RNA 5′-Triphosphatase, Nucleoside Triphosphatase, and Guanylyltransferase Activities of Baculovirus LEF-4 Protein

Autographa californica nuclear polyhedrosis virus late and very late mRNAs are transcribed by an RNA polymerase consisting of four virus-encoded polypeptides: LEF-8, LEF-9, LEF-4, and p47. The 464-amino-acid LEF-4 subunit contains the signature motifs of GTP:RNA guanylyltransferases (capping enzymes). Here, we show that the purified recombinant LEF-4 protein catalyzes two reactions involved in RNA cap formation. LEF-4 is an RNA 5′-triphosphatase that hydrolyzes the γ phosphate of triphosphate-terminated RNA and a guanylyltransferase that reacts with GTP to form a covalent protein-guanylate adduct. The RNA triphosphatase activity depends absolutely on a divalent cation; the cofactor requirement is satisfied by either magnesium or manganese. LEF-4 also hydrolyzes ATP to ADP and P_i (K_m = 43 μM ATP; V_max = 30 s⁻¹) and GTP to GDP and P_i. The LEF-4 nucleoside triphosphatase (NTPase) is activated by manganese or cobalt but not by magnesium. The RNA triphosphatase and NTPase activities of baculovirus LEF-4 resemble those of the vaccinia virus and Saccharomyces cerevisiae mRNA capping enzymes. We suggest that these proteins comprise a novel family of metal-dependent triphosphatases.     

RNA capping by HeLa cell RNA guanylyltransferase. Characterization of a covalent protein-guanylate intermediate

RNA capping by partially purified HeLa cell GTP:RNA guanylyltransferase has been shown to occur in the following sequence of two partial reactions involving a covalent protein-guanylate intermediate: (i) E(P68) + GTP in equilibrium E(P68-GMP) + PPi (ii) E(P68-GMP) + ppRNA in equilibrium GpppRNA + E(P68) Initially, the enzyme reacts with GTP in the absence of an RNA cap acceptor to form a covalent protein-guanylate complex. This complex consists of a GMP residue linked via a phosphoamide bond to a Mr = 68,000 protein. The enzyme then transfers the guanylate residue from the Mr = 68,000 polypeptide to the 5' end of diphosphate-terminated poly(a) to yield the capped derivative GpppA(pA)n. Both partial reactions have been shown to be reversible. In the reverse of Reaction i, E(P68--GMP) reacts with PPi to regenerate GTP. In the reverse of Reaction ii, the enzyme catalyzes the transfer of the 5'-GMP from capped RNA to the Mr = 68,000 protein to form protein-guanylate complex. A divalent cation is required for both partial reactions. The Mr = 68,000 protein is presumed to be a subunit of the HeLa guanylyltransferase. This interpretation is consistent with the sedimentation coefficient of 4.2 S of the native enzyme. Preliminary studies of RNA guanylyltransferase from mouse myeloma tumors suggest a similar mechanism of transguanylylation involving a Mr = 68,000 protein-guanylate complex. These data, in conjunction with previous studies of vaccinia virus guanylyltransferase (Shuman, S., and Hurwitz, J. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 187-191) suggests that covalent GMP-enzyme intermediates may be a general feature of the RNA capping reaction.     

Roles of LEF-4 and PTP/BVP RNA triphosphatases in processing of baculovirus late mRNAs

The baculovirus Autographa californica nucleopolyhedrovirus encodes two proteins with RNA triphosphatase activity. Late expression factor LEF-4, which is an essential gene, is a component of the RNA polymerase and also encodes the RNA capping enzyme guanylyltransferase. PTP/BVP is also an RNA triphosphatase, but is not essential for viral replication, possibly because its activity is redundant to that of LEF-4. To elucidate the role of these proteins in mRNA cap formation, a mutant virus that lacked both RNA triphosphatase activities was constructed. Infection studies revealed that the double-mutant virus was viable and normal with respect to the production of budded virus. Pulse-labeling studies and immunoblot analyses showed that late gene expression in the double mutant was equivalent to that in the wild type, while polyhedrin expression was slightly reduced. Direct analysis of the mRNA cap structure indicated no alteration of cap processing in the double mutant. Together, these results reveal that baculoviruses replicate and express their late genes at normal levels in the absence of its two different types of RNA triphosphatases.     

