Formation of Disulfide-Linked Complexes between the Three Minor Envelope Glycoproteins (GP _2b , GP ₃ , and GP ₄ ) of Equine Arteritis Virus

Abstract

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 5 (previously named G L ), which occur as disulfidebonded 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 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 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 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 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.Equine arteritis virus (EAV) is a single-stranded RNA virus which belongs to the genus Arterivirus. Other members of this single genus in the family Arteriviridae are lactate dehydrogenase-elevating virus (LDV), porcine reproductive and respiratory syndrome virus (PRRSV), and simian hemorrhagic fever virus. Despite marked differences in their biophysical properties, genome sizes, and structural protein compositions, the Arteriviridae were joined together with the Coronaviridae and the recently established family Roniviridae in the order Nidovirales on the basis of similarities in their genomic organizations and replication strategies.EAV virions have a diameter of 40 to 60 nm and consist of a putatively icosahedral core that is surrounded by a lipidcontaining envelope with sm...

Envelope Protein Requirements for the Assembly of Infectious Virions of Porcine Reproductive and Respiratory Syndrome Virus

“…Here, the occurrence of a covalently linked GP 2b /GP 3 /GP 4 complex in virions could be directly demonstrated. Interestingly, while the disulfide linkage between the EAV GP 2b and GP 4 proteins appeared to be formed intracellularly, the covalent association of GP 3 , presumably by linking to GP 4 (46), occurs after the assembled virus has been released from the infected cell (45). Whether or not the proteins in the PRRSV complex are also covalently linked is presently unclear.…”

“…Whereas the GP 3 protein was shown to be incorporated into virions of European-type strains (35), for North American strains, its structural nature has been questioned (11,19). For EAV, GP 2b , GP 3 , and GP 4 have been reported to exist as covalently linked heterotrimeric complexes (45). Furthermore, incorporation of these glycoproteins and of the E protein in EAV particles was shown to be interdependent (43).…”

Significance of the oligosaccharides of the porcine reproductive and respiratory syndrome virus glycoproteins GP2a and GP5 for infectious virus production

“…This discrepancy might be real or due to different analytical procedures. Recently, a study on the minor glycoproteins of EAV showed these proteins to occur in viral particles as covalently linked complexes, the incorporation of which occurs in concert with that of the E protein (Wieringa, 2003;Wieringa et al, 2003).…”

Complex Interactions between the Major and Minor Envelope Proteins of Equine Arteritis Virus Determine Its Tropism for Equine CD3+T Lymphocytes and CD14+Monocytes

“…These include four minor envelope proteins (E, GP2, GP3, and GP4, encoded by ORFs 2a, 2b, 3, and 4, respectively), two major envelope proteins (GP5 and M, encoded by ORFs 5 and 6) and the highly conserved nucleocapsid (N) protein (encoded by ORF7) (56). The two major envelope proteins GP5 and M form a disulfide-linked heterodimer (18,54), and the three minor envelope proteins GP2, GP3, and GP4 form a covalently associated heterotrimeric complex in the virion (63,65). By independently knocking out the expression of each structural protein, it was shown that all major and minor structural proteins are required for the production of infectious progeny virus (46).…”