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Journal of Virology, July 1999, p. 5274-5281, Vol. 73, No. 7
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Characterization of an Equine Arteritis Virus Replicase Mutant Defective in Subgenomic mRNA Synthesis

Guido van Marle, Leonie C. van Dinten, Willy J. M. Spaan, Willem Luytjes, and Eric J. Snijder*

Department of Virology, Leiden University Medical Center, Leiden, The Netherlands

Received 11 December 1998/Accepted 29 March 1999

Equine arteritis virus (EAV) is a positive-stranded RNA virus that synthesizes a 5'- and 3'-coterminal nested set of six subgenomic mRNAs. These mRNAs all contain a common leader sequence which is derived from the 5' end of the genome. Subgenomic mRNA transcription and genome replication are directed by the viral replicase, which is expressed in the form of two polyproteins and subsequently processed into smaller nonstructural proteins (nsps). During the recent construction of an EAV infectious cDNA clone (pEAV030 [L. C. van Dinten, J. A. den Boon, A. L. M. Wassenaar, W. J. M. Spaan, and E. J. Snijder, Proc. Natl. Acad. Sci. USA 94:991-996, 1997]), a mutant cDNA clone (pEAV030F) which carries a single replicase point mutation was obtained. This substitution (Ser-2429right-arrowPro) is located in the nsp10 subunit and renders the EAV030F virus deficient in subgenomic mRNA synthesis. To obtain more insight into the role of nsp10 in transcription and the nature of the transcriptional defect, we have now analyzed the EAV030F mutant in considerable detail. The Ser-2429right-arrowPro mutation does not affect the proteolytic processing of the replicase but apparently affects the function of nsp10 in transcription. Furthermore, our study showed that EAV030F still produces subgenomic positive and negative strands, albeit at a very low level. Both subgenomic positive-strand synthesis and negative-strand synthesis are equally affected by the Ser-2429right-arrowPro mutation, suggesting that nsp10 plays an important role in an early step of EAV mRNA transcription.

* Corresponding author. Mailing address: Department of Virology, Leiden University Medical Center, LUMC P4-26, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Phone: 31 71 5261657. Fax: 31 71 5266761. E-mail: Snijder{at}Virology.AZL.NL.

Journal of Virology, July 1999, p. 5274-5281, Vol. 73, No. 7
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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