Domain structure of vaccinia virus mRNA capping enzyme. Activity of the Mr 95,000 subunit expressed in Escherichia coli

The D1 gene encoding the large subunit of vaccinia virus mRNA capping enzyme was expressed in Escherichia coli BL21(DE3) under the control of a bacteriophage T7 promoter. Guanylyltransferase activity (assayed as the formation of a covalent enzyme-guanylate complex) was detected in soluble lysates of these bacteria. Two major species of protein-GMP complex were formed, one of Mr 95,000 (corresponding in size to the D1 gene product) and one of Mr 60,000. Partial purification of the guanylyltransferase was effected by ammonium sulfate precipitation and ion-exchange chromatography. The expressed large subunit synthesized GpppA caps when provided with 5'-triphosphate-terminated poly(A) as a cap acceptor, but was unable to catalyze cap methylation in the presence of S-adenosylmethionine. Thus, the small capping enzyme subunit was shown to be dispensable for guanylylation, but required for cap methylation of RNA. The Mr 95,000 and Mr 60,000 protein-GMP forming activities were resolved during centrifugation in a glycerol gradient; the two forms sedimented at 5.5 S and 4.4 S, respectively, consistent with each enzyme form being a monomer. Either species catalyzed GMP transfer to an RNA acceptor. The isolated Mr 95,000 guanylyltransferase could be converted to an active Mr 60,000 form in vitro by limited proteolysis with trypsin. Expression of carboxyl-deleted forms of the D1 gene product in E. of carboxyl-deleted forms of the D1 gene product in E. coli further localized the guanylyltransferase domain to the amino two-thirds of the Mr 95,000 polypeptide.     

Purification and characterization of the messenger ribonucleic acid capping enzyme GTP:RNA guanylyltransferase from wheat germ

A GTP:RNA guanylyltransferase or capping enzyme has been purified approximately 2000-fold from wheat germ. The enzyme catalyzes the transfer of the GMP residue from GTP to the 5' end of RNA or synthetic polyribonucleotides. Diphosphate-ended polymers were capped more efficiently than molecules with triphosphate ends, and molecules with monophosphate ends were not capped at all. There appears to be little specificity since RNAs with purine or pyrimidine ends served as acceptors. Other features of the wheat germ RNA guanylyltransferase include relatively low Km values for GTP (2.7 microM) and ppA (pA)n (14.2 nM), a divalent cation requirement satisfied by low (0.5 mM) concentrations of MnCl2 or higher (5 mM) concentrations of MgCl2, and a pH optimum around neutrality.     

Expression of baculovirus late and very late genes depends on LEF-4, a component of the viral RNA polymerase whose guanyltransferase function is essential

Baculovirus lef-4 encodes one subunit of the viral RNA polymerase. Here, we demonstrate the essential nature of LEF-4 by RNA interference and bacmid knockout technology. Silencing of LEF-4 in wild-type virus-infected cells suppressed expression of structural genes, while early expression was unaffected, demonstrating its essential role in late gene expression. After transfection of insect cells with lef-4 mutant bacmid, no viral progeny was produced, further defining its central role in infection. Cotransfection with wild-type lef-4 plasmid restored normal replication, but plasmid encoding a guanyltransferase-deficient version failed to rescue. These results emphasize the importance of the mRNA capping function of LEF-4.     

Catalytic activity of vaccinia mRNA capping enzyme subunits coexpressed in Escherichia coli

RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-7)-methyltransferase activities are associated with the vaccinia virus mRNA capping enzyme, a heterodimeric protein containing polypeptides of Mr 95,000 and Mr 31,000. The genes encoding the large and small subunits (corresponding to the D1 and the D12 ORFs, respectively, of the viral genome) were coexpressed in Escherichia coli BL21 (DE3) under the control of a bacteriophage T7 promoter. Guanylyltransferase activity (assayed as the formation of a covalent enzyme-guanylate complex) was detected in soluble lysates of these bacteria. A 1000-fold purification of the guanylyltransferase was achieved by ammonium sulfate precipitation and chromatography using phosphocellulose and SP5PW columns. Partially purified guanylytransferase synthesized GpppA caps when provided with 5'-triphosphate-terminated poly(A) as a cap acceptor. In the presence of AdoMet the enzyme catalyzed concomitant cap methylation with 99% efficiency. Inclusion of S-adenosyl methionine increased both the rate and extent of RNA capping, permitting quantitative modification of RNA 5' ends. Guanylyltransferase sedimented as a single component of 6.5 S during further purification in a glycerol gradient; this S value is identical with that of the heterodimeric capping enzyme from vaccinia virions. Electrophoretic analysis showed a major polypeptide of Mr 95,000 cosedimenting with the guanylyltransferase. RNA triphosphatase activity cosedimented exactly with guanylyltransferase. Methyltransferase activity was associated with guanylyltransferase and was also present in less rapidly sedimenting fractions. The methyltransferase activity profile correlated with the presence of a Mr 31,000 polypeptide. These results indicate that the D1 and D12 gene products are together sufficient to catalyze all three enzymatic steps in cap synthesis. A model for the domain structure of this enzyme is proposed.     

