Folding and Oligomerization of the gp2b/gp3/gp4 Spike Proteins of Equine Arteritis Virus in Vitro

Abstract

Equine arteritis virus (EAV) is a small, positive-stranded RNA virus. The glycoproteins gp2b, gp3 and gp4 form a heterotrimer in the viral envelope, which is required for cell entry of EAV. We describe expression of the ectodomains of the proteins in E. coli and their refolding from inclusion bodies. After extraction of inclusion bodies and dialysis, Gst-, but not His-tagged proteins, refold into a soluble conformation. However, when dialyzed together with Gst-gp3 or with Gst-gp4, His-gp2b and His-gp4 remain soluble and oligomers are obtained by affinity-chromatography. Thus, folding and oligomerization of gp2b, gp3 and gp4 in vitro are interdependent processes.

Equine arteritis virus

“…Similarly, the cysteine residue of GP4 that interacts with Cys-102 of GP2 has not been identified. It has been shown that folding and oligomerization of GP2, GP3, and GP4 expressed in E. coli are independent processes (Kabatek, 2013). However, it remains to be shown whether this is true in virus-infected cells.…”

Membrane proteins of arterivirus particles: Structure, topology, processing and function

“…Since two cysteine residues must be in very close vicinity to form a covalent bond, Gp3 is likely to be non-covalently associated with the Gp2/4 dimer already in budding virus particles (Wieringa et al, 2003b). Accordingly, the minor glycoproteins can associate with each other independently of glycosylation and disulphide bonds, as demonstrated by refolding experiments with the ectodomains of these proteins purified from E. coli (Kabatek and Veit, 2012).…”

“…This is followed by association of both proteins and stabilization of the resulting dimer by an intermolecular disulphide bond (Wieringa et al, 2003a). Since Gp3 is capable to associate with Gp2/4 in virus particles and also with Gp2 and Gp4 in vitro (Kabatek and Veit, 2012), the question arises what prevents formation of the Gp2/3/4 trimer already in the ER and what triggers its build-up later on. One might speculate that a conformational rearrangement occurs in Gp2/4 and Gp3 after transport from the ER to the Golgi, which might be triggered by the different milieu present in the Golgi lumen.…”

“…One characteristic hallmark of viral fusion proteins is the presence of a fusion peptide, a hydrophobic region within the protein sequence that -after a conformational change of the fusion protein -inserts into the membrane of the target cell to initiate fusion (Earp et al, 2005;Kielian and Rey, 2006). A Kyte-Doolittle plot reveals that Gp2, Gp3 and Gp4 (but not Gp5 and M) contain an additional region with similar hydrophobicity in the middle of each protein besides the N-terminal signal peptide and the hydrophobic C-terminus (Kabatek and Veit, 2012). These proteins thus resemble classical viral fusion proteins in this respect.…”