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Journal of Virology, April 2001, p. 3111-3120, Vol. 75, No. 7
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.7.3111-3120.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Phenotypic Characterization of Three Phylogenetically Conserved Stem-Loop Motifs in the Mengovirus 3' Untranslated Region

Hernando Duque and Ann C. Palmenberg*

Institute for Molecular Virology and Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706

Received 28 June 1999/Accepted 4 January 2001

An alignment of cardiovirus sequences led to the prediction of three conserved stem-loops in the 3' untranslated region (UTR) of mengovirus. Deletions of each stem were engineered in mengovirus cDNAs and also in mengovirus replicons, in which part of the viral capsid sequences were replaced with the firefly luciferase gene. The effect of deletion on RNA infectivity and plaque phenotype was evaluated after transfection of viral transcripts into HeLa cells or by luciferase assays of cellular extracts after transfection with RNA replicons. Stem I (mengovirus bases 7666 to 7687) was found to be dispensable for viral growth or exponential luciferase expression. Deletion of stem III (bases 7711 to 7721) was lethal to the virus, and the replicons were incapable of RNA synthesis. Deletion of stem II (Delta II; bases 7692 to 7705) produced an intermediate phenotype, in that replicons had marginal RNA synthesis activity but transfection with genomic RNA usually failed to produce plaques after normal incubation times (31 h, 37°C). In a few of the Delta II transfections, however, plaques were observed after long incubation, especially if the cells received large amounts of RNA (3 µg per 3 × 106 cells). Viruses from two Delta II-derived plaques were isolated and amplified. Their RNAs were converted into cDNA, sequenced, and mapped for genotype. Each maintained the Delta II deletion and, in addition, had one or two reversion mutations, which were characterized by reverse genetics as responsible for the phenotypes. One reversion caused an amino acid change in the polymerase (3Dpol), and the other was localized to the 3' UTR, upstream of stem I.

* Corresponding author. Mailing address: Department of Biochemistry, 433 Babcock Dr., Madison, WI 53706. Phone: (608) 262-7519. Fax: (608) 262-6690. E-mail: acpalmen{at}facstaff.wisc.edu.

Journal of Virology, April 2001, p. 3111-3120, Vol. 75, No. 7
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.7.3111-3120.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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