The open reading frame 3 of equine arteritis virus encodes an immunogenic glycosylated, integral membrane protein.

Open reading frame 3 (ORF 3) of equine arteritis virus (EAV) is predicted to encode a glycosylated membrane protein (GP3) that is uncharacterized. ORF 3 of the American Type Culture Collection strain of EAV was in vitro transcribed and the encoded GP3 protein was in vitro translated with and without canine microsomal membranes. The GP3 protein was approximately 17 kDa after in vitro translation without canine microsomal membranes whereas the glycosylated form, after translation with microsomal membranes, was a diffuse band of 36-42 kDa, indicating that the GP3 protein is extensively glycosylated. Deglycosylation reduced the GP3 protein to approximately 17 kDa, the same size as that translated without microsomal membranes, indicating that the signal sequence was not cleaved. The EAV GP3 protein was membrane associated and not released as a soluble protein, in marked contrast to the ORF 3-encoded proteins of some other arteriviruses. The GP3 protein was protected from protease digestion in closed membrane vesicles, suggesting that the protein extends into the membrane vesicles and is anchored by the N-terminal signal sequence, a C-terminal hydrophobic domain, or both, but does not span the membrane three times. A GP3 protein lacking the C-terminal transmembrane domain remained membrane associated, indicating that this terminus is not a necessary membrane anchor. Sera from stallions persistently infected with EAV and horses immunized repeatedly with the modified live EAV vaccine contained antibodies specific for the GP3 protein. The data indicate that the GP3 protein is an extensively glycosylated membrane protein that is immunogenic during some EAV infections. Copyright 1999 Academic Press.