Formation of disulfide-linked complexes between the three minor envelope glycoproteins (GP2b, GP3, and GP4) of equine arteritis virus

Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order Nidovirales. Six transmembrane proteins have been identified in EAV particles: the nonglycosylated membrane protein M and the glycoprotein GP(s) (previously named G(L)), which occur as disulfide-bonded heterodimers and are the major viral envelope proteins; the unglycosylated small envelope protein E; and the minor glycoproteins GP(2b) (formerly designated G(s)), GP(3), and GP(4). Analysis of the appearance of the GP(2b), GP(3), and GP(4) proteins in viral particles by gel electrophoresis under reducing and nonreducing conditions revealed the occurrence of two different covalently linked oligomeric complexes between these proteins, i.e., heterodimers of GP(2b) and GP(4) and heterotrimers of GP(2b), GP(3), and GP(4). Shortly after their release from infected cells, virions contained mainly cystine-linked GP(2b)/GP(4) heterodimers, which were subsequently converted into disulfide-bonded GP(2b)/GP(3)/GP(4) trimers through the covalent recruitment of GP(3). This process occurred faster at a higher pH but was arrested at 4degreesC. Furthermore, the conversion was almost instantaneous in the presence of the thiol oxidant diamide. In contrast, the sulfhydryl-modifying agent N-ethylmaleimide inhibited the formation of disulfide-bonded GP(2b)/GP(3)/GP(4) trimers. Using sucrose density gradients, we could not demonstrate a noncovalent association of GP(3) with the cystine-linked GP(2b)/GP(4) dimer in freshly released virions, nor did we observe higher-order structures of the GP(2b)/GP(4) or GP(2b)/GP(3)/GP(4) complexes. Nevertheless, the instantaneous diamide-induced formation of disulfide-bonded GP(2b)/GP(3)/GP(4) heterotrimers at 4degreesC suggests that the three minor glycoproteins of EAV are assembled as trimeric complexes. The existence of a noncovalent interaction between the cystine-linked GP(2b)/GP(4) dimer and GP(3) was also inferred from coexpression experiments showing that the presence of GP(3) increased the electrophoretic mobility of the disulfide-bonded GP(2b)/ GP(4) dimers. Our study reveals that the minor envelope proteins of arteriviruses enter into both covalent and noncovalent interactions, the function of which has yet to be established.