RESUMEN
Glycoprotein 3 (GP3) serves as a structural protein in equine arteritis virus (EAV), forming a heterotrimeric complex that plays a pivotal role in virus tropism. In this study, we tested the membrane topology of GP3, both when expressed separately and during infection with recombinant tagged EAV GP3-HA. In our antibody accessibility experiment, we made a noteworthy discovery: GP3, when expressed separately, exhibits a dual topology. We introduced an additional N-glycosylation site, which was only partially used, providing further evidence for the dual topology of GP3. Intriguingly, this mutated GP3 was secreted into the medium, a result of the disruption of the ER retention motif RXR. The additional glycosylation site was not used when we examined the recombinant EAV virus with the same mutation. Despite the fact of higher expression levels of mutant GP3-HA, the protein was not secreted, and the recombinant mutant virus did not have growth delay compared to the EAV wild-type virus. This finding suggests that GP3 has a single type one membrane topology in virus infected cells, whereas the expression of GP3 in trans results in the dual topology of this protein. The RXR motif in the C-terminus is a co-factor of ER retention of the protein, but the main retention signal remains elusive.
