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Journal of Virology, November 2007, p. 12485-12495, Vol. 81, No. 22
0022-538X/07/$08.00+0     doi:10.1128/JVI.00972-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Human Rhinovirus Type 14 Gain-of-Function Mutants for oriI Utilization Define Residues of 3C(D) and 3Dpol That Contribute to Assembly and Stability of the Picornavirus VPg Uridylylation Complex{triangledown}

Miaoqing Shen,1,{dagger} Qixin Wang,1 Yan Yang,2,{ddagger} Harsh B. Pathak,1,§ Jamie J. Arnold,1 Christian Castro,1 Stanley M. Lemon,2 and Craig E. Cameron1*

Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802,1 Institute for Human Infections and Immunity and Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas 77555-10732

Received 5 May 2007/ Accepted 30 August 2007

VPg linkage to the 5' ends of picornavirus RNAs requires production of VPg-pUpU. VPg-pUpU is templated by an RNA stem-loop (the cre or oriI) found at different locations in picornavirus genomes. At least one adaptive mutation is required for human rhinovirus type 14 (HRV-14) to use poliovirus type 3 (PV-3) or PV-1 oriI efficiently. One mutation changes Leu-94 of 3C to Pro; the other changes Asp-406 of 3Dpol to Asn. By using an in vitro VPg uridylylation system for HRV-14 that recapitulates biological phenotypes, we show that the 3C adaptive mutation functions at the level of 3C(D) and the 3D adaptive mutation functions at the level of 3Dpol. Pro-94 3C(D) has an expanded specificity and enhanced stability relative to wild-type 3C(D) that leads to production of more processive uridylylation complexes. PV-1/HRV-14 oriI chimeras reveal sequence specificity in 3C(D) recognition of oriI that resides in the upper stem. Asn-406 3Dpol is as active as wild-type 3Dpol in RNA-primed reactions but exhibits greater VPg uridylylation activity due to more efficient recruitment to and retention in the VPg uridylylation complex. Asn-406 3Dpol from PV-1 exhibits identical behavior. These studies suggest a two-step binding mechanism in the assembly of the 3C(D)-oriI complex that leads to unwinding of at least the upper stem of oriI and provide additional support for a direct interaction between the back of the thumb of 3Dpol and 3C that is required for 3Dpol recruitment to and retention in the uridylylation complex.

* Corresponding author. Mailing address: Pennsylvania State University, Department of Biochemistry and Molecular Biology, 201 Althouse Laboratory, University Park, PA 16802. Phone: (814) 863-8705. Fax: (814) 865-7927. E-mail: cec9{at}psu.edu

{triangledown} Published ahead of print on 12 September 2007.

{dagger} Present address: Department of Biomedical Sciences, Cornell University, Ithaca, NY 14853.

{ddagger} Present address: Department of Molecular and Cellular Oncology, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030.

§ Present address: Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, PA 19111.

Journal of Virology, November 2007, p. 12485-12495, Vol. 81, No. 22
0022-538X/07/$08.00+0     doi:10.1128/JVI.00972-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Oh, H. S., Pathak, H. B., Goodfellow, I. G., Arnold, J. J., Cameron, C. E. (2009). Insight into Poliovirus Genome Replication and Encapsidation Obtained from Studies of 3B-3C Cleavage Site Mutants. J. Virol. 83: 9370-9387 [Abstract] [Full Text]  
  • 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]  
  • Yang, Y., Yi, M., Evans, D. J., Simmonds, P., Lemon, S. M. (2008). Identification of a Conserved RNA Replication Element (cre) within the 3Dpol-Coding Sequence of Hepatoviruses. J. Virol. 82: 10118-10128 [Abstract] [Full Text]  
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