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

1998 ◽  
Vol 72 (12) ◽  
pp. 10011-10019 ◽  
Author(s):  
Jianping Jin ◽  
Wen Dong ◽  
Linda A. Guarino
Keyword(s):  
Rna Polymerase ◽  
Nuclear Polyhedrosis Virus ◽  
Autographa Californica ◽  
Temperature Sensitive ◽  
Late Gene Expression ◽  
Virus Rna ◽  
Rna Triphosphatase ◽  
Full Complement ◽  
Nuclear Polyhedrosis ◽  
Capping Enzymes

ABSTRACT The baculovirus Autographa californica nuclear polyhedrosis virus encodes a DNA-dependent RNA polymerase that is required for transcription of viral late genes. This polymerase is composed of four equimolar subunits, LEF-8, LEF-4, LEF-9, and p47. The LEF-4 subunit has guanylyltransferase activity, suggesting that baculoviruses may encode a full complement of capping enzymes. Here we show that LEF-4 is a bifunctional enzyme that hydrolyzes the gamma phosphates of triphosphate-terminated RNA and also hydrolyzes ATP and GTP to the respective diphosphate forms. Alanine substitution of five residues previously shown to be essential for vaccinia virus RNA triphosphatase activity inactivated the triphosphatase component of LEF-4 but not the guanylyltransferase domain. Conversely, mutation of the invariant lysine in the guanylyltransferase domain abolished the guanylyltransferase activity without affecting triphosphatase function. We also investigated the effects of substituting phenylalanine for leucine at position 105, a mutation that results in a virus that is temperature sensitive for late gene expression. We found that this mutation had no significant effect on the ATPase or guanylyltransferase activity of LEF-4 but resulted in a modest decrease in RNA triphosphatase activity.

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

1998 ◽  
Vol 72 (12) ◽  
pp. 10020-10028 ◽  
Author(s):  
Christian H. Gross ◽  
Stewart Shuman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Divalent Cation ◽  
Nuclear Polyhedrosis Virus ◽  
Autographa Californica ◽  
Mrna Capping ◽  
Rna Triphosphatase ◽  
Nuclear Polyhedrosis ◽  
Capping Enzymes ◽  
Nucleoside Triphosphatase

ABSTRACT 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 Pi(Km = 43 μM ATP;V max = 30 s−1) and GTP to GDP and Pi. 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 cerevisiaemRNA capping enzymes. We suggest that these proteins comprise a novel family of metal-dependent triphosphatases.

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

1998 ◽  
Vol 72 (12) ◽  
pp. 10003-10010 ◽  
Author(s):  
Linda A. Guarino ◽  
Jianping Jin ◽  
Wen Dong
Keyword(s):  
Rna Polymerase ◽  
Divalent Cation ◽  
Nuclear Polyhedrosis Virus ◽  
Autographa Californica ◽  
Nuclear Targeting ◽  
Late Genes ◽  
Absolute Requirement ◽  
Infected Cells ◽  
Nuclear Polyhedrosis ◽  
Single Subunit

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.

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