Guanylyltransferase Activity of the LEF-4 Subunit of Baculovirus RNA Polymerase

Abstract

The baculovirus Autographa californica nuclear polyhedrosis virus encodes a DNA-dependent RNA polymerase that transcribes viral late genes. This polymerase is composed of four equimolar subunits, LEF-4, LEF-8, LEF-9, and p47. Here we present data indicating that the LEF-4 subunit of RNA polymerase is a guanylyltransferase. Incubation of RNA polymerase in the presence of divalent cation and radiolabeled GTP resulted in the formation of a covalent enzyme-guanylate complex that comigrated with the LEF-4 subunit. The label transfer assay showed an absolute requirement for divalent cation which could be satisfied by either manganese or magnesium. The reaction was specific for guanine nucleotides, and GTP was more effective than dGTP in the formation of enzyme-guanylate complex. To demonstrate that LEF-4 was the guanylyltransferase, the single subunit was overexpressed in baculovirus-infected cells. The overexpressed protein was primarily cytosolic, indicating that other proteins in the RNA polymerase complex were responsible for nuclear targeting of LEF-4. LEF-4 alone was able to covalently bind GMP, although less efficiently than viral RNA polymerase.

The LEF-4 Subunit of Baculovirus RNA Polymerase Has RNA 5′-Triphosphatase and ATPase Activities

“…The specific activities of the mutant and wild-type enzymes were determined within the linear range of enzymatic activity. With the wild-type enzyme, 0.5% of the available binding sites were radiolabeled, comparable to the level observed with native LEF-4 produced in baculovirus-infected cells (12). The activities of the triphosphatase mutants were equivalent to that of the wild-type protein, with the exception of that of E9/11A, which was 2.5-fold higher than the activity of the wild-type protein.…”

“…RNA triphosphatase activity of RNA polymerase and purified LEF-4. We have previously shown that the LEF-4 subunit of AcNPV RNA polymerase has guanylyltransferase activity, suggesting that LEF-4 is an RNA capping enzyme (12). Furthermore, analysis of the LEF-4 amino acid sequence revealed homology to six motifs that are shared among viral and cellular guanylyltransferases in the C-terminal half of the protein.…”

“…3). LEF-4 was overexpressed in baculovirus-infected cells and purified to near homogeneity by heparin-agarose and Mono Q chromatography as previously described (12). Purified LEF-4 was then filtered through a Superdex 200 gel exclusion column.…”

“…To confirm that the RNA triphosphatase and ATPase activities observed were attributable to LEF-4, we expressed three mutated versions of LEF-4. Four glutamates and one arginine corresponding to residues previously shown to be essential for vaccinia virus triphospha- (12). Fractions corresponding to the peak of absorbance at 280 nm were assayed for guanylyltransferase, RNA triphosphatase (RTPase), and ATPase activities as described in the legend to Fig.…”

“…The accompanying report (12) provides a partial answer to this question with its demonstration that the LEF-4 subunit of Autographa californica nuclear polyhedrosis virus (AcNPV) RNA polymerase has guanylyltransferase activity. Further-more, analysis of the LEF-4 amino acid sequence revealed the presence of a conserved KxDG motif and homology to five additional motifs that are common to viral and cellular guanylyltransferases, which strongly supports our enzymatic data.…”