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Journal of Virology, May 2001, p. 4226-4238, Vol. 75, No. 9
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.9.4226-4238.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Efficient Translation Initiation Is Required for Replication of Bovine Viral Diarrhea Virus Subgenomic Replicons

Tina M. Myers,1 Victoria G. Kolupaeva,2 Ernesto Mendez,1 Scott G. Baginski,1 Ilya Frolov,1 Christopher U. T. Hellen,2 and Charles M. Rice1,*

Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110,1 and Department of Microbiology and Immunology, State University of New York Health Science Center at Brooklyn, Brooklyn, New York 112032

Received 9 October 2000/Accepted 13 February 2001

An internal ribosome entry site (IRES) mediates translation initiation of bovine viral diarrhea virus (BVDV) RNA. Studies have suggested that a portion of the Npro open reading frame (ORF) is required, although its exact function has not been defined. Here we show that a subgenomic (sg) BVDV RNA in which the NS3 ORF is preceded only by the 5' nontranslated region did not replicate to detectable levels following transfection. However, RNA synthesis and cytopathic effects were observed following serial passage in the presence of a noncytopathic helper virus. Five sg clones derived from the passaged virus contained an identical, silent substitution near the beginning of the NS3 coding sequence (G400U), as well as additional mutations. Four of the reconstructed mutant RNAs replicated in transfected cells, and in vitro translation showed increased levels of NS3 for the mutant RNAs compared to that of wild-type (wt) MetNS3. To more precisely dissect the role of these mutations, we constructed two sg derivatives: ad3.10, which contains only the G400U mutation, and ad3.7, with silent substitutions designed to minimize RNA secondary structure downstream of the initiator AUG. Both RNAs replicated and were translated in vitro to similar levels. Moreover, ad3.7 and ad3.10, but not wt MetNS3, formed toeprints downstream of the initiator AUG codon in an assay for detecting the binding of 40S ribosomal subunits and 43S ribosomal complexes to the IRES. These results suggest that a lack of stable RNA secondary structure(s), rather than a specific RNA sequence, immediately downstream of the initiator AUG is important for optimal translation initiation of pestivirus RNAs.

* Corresponding author. Present address: Center for the Study of Hepatitis C, The Rockefeller University, Box 64, 1230 York Ave., New York, NY 10021. Phone: (212) 327-7046. Fax: (212) 327-7048. E-mail: ricec{at}rockefeller.edu.

Journal of Virology, May 2001, p. 4226-4238, Vol. 75, No. 9
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.9.4226-4238.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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