Production of Recombinant EAV with Tagged Structural Protein Gp3 to Study Artervirus Minor Protein Localization in Infected Cells.

Anna Karolina Matczuk,Grzegorz Chodaczek,Maciej Ugorski

doi:10.3390/v11080735

Abstract

Equine arteritis virus (EAV) is a prototype member of the Arterivirus family, comprising important pathogens of domestic animals. Minor glycoproteins of Arteriviruses are responsible for virus entry and cellular tropism. The experimental methods for studying minor Arterivirus proteins are limited because of the lack of antibodies and nested open reading frames (ORFs). In this study, we generated recombinant EAV with separated ORFs 3 and 4, and Gp3 carrying HA-tag (Gp3-HA). The recombinant viruses were stable on passaging and replicated in titers similar to the wild-type EAV. Gp3-HA was incorporated into the virion particles as monomers and as a Gp2/Gp3-HA/Gp4 trimer. Gp3-HA localized in ER and, to a lesser extent, in the Golgi, it also co-localized with the E protein but not with the N protein. The co-localization of Gp3-HA and the E protein with ERGIC was reduced. Moreover, EAV with Gp3-HA could become a valuable research tool for identifying host cell factors during infection and the role of Gp3 in virus attachment and entry.

Highlights

Equine arteritis virus (EAV) is a prototype member of Arteriviridae, a family of enveloped positive-stranded RNA viruses comprising porcine reproductive and respiratory syndrome virus (PRRSV), a major pathogen in the swine industry [1]
For recombinant EAV with a tagged Gp3, we selected the junction between ORF3 and ORF4 because, in the pEAV211 vector, the overlap between the ORF3 and ORF4 consists of 98 nt, while that between ORF2 and ORF3 is 202 nt
The expression of Gp3 carrying HA-tag (Gp3-HA) was only observed in cells transfected with in vitro-transcribed RNA produced from pEAV211Gp3-HA; it was not observed in pEAV211, pEAV211s3/4, or mock-transfected cells

Summary

Introduction

Equine arteritis virus (EAV) is a prototype member of Arteriviridae, a family of enveloped positive-stranded RNA viruses comprising porcine reproductive and respiratory syndrome virus (PRRSV), a major pathogen in the swine industry [1]. EAV infects horses and donkeys and leads to abortions in pregnant mares and respiratory illness with flu-like symptoms, which can even lead to death in young animals. The genes for the structural virion proteins, which are located at the 3’-end of the arterivirus genome, overlap with each other and are expressed from the 3’ co-terminal nested set of six leader-containing subgenomic RNAs [4]. Gp4 start codon mutation (1, green star) and the nucleotide sequence (2, arrow). The ORF4 Gp4 start codon mutation (1, green star) and the AscI nucleotide sequence (2, arrow).

Methods

Results

Discussion

Conclusion