Formation of Disulfide-Linked Complexes between the Three Minor Envelope Glycoproteins (GP2b, GP3, and GP4) of Equine Arteritis Virus

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American Society for Microbiology

RESUMO

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 GP5 (previously named GL), which occur as disulfide-bonded heterodimers and are the major viral envelope proteins; the unglycosylated small envelope protein E; and the minor glycoproteins GP2b (formerly designated GS), GP3, and GP4. Analysis of the appearance of the GP2b, GP3, and GP4 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 GP2b and GP4 and heterotrimers of GP2b, GP3, and GP4. Shortly after their release from infected cells, virions contained mainly cystine-linked GP2b/GP4 heterodimers, which were subsequently converted into disulfide-bonded GP2b/GP3/GP4 trimers through the covalent recruitment of GP3. This process occurred faster at a higher pH but was arrested at 4°C. 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 GP2b/GP3/GP4 trimers. Using sucrose density gradients, we could not demonstrate a noncovalent association of GP3 with the cystine-linked GP2b/GP4 dimer in freshly released virions, nor did we observe higher-order structures of the GP2b/GP4 or GP2b/GP3/GP4 complexes. Nevertheless, the instantaneous diamide-induced formation of disulfide-bonded GP2b/GP3/GP4 heterotrimers at 4°C suggests that the three minor glycoproteins of EAV are assembled as trimeric complexes. The existence of a noncovalent interaction between the cystine-linked GP2b/GP4 dimer and GP3 was also inferred from coexpression experiments showing that the presence of GP3 increased the electrophoretic mobility of the disulfide-bonded GP2b/GP4 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.

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