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Journal of Virology, February 2008, p. 1214-1228, Vol. 82, No. 3
0022-538X/08/$08.00+0     doi:10.1128/JVI.01690-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Genetic Interactions between an Essential 3' cis-Acting RNA Pseudoknot, Replicase Gene Products, and the Extreme 3' End of the Mouse Coronavirus Genome{triangledown}

Roland Züst,1 Timothy B. Miller,2 Scott J. Goebel,2 Volker Thiel,1 and Paul S. Masters2*

Research Department, Kantonal Hospital St. Gallen, St. Gallen, Switzerland,1 Wadsworth Center, New York State Department of Health, Albany, New York 122012

Received 3 August 2007/ Accepted 11 November 2007

The upstream end of the 3' untranslated region (UTR) of the mouse hepatitis virus genome contains two essential and overlapping RNA secondary structures, a bulged stem-loop and a pseudoknot, which have been proposed to be elements of a molecular switch that is critical for viral RNA synthesis. It has previously been shown that a particular six-base insertion in loop 1 of the pseudoknot is extremely deleterious to the virus. We have now isolated multiple independent second-site revertants of the loop 1 insertion mutant, and we used reverse-genetics methods to confirm the identities of suppressor mutations that could compensate for the original insertion. The suppressors were localized to two separate regions of the genome. Members of one class of suppressor were mapped to the portions of gene 1 that encode nsp8 and nsp9, thereby providing the first evidence for specific interactions between coronavirus replicase gene products and a cis-acting genomic RNA element. The second class of suppressor was mapped to the extreme 3' end of the genome, a result which pointed to the existence of a direct base-pairing interaction between loop 1 of the pseudoknot and the genomic terminus. The latter finding was strongly supported by phylogenetic evidence and by the construction of a deletion mutant that reduced the 3' UTR to its minimal essential elements. Taken together, the interactions revealed by the two classes of suppressors suggest a model for the initiation of coronavirus negative-strand RNA synthesis.

* Corresponding author. Mailing address: David Axelrod Institute, Wadsworth Center, NYSDOH, New Scotland Avenue, P.O. Box 22002, Albany, New York 12201-2002. Phone: (518) 474-1283. Fax: (518) 473-1326. E-mail: masters{at}wadsworth.org

{triangledown} Published ahead of print on 21 November 2007.

Journal of Virology, February 2008, p. 1214-1228, Vol. 82, No. 3
0022-538X/08/$08.00+0     doi:10.1128/JVI.01690-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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