Session topic

17:30–17:35

Title: Production of a recombinant minor protein spike GP2/GP3/GP4 of equine arteritis virus, a prototype arterivirus EAV.
ID: PS 21
Type: Poster session
Talk time: 3 + 2 min
Session: Poster session 1
Receptors and entry

Speaker: Anna Matczuk (Wrocław/PL)


Abstract - Text

Abstract text (incl. references and figure legends)

Introduction


Equine arteritis virus (EAV) is a prototype member of Arteriviridae, a family of viruses comprising important pathogens of domestic animals. Structural envelope proteins of EAV form heterologous complexes. The spike composed of the glycoproteins GP2, GP3 and GP4 and functionally connected protein E (minor proteins of EAV) are responsible for virus entry and cellular tropism.


Objectives


Arteriviruses have unusual form of spike composed of three different glycoproteins. It is unknown which protein posses fusion peptide and receptor binding activity. Simultaneous expression of the GP2/GP3/GP4 trimer will facilitate research on arteriviral spike. Mammalian expression is necessary for glycosylation and proper folding of the viral proteins.


Materials and methods


The tagged sequences of minor EAV glycoproteins were created by overlap extension PCR from the reverse genetics plasmid pEAV211 template. The GP2 was tagged with myc tag, GP3 with HA tag and GP4 with either FLAG tag, linker and FLAG tag or linker and V5 tag. The PCR products were cloned into MultiMam (Geneva Biotech, Geneva, Switzerland). The acceptor and donor vectors were combined together with Cre-lox recombination (New England BioLabs, Germany). Transient trimer expression with lipofectamine 2000 was performed in BHK-21, Vero and CHO-K1 cells. Expression of each protein of the trimer was was assessed with western blot, immunofluorescence, the complex formation was assessed with immunoprecipitations with antibodies directed against all of the tags.


Results


In this study, tagged recombinant GP2/GP3/GP4 trimer was generated in mammalian expression system. GP4 with FLAG tag and GP4 with linker and FLAG tag were not expressed, but the GP4 linekr V5 tag was expressed well. Expression of GP2-myc was possible under CAG promoter, while expression under CMV was minimal. The expression of GP2 and GP4 was achieved in different cell lines, while Gp3-HA was expressed only in BHK-21 cells. In immunoprecipitation experiments, with antibodies directed towards one tag, it was possible to pull all of remaining proteins, meaning that the proteins form a multimeric complexes. Recombinant GP2/GP3/GP4 trimer could become a valuable research tool to study viral membrane fusion.


 


This work was supported by grant UMO-2014/12/S/NZ6/00732 from the National Science Centre.