Structure and function analysis of the poliovirus cis-acting replication element (CRE)

  1. IAN G. GOODFELLOW,
  2. DAVID KERRIGAN, and
  3. DAVID J. EVANS
  1. Faculty of Biomedical and Life Sciences, Division of Virology, University of Glasgow, Church Street, Glasgow, G11 5JR UK

Abstract

The poliovirus cis-acting replication element (CRE) templates the uridylylation of VPg, the protein primer for genome replication. The CRE is a highly conserved structural RNA element in the enteroviruses and located within the polyprotein-coding region of the genome. We have determined the native structure of the CRE, defined the regions of the structure critical for activity, and investigated the influence of genomic location on function. Our results demonstrate that a 14-nucleotide unpaired terminal loop, presented on a suitably stable stem, is all that is required for function. These conclusions complement the recent analysis of the 14-nucleotide terminal loop in the CRE of human rhinovirus type 14. The CRE can be translocated to the 5′ noncoding region of the genome, at least 3.7-kb distant from the native location, without adversely influencing activity, and CRE duplications do not adversely influence replication. We do not have evidence for a specific interaction between the CRE and the RNA-binding 3CDpro complex, an essential component of the uridylylation reaction, and the mechanism by which the CRE is coordinated and orientated during the reaction remains unclear. These studies provide a detailed overview of the structural determinants required for CRE function, and will facilitate a better understanding of the requirements for picornavirus replication.

Keywords

Footnotes

  • 1 The nomenclature used to define bases within the CRE is in the form of native nucleotide, position, substituted nucleotide, with all numbering starting at the base of the CRE, which corresponds to nucleotide 4435 of the genomic sequence of poliovirus type 3 (P3/Leon/37), GenBank accession no. K01392.

  • 2 Standard naming conventions: A pT7/prefix indicates a plasmid with a T7 promoter containing the virus or subgenomic replicon cDNA, pT7/Rep3 was derived from pT7/FLC (Barclay et al. 1998), a ‘‘full-length (infectious) clone’’ of poliovirus type 3 Leon. The resulting replicon or virus generated following transfection are designated Rep3 or FLC, respectively.

  • Article and publication are at http://www.rnajournal.org/cgi/doi/10.1261/rna.2950603.

    • Accepted September 25, 2002.
    • Received July 8, 2002.
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