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Journal of Virology, May 2000, p. 4590-4600, Vol. 74, No. 10
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Identification of a cis-Acting Replication Element within the Poliovirus Coding Region

Ian Goodfellow,1 Yasmin Chaudhry,1 Andrew Richardson,2 Janet Meredith,2 Jeffrey W. Almond,2,dagger Wendy Barclay,2 and David J. Evans1,*

Division of Virology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow,1 and School of Animal and Microbial Science, The University of Reading, Whiteknights, Reading,2 United Kingdom

Received 4 October 1999/Accepted 16 February 2000

The replication of poliovirus, a positive-stranded RNA virus, requires translation of the infecting genome followed by virus-encoded VPg and 3D polymerase-primed synthesis of a negative-stranded template. RNA sequences involved in the latter process are poorly defined. Since many sequences involved in picornavirus replication form RNA structures, we searched the genome, other than the untranslated regions, for predicted local secondary structural elements and identified a 61-nucleotide (nt) stem-loop in the region encoding the 2C protein. Covariance analysis suggested the structure was well conserved in the Enterovirus genus of the Picornaviridae. Site-directed mutagenesis, disrupting the structure without affecting the 2C product, destroyed genome viability and suggested that the structure was required in the positive sense for function. Recovery of revertant viruses suggested that integrity of the structure was critical for function, and analysis of replication demonstrated that nonviable mutants did not synthesize negative strands. Our conclusion, that this RNA secondary structure constitutes a novel poliovirus cis-acting replication element (CRE), is supported by the demonstration that subgenomic replicons bearing lethal mutations in the native structure can be restored to replication competence by the addition of a second copy of the 61-nt wild-type sequence at another location within the genome. This poliovirus CRE functionally resembles an element identified in rhinovirus type 14 (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 1998) and the cardioviruses (P. E. Lobert, N. Escriou, J. Ruelle, and T. Michiels, Proc. Natl. Acad. Sci. USA 96:11560-11565, 1999) but differs in sequence, structure, and location. The functional role and evolutionary significance of CREs in the replication of positive-sense RNA viruses is discussed.

* Corresponding author. Mailing address: Institute of Virology, University of Glasgow, Church Street, Glasgow G11 5JR. Phone and fax: 44 (0)141 330 6249. E-mail: David.Evans{at}vir.gla.ac.uk.

dagger Present address: Aventis Pasteur SA, Campus Mérieux, F-69280 Marcy-L'Etoile, France.

Journal of Virology, May 2000, p. 4590-4600, Vol. 74, No. 10
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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