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Journal of Virology, June 2008, p. 5835-5846, Vol. 82, No. 12
0022-538X/08/$08.00+0     doi:10.1128/JVI.01513-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Poly(rC) Binding Proteins and the 5' Cloverleaf of Uncapped Poliovirus mRNA Function during De Novo Assembly of Polysomes{triangledown}

Brian J. Kempf1 and David J. Barton1,2*

Department of Microbiology,1 Program in Molecular Biology, University of Colorado Denver, School of Medicine, Aurora, Colorado 800452

Received 10 July 2007/ Accepted 28 March 2008

Poliovirus (PV) mRNA is unusual because it possesses a 5'-terminal monophosphate rather than a 5'-terminal cap. Uncapped mRNAs are typically degraded by the 5' exonuclease XRN1. A 5'-terminal cloverleaf RNA structure interacts with poly(rC) binding proteins (PCBPs) to protect uncapped PV mRNA from 5' exonuclease (K. E. Murray, A. W. Roberts, and D. J. Barton, RNA 7:1126-1141, 2001). In this study, we examined de novo polysome formation using HeLa cell-free translation-replication reactions. PV mRNA formed polysomes coordinate with the time needed for ribosomes to traverse the viral open reading frame (ORF). Nascent PV polypeptides cofractionated with viral polysomes, while mature PV proteins were released from the polysomes. Alterations in the size of the PV ORF correlated with alterations in the size of polysomes with ribosomes present every 250 to 500 nucleotides of the ORF. Eukaryotic initiation factor 4GI (eIF4GI) was cleaved rapidly as viral polysomes assembled and the COOH-terminal portion of eIF4GI cofractionated with viral polysomes. Poly(A) binding protein, along with PCBP 1 and 2, also cofractionated with viral polysomes. A C24A mutation that inhibits PCBP-5'-terminal cloverleaf RNA interactions inhibited the formation and stability of nascent PV polysomes. Kinetic analyses indicated that the PCBP-5' cloverleaf RNA interaction was necessary to protect PV mRNA from 5' exonuclease immediately as ribosomes initially traversed the viral ORF, before viral proteins could alter translation factors within nascent polysomes or contribute to ribonucleoprotein complexes at the termini of the viral mRNA.

* Corresponding author. Mailing address: Department of Microbiology, University of Colorado Denver, School of Medicine, Mail Stop 8333, RC1-North, Room P18-9116, 12800 E. 19th Ave., Aurora, CO 80045. Phone: (303) 724-4215. Fax: (303) 724-4226. E-mail: david.barton{at}uchsc.edu

{triangledown} Published ahead of print on 9 April 2008.

Journal of Virology, June 2008, p. 5835-5846, Vol. 82, No. 12
0022-538X/08/$08.00+0     doi:10.1128/JVI.01513-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Kempf, B. J., Barton, D. J. (2008). Poliovirus 2APro Increases Viral mRNA and Polysome Stability Coordinately in Time with Cleavage of eIF4G. J. Virol. 82: 5847-5859 [Abstract] [Full Text]  


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