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Journal of Virology, January 2003, p. 891-904, Vol. 77, No. 2
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.2.891-904.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Biochemical and Genetic Studies of the VPg Uridylylation Reaction Catalyzed by the RNA Polymerase of Poliovirus

Aniko V. Paul,1 Julia Peters,1 JoAnn Mugavero,1 Jiang Yin,1 Jacques H. van Boom,2 and E. Wimmer1*

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

Received 23 May 2002/ Accepted 7 October 2002

The first step in poliovirus (PV) RNA synthesis is the covalent linkage of UMP to the terminal protein VPg. This reaction can be studied in vitro with two different assays. The simpler assay is based on a poly(A) template and requires synthetic VPg, purified RNA polymerase 3Dpol, UTP, and a divalent cation. The other assay uses specific viral sequences [cre(2C)] as a template for VPg uridylylation and requires the addition of proteinase 3CDpro. Using one or both of these assays, we analyzed the VPg specificities and metal requirements of the uridylylation reactions. We determined the effects of single and double amino acid substitutions in VPg on the abilities of the peptides to serve as substrates for 3Dpol. Mutations in VPg, which interfered with uridylylation in vitro, were found to abolish viral growth. A chimeric PV containing the VPg of human rhinovirus 14 (HRV14) was viable, but substitutions of HRV2 and HRV89 VPgs for PV VPg were lethal. Of the three rhinoviral VPgs tested, only the HRV14 peptide was found to function as a substrate for PV1(M) 3Dpol in vitro. We also examined the metal specificity of the VPg uridylylation reaction on a poly(A) template. Our results show a strong preference of the RNA polymerase for Mn2+ as a cofactor compared to Mg2+ or other divalent cations.

* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook, Stony Brook, NY 11794. Phone: (631) 632-8787. Fax: (631) 632-8891. E-mail: ewimmer{at}ms.cc.sunysb.edu.

Journal of Virology, January 2003, p. 891-904, Vol. 77, No. 2
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.2.891-904.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Puustinen, P., Makinen, K. (2004). Uridylylation of the Potyvirus VPg by Viral Replicase NIb Correlates with the Nucleotide Binding Capacity of VPg. J. Biol. Chem. 279: 38103-38110 [Abstract] [Full Text]  
  • Thiviyanathan, V., Yang, Y., Kaluarachchi, K., Rijnbrand, R., Gorenstein, D. G., Lemon, S. M. (2004). High-resolution structure of a picornaviral internal cis-acting RNA replication element (cre). Proc. Natl. Acad. Sci. USA 101: 12688-12693 [Abstract] [Full Text]  


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