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Journal of Virology, December 1998, p. 10003-10010, Vol. 72, No. 12
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

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

Linda A. Guarino,1,2,3,* Jianping Jin,1,3 and Wen Dong2,3

Departments of Biochemistry and Biophysics1 and Entomology2 and Center for Advanced Invertebrate Molecular Sciences,3 Texas A&M University, College Station, Texas 77843-2128

Received 8 June 1998/Accepted 9 September 1998

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.

* Corresponding author. Mailing address: Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843-2128. Phone: (409) 845-7556. Fax: (409) 845-9274. E-mail: lguarino{at}tamu.edu.

Journal of Virology, December 1998, p. 10003-10010, Vol. 72, No. 12
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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