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Journal of Virology, December 2003, p. 12996-13004, Vol. 77, No. 24
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.24.12996-13004.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Intra- and Intermolecular Disulfide Bonds of the GP2b Glycoprotein of Equine Arteritis Virus: Relevance for Virus Assembly and Infectivity

Roeland Wieringa,{dagger} Antoine A. F. de Vries,{dagger} Sabine M. Post,{ddagger} and Peter J. M. Rottier*

Department of Infectious Diseases and Immunology, Virology Division, Faculty of Veterinary Medicine, and Institute of Biomembranes, Utrecht University, 3584 CL Utrecht, The Netherlands

Received 23 April 2003/ Accepted 14 September 2003

Equine arteritis virus (EAV) is an enveloped, positive-strand RNA virus belonging to the family Arteriviridae of the order Nidovirales. EAV virions contain six different envelope proteins. The glycoprotein GP5 (previously named GL) and the unglycosylated membrane protein M are the major envelope proteins, while the glycoproteins GP2b (previously named GS), GP3, and GP4 are minor structural proteins. The unglycosylated small hydrophobic envelope protein E is present in virus particles in intermediate molar amounts compared to the other transmembrane proteins. The GP5 and M proteins are both essential for particle assembly. They occur as covalently linked heterodimers that constitute the basic protein matrix of the envelope. The GP2b, GP3, and GP4 proteins occur as a heterotrimeric complex in which disulfide bonds play an important role. The function of this complex has not been established yet, but the available data suggest it to be involved in the viral entry process. Here we investigated the role of the four cysteine residues of the mature GP2b protein in the assembly of the GP2b/GP3/GP4 complex. Open reading frames encoding cysteine-to-serine mutants of the GP2b protein were expressed independently or from a full-length infectious EAV cDNA clone. The results of these experiments support a model in which the cysteine residue at position 102 of GP2b forms an intermolecular cystine bridge with one of the cysteines of the GP4 protein, while the cysteine residues at positions 48 and 137 of GP2b are linked by an intrachain disulfide bond. In this model, another cysteine residue in the GP4 protein is responsible for the covalent association of GP3 with the disulfide-linked GP2b/GP4 heterodimer. In addition, our data highlight the importance of the correct association of the minor EAV envelope glycoproteins for their efficient incorporation into viral particles and for virus infectivity.

* Corresponding author. Mailing address: Department of Infectious Diseases and Immunology, Virology Division, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands. Phone: 31-30-2532462. Fax: 31-30-2536723. E-mail: p.rottier{at}vet.uu.nl.

{dagger} Present address: Gene Therapy Section, Department of Molecular Cell Biology, Leiden University Medical Center (LUMC), 2333 AL Leiden, The Netherlands.

{ddagger} Present address: Gaubius Laboratory, TNO-PG, 2333 CK Leiden, The Netherlands.

Journal of Virology, December 2003, p. 12996-13004, Vol. 77, No. 24
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.24.12996-13004.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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