Replication of RNA viruses: specific binding of the Q RNA polymerase to Q RNA

The specific binding in vitro of the Qβ RNA polymerase to Qβ RNA has been detected by the formation of an enzyme-Qβ RNA complex that did not exchange bound RNA molecules and was not dissociated by 0.8 m NaCl. Formation of this nondissociating complex required GTP and two host protein factors, but not ATP, CTP, UTP, or Mg²⁺ ions. GDP, GMP, dGTP, ITP, and β,γ-methylene GTP did not replace GTP in the reaction. Complex formation at 0 °C was not observed, and the rates of the reaction at 30 °C and 25 °C were 41% and 23%, respectively, of the rate at 37 °C. The reaction occurred with intact Qβ RNA and with polycytidylic acid template but not with bacterial or other bacteriophage RNA. With limiting amounts of enzyme, the amount of Qβ RNA bound in the nondissociating complex was the same as the amount of [γ-³²P]GTP incorporated into nascent RNA chains, indicating a close relationship between complex formation and the initiation of RNA synthesis. The two reactions appear to be separate, however, because in the absence of Mg²⁺ ions, when complex formation occurred readily, no RNA synthesis could be detected either by incorporation of labeled substrate into acid-insoluble material or by formation of short RNA chains still attached to the enzyme. In the presence of factor protein and GTP, a maximum of one active enzyme molecule was bound per molecule of Qβ RNA template, as determined by a liquid polymer phase-separation procedure. These results suggest that formation of the nondissociating complex measures recognition by the Qβ RNA polymerase of a single Qβ RNA site utilized for the initiation of synthesis.     

Modification of RNA by mRNA guanylyltransferase and mRNA (guanine-7-)methyltransferase from vaccinia virions.

A purified enzyme system isolated from vaccinia virus cores has been shown to modify the 5' termini of viral mRNA and synthetic poly(A) and poly(G) to form the structures m7G(5')pppA- and m7G(5')pppG-. The enzyme system has both guanylyltransferase and methyltransferase activities. The GTP:mRNA guanylyltransferase activity incorporates GMP into the 5' terminus via a 5'-5' triphosphate bond. The properties of this reaction are: (a) of the four nucleoside triphosphates only GTP is a donor, (b) mRNA with two phosphates at the 5' terminus is an acceptor while RNA with a single 5'-terminal phosphate is not, (c) Mg2+ is required, (d) the pH optimum is 7.8, (e) PP1 is a strong inhibitor, and (f) the reverse reaction, namely the formation of GTP from PP1 and RNA containing the 5'-terminal structure G(5')pppN-, readily occurs. The S-adenosylmethionine:mRNA(guanine-7-)methyltransferase activity catalyzes the methylation of the 5'-terminal guanosine. This reaction exhibits the following characteristics: (a) mRNA with the 5'-terminal sequences G(5')pppA- and G(5')pppG- are acceptors, (b) only position 7 of the terminal guanosine is methylated; internal or conventional 5'-terminal guanosine residues are not methylated, (c) the reaction is not dependent upon GTP or divalent cations, (d) optimal activity is observed in a broad pH range around neutrality, (e) the reaction is inhibited by S-adenosylhomocysteine. Both the guanylyltransferase and methyltransferase reactions exhibit bisubstrate kinetics and proceed via a sequential mechanism. The reactions may be summarized: (see article).     

Heterologous production, purification and characterization of enzymatically active Sindbis virus nonstructural protein nsP1

Alphavirus nonstructural protein nsP1 possesses distinct methyltransferase (MTase) and guanylyltransferase (GTase) activities involved in the capping of viral RNAs. In alphaviruses, the methylation of GTP occurs before RNA transguanylation and nsP1 forms a covalent complex with m(7)GMP unlike the host mRNA guanylyltransferase which forms GMP-enzyme complex. In this study, full length SINV nsP1 was expressed in a soluble form with an N-terminal histidine tag in Escherichia coli and purified to homogeneity. The purified protein is enzymatically active and contains both MTase and GTase activity indicating that SINV nsP1 does not require membrane association for its enzymatic function. Biochemical analysis shows that detergents abolish nsP1 GTase activity, whereas nonionic detergents do not affect MTase activity. Furthermore, SINV nsP1 contains the metal-ion dependent GTase, whereas MTase does not require a metal ion. Circular dichroism spectroscopic analysis of purified protein indicate that nsP1 has a mixed α/β structure and is in the folded native conformation.     

Studies of the silencing of baculovirus DNA binding protein

Baculovirus DNA binding protein (DBP) binds preferentially single-stranded DNA in vitro and colocalizes with viral DNA replication sites. Here, its putative role as viral replication factor has been addressed by RNA interference. Silencing of DBP in Autographa californica multiple nucleopolyhedrovirus-infected cells increased expression of LEF-3, LEF-4, and P35. In contrast, expression of the structural genes coding for P39 and polyhedrin was suppressed while expression of genes coding for P10 and GP64 was unaffected. In the absence of DBP, viral DNA replication sites were formed, indicating replication of viral DNA. Electron microscopy studies, however, revealed a loss of formation of polyhedra and virus envelopment, suggesting that the primary role of DBP is viral formation rather than viral DNA replication.