analog of phosphate, we surmise that the side chain interactions of the sulfate reflect contacts made by the enzyme with the  phosphate of the triphosphate-terminated RNA and nucleoside triphosphate substrates. A manganese ion within the tunnel cavity is coordinated with octahedral geometry to the sulfate, to the side chain carboxylates of the two essential glutamates in motif A, and to one of the glutamates in motif C. Glutamine substitutions for any of the three Cet1p glutamates that directly coordinate the manganese result in a complete loss of catalytic activity (37). By analogy with Cet1p, we propose that Glu37, Glu39, and Glu192 of vaccinia virus capping enzyme interact directly with a divalent cation. The active site of Cet1p may be less flexible than that of vD1(1-545)p, insofar as none of the three metal-binding glutamates of Cet1p could be functionally replaced by aspartic acid.

A second essential glutamate in motif C of Cet1p coordinates a water molecule bound to manganese. We invoke a similar role for the Glu194 in motif C of vaccinia virus capping enzyme. Coordination of solvent by polar interactions of vaccinia virus side chain 194 would be consistent with the retention of catalytic function by the E194Q mutant. Whereas glutamine may suffice for coordinating solvent bound to magnesium, phosphohydrolase activity in manganese and cobalt was strictly dependent on a glutamate side chain. Spatial differences in the coordination spheres of these metals may account for the distinct mutational effects with different metal cofactors.

Motifs A and C are located within  strands of yeast Cet1p that are widely separated in the primary structure but closely approximated in the tertiary structure (28). Motifs A and C are located on the tunnel "floor" that abuts the globular core of the protein. In motifs A and C of the fungal RNA triphosphatases, alternating charged side chains are interdigitated with alternating aliphatic or aromatic side chains (Fig. 3). This sequence pattern is reprised in motifs A and C of the poxvirus, African swine fever virus, and baculovirus RNA triphosphatases, suggesting that the metal-binding residues of the viral enzymes may also be located within  strands.

Although we propose that yeast and vaccinia virus RNA triphosphatases share a common metal-binding site, we suspect that the active sites of the yeast and viral enzymes may adopt different tertiary structures, i.e., that the vaccinia virus active site does not residue within an enclosed tunnel, because the motif A and C glutamates of vaccinia virus D1(1-545)p are accessible to limited digestion with V8 protease (60). A definitive assessment of the similarities between fungal and viral RNA triphosphatases will hinge on successful crystallization of vD1(1-545)p.

Finally, the availability of isogenic yeast strains containing mammalian versus viral capping systems provides a means of drug discovery aimed at blocking viral cap formation. For example, any compound that is selectively cytotoxic to the vD1(1-549)-MCE1(211-597)-K294A strain but not to the MCE1 strain is a strong candidate for being a specific inhibitor of vaccinia virus capping. Identification of effective antipoxviral drugs is a reasonable goal in light of the high incidence of molluscum contagiosum infection as a sequela of AIDS and the potential threat of smallpox as a biological weapon. We anticipate extending the yeast complementation approach to the methyltransferase component of vaccinia virus capping enzyme (19, 32, 33) with the goal of generating yeast strains in which the entire capping apparatus is of viral origin. It will also be of interest to apply the yeast complementation test to the capping enzymes of other viruses that affect human health, e.g., rotavirus (7, 40). Genetic complementation by viral polypeptides fused to the mammalian delivery vehicle also provides a means to identify the capping enzymes of viruses in cases where the biochemical activities are refractory to purification.