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Intra- and Intermolecular Disulfide Bonds of the GP_2b Glycoprotein of Equine Arteritis Virus: Relevance for Virus Assembly and Infectivity

Roeland Wieringa, Antoine A. F. de Vries, Sabine M. Post, 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 G_L) and the unglycosylated membrane protein M are the major envelope proteins, while the glycoproteins GP_2b (previously named G_S), 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 GP_2b, 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 GP_2b protein in the assembly of the GP_2b/GP₃/GP₄ complex. Open reading frames encoding cysteine-to-serine mutants of the GP_2b 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 GP_2b forms an intermolecular cystine bridge with one of the cysteines of the GP₄ protein, while the cysteine residues at positions 48 and 137 of GP_2b 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 GP_2b/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.

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

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

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