The Accessory Protein ORF3 Contributes to Porcine Epidemic Diarrhea Virus Replication by Direct Binding to the Spike Protein.

Challika Kaewborisuth,Anan Jongkaewwattana,Qigai He

doi:10.3390/v10080399

Abstract

The porcine epidemic diarrhea virus (PEDV) is an important swine pathogen responsible for severe watery diarrhea, particularly in neonatal piglets. Despite extensive studies performed to elucidate the function of several viral proteins, the contribution of an accessory protein ORF3 in PEDV replication is still largely unknown. Here, we constructed expression plasmids as well as recombinant PEDV carrying myc-tagged ORF3 to assess their expression and subcellular localization in both transfected and infected cells. In PEDV-infected cells, ORF3 was predominantly localized in the cytoplasm, partially in the endoplasmic reticulum (ER) and the Golgi apparatus (Golgi). Interestingly, ORF3 with the N-terminal Flag tag was also detected on the cell surface concomitant with the spike (S) protein as determined by flow cytometry and confocal microscopy. ORF3 and S proteins were also co-localized at perinuclear compartments and in the vesicle-like structures in transfected and infected cells. We also demonstrated that both full-length and naturally truncated ORF3 proteins could interact with the S protein but with different binding affinity, which correlate with the ability of the protein to regulate virus replication in cell culture. Collectively, our results underscore the unprecedented role of the ORF3, which involves the interaction of ORF3 with S and, possibly, other structural protein during PEDV replication.

Highlights

The porcine epidemic diarrhea virus (PEDV), a member of the family Coronaviridae, is an enveloped, single-stranded, positive-sense RNA virus that causes severe enteric diseases with a high mortality rate in suckling piglets [1]
We found that open reading frame 3 (ORF3) and S are co-localized in several cellular compartments in infected cells and the ORF3-S interaction was confirmed
When each virus was inoculated onto VeroE6-APN cells and harvested at 24, 48 and 60 hpi, we found that titers of all viruses were comparable early after infection, but different growth characteristics could be noted at the later time point with the PEDVAV12 _ORF3-Trnc titer significantly higher than that of the PEDVAV12 _ORF3-FL

Summary

Introduction

The porcine epidemic diarrhea virus (PEDV), a member of the family Coronaviridae, is an enveloped, single-stranded, positive-sense RNA virus that causes severe enteric diseases with a high mortality rate in suckling piglets [1]. Pathogenic strains of PEDV have caused recurrent outbreaks with enormous economic losses worldwide. The 28-kb PEDV genome has a gene organization typical of an Alphacoronavirus, i.e., ORF 1a/b, spike (S), open reading frame 3 (ORF3), envelope (E), membrane (M) and the nucleocapsid (N) genes [2]. It is assumed that PEDV replication is initiated by the interaction of the S protein to cellular receptors, followed by endocytosis-mediated viral internalization [3]. After the release of viral genomic RNA into the cytoplasm, replicase proteins undergo extensive intracellular membranes modification to promote viral RNA synthesis. After synthesis of structural proteins, nascent virions are assembled in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), before being

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Conclusion