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Journal of Virology, March 1999, p. 2027-2037, Vol. 73, No. 3
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Proteolytic Processing of the Open Reading Frame 1b-Encoded Part of Arterivirus Replicase Is Mediated by nsp4 Serine Protease and Is Essential for Virus Replication

Leonie C. van Dinten,1 Sietske Rensen,1 Alexander E. Gorbalenya,1,2,3 and Eric J. Snijder1,*

Department of Virology, Leiden University Medical Center, Leiden, The Netherlands1; M. P. Chumakov Institute of Poliomyelitis and Viral Encephalitides, Russian Academy of Medical Sciences, 142782 Moscow Region, Russia2; and Advanced Biomedical Computer Center, SAIC/NCI-FCRDC, Frederick, Maryland 21702-12013

Received 2 September 1998/Accepted 10 December 1998

The open reading frame (ORF) 1b-encoded part of the equine arteritis virus (EAV) replicase is expressed by ribosomal frameshifting during genome translation, which results in the production of an ORF1ab fusion protein (345 kDa). Four ORF1b-encoded processing products, nsp9 (p80), nsp10 (p50), nsp11 (p26), and nsp12 (p12), have previously been identified in EAV-infected cells (L. C. van Dinten, A. L. M. Wassenaar, A. E. Gorbalenya, W. J. M. Spaan, and E. J. Snijder, J. Virol. 70:6625-6633, 1996). In the present study, the generation of these four nonstructural proteins was shown to be mediated by the nsp4 serine protease, which is the main viral protease (E. J. Snijder, A. L. M. Wassenaar, L. C. van Dinten, W. J. M. Spaan, and A. E. Gorbalenya, J. Biol. Chem. 271:4864-4871, 1996). Mutagenesis of candidate cleavage sites revealed that Glu-2370/Ser, Gln-2837/Ser, and Glu-3056/Gly are the probable nsp9/10, nsp10/11, and nsp11/12 junctions, respectively. Mutations which abolished ORF1b protein processing were introduced into a recently developed infectious cDNA clone (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 94:991-997, 1997). An analysis of these mutants showed that the selective blockage of ORF1b processing affected different stages of EAV reproduction. In particular, the mutant with the nsp10/11 cleavage site mutation Gln-2837right-arrowPro displayed an unusual phenotype, since it was still capable of RNA synthesis but was incapable of producing infectious virus.

* 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, March 1999, p. 2027-2037, Vol. 73, No. 3
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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