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

doi:10.1128/JVI.75.22.10969-10978.2001

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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 3D^pol

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-encodedRNA polymerase. We have expressed in Escherichia coli and purifiedboth a glutathione S-transferase fusion polypeptide and an untaggedform of the HRV2 RNA polymerase 3D^pol. Using in vitro assay systems previously described for poliovirusRNA polymerase 3D^pol (J. B. Flanegan and D. Baltimore, Proc. Natl. Acad. Sci. USA74:3677-3680, 1977; A. V. Paul, J. H. van Boom, D. Filippov, andE. Wimmer, Nature 393:280-284, 1998), we have analyzed the biochemicalproperties of the two different enzyme preparations. HRV2 3D^pol is both template and primer dependent, and it catalyzes two typesof synthetic reactions in the presence of UTP, Mn²⁺, and a poly(A) template. The first consists of an elongationreaction of an oligo(dT)₁₅ primer into poly(U). The second isa protein-priming reaction in which the enzyme covalently linksUMP to the hydroxyl group of tyrosine in the terminal proteinVPg, yielding VPgpU. This precursor is elongated first into VPgpUpUand then into VPg-linked poly(U), which is identical to the 5'end of picornavirus minus strands. The two forms of the enzymeare about equally active both in the oligonucleotide elongationand in the VPg-primed reaction. Various synthetic mutant VPgswere tested as substrates in the VPg uridylylationreaction.

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

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