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Journal of Virology, May 2008, p. 4363-4370, Vol. 82, No. 9
0022-538X/08/$08.00+0     doi:10.1128/JVI.02087-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Identification of the oriI-Binding Site of Poliovirus 3C Protein by Nuclear Magnetic Resonance Spectroscopy{triangledown}

C. D. Amero,1 J. J. Arnold,2 I. M. Moustafa,2 C. E. Cameron,2* and M. P. Foster1,3*

Biophysics Program, Ohio State University, Columbus, Ohio 43210,1 Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802,2 Department of Biochemistry, Ohio State University, Columbus, Ohio 432103

Received 19 September 2007/ Accepted 21 February 2008

Replication of picornaviral genomes requires recognition of at least three cis-acting replication elements: oriL, oriI, and oriR. Although these elements lack an obvious consensus sequence or structure, they are all recognized by the virus-encoded 3C protein. We have studied the poliovirus 3C-oriI interaction in order to begin to decipher the code of RNA recognition by picornaviral 3C proteins. oriI is a stem-loop structure that serves as the template for uridylylation of the peptide primer VPg by the viral RNA-dependent RNA polymerase. In this report, we have used nuclear magnetic resonance (NMR) techniques to study 3C alone and in complex with two single-stranded RNA oligonucleotides derived from the oriI stem. The 1H-15N spectra of 3C recorded in the presence of these RNAs revealed site-specific chemical shift perturbations. Residues that exhibit significant perturbations are primarily localized in the amino terminus and in a highly conserved loop between residues 81 and 89. In general, the RNA-binding site defined in this study is consistent with predictions based on biochemical and mutagenesis studies. Although some residues implicated in RNA binding by previous studies are perturbed in the 3C-RNA complex reported here, many are unique. These studies provide unique site-specific insight into residues of 3C that interact with RNA and set the stage for detailed structural investigation of the 3C-RNA complex by NMR. Interpretation of our results in the context of an intact oriI provides insight into the architecture of the picornavirus VPg uridylylation complex.

* Corresponding author. Mailing address for Craig E. Cameron: Department of Biochemistry and Molecular Biology, Pennsylvania State University, 201 Althouse Laboratory, University Park, PA 16802. Phone: (814) 863-8705. Fax: (814) 865-7927. E-mail: cec9{at}psu.edu. Mailing address for Mark P. Foster: Department of Biochemistry, Ohio State University, 734 Riffe Building, 484 West 12th Ave., Columbus, OH 43210. Phone: (614) 292-1377. Fax: (614) 292-6773. E-mail: foster.281{at}osu.edu

{triangledown} Published ahead of print on 27 February 2008.

Journal of Virology, May 2008, p. 4363-4370, Vol. 82, No. 9
0022-538X/08/$08.00+0     doi:10.1128/JVI.02087-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Pathak, H. B., Oh, H. S., Goodfellow, I. G., Arnold, J. J., Cameron, C. E. (2008). Picornavirus Genome Replication: ROLES OF PRECURSOR PROTEINS AND RATE-LIMITING STEPS IN oriI-DEPENDENT VPg URIDYLYLATION. J. Biol. Chem. 283: 30677-30688 [Abstract] [Full Text]  


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