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Journal of Virology, November 2001, p. 10969-10978, Vol. 75, No. 22
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.22.10969-10978.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Biochemical and Genetic Studies of the Initiation of Human Rhinovirus 2 RNA Replication: Purification and Enzymatic Analysis of the RNA-Dependent RNA Polymerase 3Dpol

Kinga Gerber, Eckard Wimmer, and Aniko V. Paul*

Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook, Stony Brook, New York 11794

Received 5 April 2001/Accepted 9 August 2001

The replication of human rhinovirus 2 (HRV2), a positive-stranded RNA virus belonging to the Picornaviridae, requires a virus-encoded RNA polymerase. We have expressed in Escherichia coli and purified both a glutathione S-transferase fusion polypeptide and an untagged form of the HRV2 RNA polymerase 3Dpol. Using in vitro assay systems previously described for poliovirus RNA polymerase 3Dpol (J. B. Flanegan and D. Baltimore, Proc. Natl. Acad. Sci. USA 74:3677-3680, 1977; A. V. Paul, J. H. van Boom, D. Filippov, and E. Wimmer, Nature 393:280-284, 1998), we have analyzed the biochemical properties of the two different enzyme preparations. HRV2 3Dpol is both template and primer dependent, and it catalyzes two types of synthetic reactions in the presence of UTP, Mn2+, and a poly(A) template. The first consists of an elongation reaction of an oligo(dT)15 primer into poly(U). The second is a protein-priming reaction in which the enzyme covalently links UMP to the hydroxyl group of tyrosine in the terminal protein VPg, yielding VPgpU. This precursor is elongated first into VPgpUpU and then into VPg-linked poly(U), which is identical to the 5' end of picornavirus minus strands. The two forms of the enzyme are about equally active both in the oligonucleotide elongation and in the VPg-primed reaction. Various synthetic mutant VPgs were tested as substrates in the VPg uridylylation reaction.

* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook, Stony Brook, NY 11794. Phone: (631) 632-9777. Fax: (631) 632-8891. E-mail: apaul{at}ms.cc.sunysb.edu.

Journal of Virology, November 2001, p. 10969-10978, Vol. 75, No. 22
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.22.10969-10978.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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