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Journal of Virology, August 1999, p. 6335-6345, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Identification of a Novel Structural Protein of Arteriviruses

Eric J. Snijder,1,* Hans van Tol,1 Ketil W. Pedersen,2 Martin J. B. Raamsman,3 and Antoine A. F. de Vries3,dagger

Department of Virology, Leiden University Medical Center, Leiden,1 and Virology Unit, Department of Infectious Diseases and Immunology, Veterinary Faculty, Utrecht University, Utrecht,3 The Netherlands, and Division of Electron Microscopy, Department of Biology, University of Oslo, Oslo, Norway2

Received 24 November 1998/Accepted 12 April 1999

Arteriviruses are positive-stranded RNA viruses with an efficiently organized, polycistronic genome. A short region between the replicase gene and open reading frame (ORF) 2 of the equine arteritis virus (EAV) genome was previously assumed to be untranslated. However, here we report that this segment of the EAV genome contains the 5' part of a novel gene (ORF 2a) which is conserved in all arteriviruses. The 3' part of EAV ORF 2a overlaps with the 5' part of the former ORF 2 (now renamed ORF 2b), which encodes the GS glycoprotein. Both ORF 2a and ORF 2b appear to be expressed from mRNA 2, which thereby constitutes the first proven example of a bicistronic mRNA in arteriviruses. The 67-amino-acid protein encoded by EAV ORF 2a, which we have provisionally named the envelope (E) protein, is very hydrophobic and has a basic C terminus. An E protein-specific antiserum was raised and used to demonstrate the expression of the novel gene in EAV-infected cells. The EAV E protein proved to be very stable, did not form disulfide-linked oligomers, and was not N-glycosylated. Immunofluorescence and immunoelectron microscopy studies showed that the E protein associates with intracellular membranes both in EAV-infected cells and upon independent expression. An analysis of purified EAV particles revealed that the E protein is a structural protein. By using reverse genetics, we demonstrated that both the EAV E and GS proteins are essential for the production of infectious progeny 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.

dagger Present address: Center for Biotechnology (CBT), Department of Biosciences at Novum, Karolinska Institutet, 14157 Huddinge, Sweden.

Journal of Virology, August 1999, p. 6335-6345, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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