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Journal of Virology, November 2001, p. 10979-10990, Vol. 75, No. 22
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.22.10979-10990.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Biochemical and Genetic Studies of the Initiation of Human Rhinovirus 2 RNA Replication: Identification of a cis-Replicating Element in the Coding Sequence of 2Apro

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

We have previously shown that the RNA polymerase 3Dpol of human rhinovirus 2 (HRV2) catalyzes the covalent linkage of UMP to the terminal protein (VPg) using poly(A) as a template (K. Gerber, E. Wimmer, and A. V. Paul, J. Virol. 75:10969-10978, 2001). The products of this in vitro reaction are VPgpU, VPgpUpU, and VPg-poly(U), the 5' end of minus-strand RNA. In the present study we used an assay system developed for poliovirus 3Dpol (A. V. Paul, E. Rieder, D. W. Kim, J. H. van Boom, and E. Wimmer, J. Virol. 74: 10359-10370, 2000) to search for a viral sequence or structure in HRV2 RNA that would provide specificity to this reaction. We now show that a small hairpin in HRV2 RNA [cre(2A)], located in the coding sequence of 2Apro, serves as the primary template for HRV2 3Dpol in the uridylylation of HRV2 VPg, yielding VPgpU and VPgpUpU. The in vitro reaction is strongly stimulated by the addition of purified HRV2 3CDpro. Our analyses suggest that HRV2 3Dpol uses a "slide-back" mechanism during synthesis of the VPg-linked precursors. The corresponding cis- replicating RNA elements in the 2CATPase coding region of poliovirus type 1 Mahoney (I. Goodfellow, Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans, J. Virol. 74:4590-4600, 2000) and VP1 of HRV14 (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 1998) can be functionally exchanged in the assay with cre(2A) of HRV2. Mutations of either the first or the second A in the conserved A1A2A3CA sequence in the loop of HRV2 cre(2A) abolished both viral growth and the RNA's ability to serve as a template in the in vitro 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. 10979-10990, Vol. 75, No. 22
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.22.10979-10990.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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