Identification of an RNA Hairpin in Poliovirus RNA That Serves as the Primary Template in the In Vitro Uridylylation of VPg

doi:10.1128/JVI.74.22.10359-10370.2000

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Journal of Virology, November 2000, p. 10359-10370, Vol. 74, No. 22
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Identification of an RNA Hairpin in Poliovirus RNA That Serves as the Primary Template in the In Vitro Uridylylation of VPg

Aniko V. Paul,¹^,^* Elizabeth Rieder,¹ Dong Wook Kim,¹ Jacques H. van Boom,² and Eckard Wimmer¹

Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook, Stony Brook, New York 11794,¹ and Gorlaeus Laboratory, Leiden University, 2300 RA Leiden, The Netherlands²

Received 10 May 2000/Accepted 17 August 2000

The first step in the replication of the plus-stranded poliovirus RNA is the synthesis of a complementary minus strand. Thisprocess is initiated by the covalent attachment of UMP to theterminal protein VPg, yielding VPgpU and VPgpUpU. We have previouslyshown that these products can be made in vitro in a reaction thatrequires only synthetic VPg, UTP, poly(A), purified poliovirusRNA polymerase 3D^pol, and Mg²⁺ (A. V. Paul, J. H. van Boom, D. Filippov, and E. Wimmer, Nature393:280-284, 1998). Since such a poly(A)-dependent process cannotconfer sufficient specificity to poliovirus RNA replication, wehave developed a new assay to search for a viral RNA templatein conjunction with viral or cellular factors that could providethis function. We have now discovered a small RNA hairpin in thecoding region of protein 2C as the site in PV1(M) RNA that isused as the primary template for the in vitro uridylylation ofVPg. This hairpin has recently been described in poliovirus RNAas being an essential structure for the initiation of minus strandRNA synthesis (I. Goodfellow, Y. Chaudhry, A. Richardson, J. Meredith,J. W. Almond, W. Barclay, and D. J. Evans, J. Virol. 74:4590-4600,2000). The uridylylation reaction either with transcripts of cre(2C)RNA or with full-length PV1(M) RNA as the template is stronglystimulated by the addition of purified viral protein 3CD^pro. Deletion of the cre(2C) RNA sequences from minigenomes eliminatestheir ability to serve as template in the reaction. A similarsignal in the coding region of VP1 in HRV14 RNA (K. L. McKnightand S. M. Lemon, RNA 4:1569-1584, 1998) and the poliovirus cre(2C)can be functionally exchanged in the assay. The mechanism by whichthe VPgpUpU precursor, made specifically on the cre(2C) template,might be transferred to the site where it serves as primer forpoliovirus RNA synthesis, remains to bedetermined.

^* 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.

Journal of Virology, November 2000, p. 10359-10370, Vol. 74, No. 22
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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