Heterodimerization of the Two Major Envelope Proteins Is Essential for Arterivirus Infectivity

Abstract

The two major envelope proteins of arteriviruses, the membrane protein (M) and the major glycoprotein (GP₅), associate into a disulfide-linked heterodimer that is incorporated into the virion and has been assumed to be a prerequisite for virus assembly. Using an equine arteritis virus (EAV) infectious cDNA clone, we have analyzed the requirement for GP₅-M heterodimerization and have identified the Cys residues involved in the formation of the GP₅-M disulfide bond. The single Cys residue (Cys-8) in the M ectodomain was crucial for heterodimerization and virus infectivity. Mutagenesis of any of the five Cys residues in the GP₅ ectodomain or removal of the single GP₅ N-glycosylation site also rendered the full-length clone noninfectious. However, an analysis of revertants yielded an exceptional pseudorevertant in which residues 52 to 79 of the GP₅ ectodomain had been deleted and the original Cys-80→Ser mutation had been maintained. Consequently, this revertant lacked the GP₅ N-glycosyation site (Asn-56) and retained only a single cysteine residue (Cys-34). By using this GP₅ deletion, we confirmed that Cys-34 of GP₅ and Cys-8 of M are essential for GP₅-M heterodimerization, a key event in the assembly of the EAV envelope.