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

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.

Porcine epidemic diarrhea virus ORF3 protein causes endoplasmic reticulum stress to facilitate autophagy

“…In our study, we firstly constructed different eukaryotic expression vectors to expression ORF3 protein, and found ORF3 protein distributed in perinuclear cytoplasm, and then shifted for aggregation and concentrated in the nucleate of cytoplasm to form punctate patter. This distribution manner of PEDV ORF3 is similar with one study that ORF3 proteins localizes in the cytoplasm in punctate manner (Ye et al, 2015), but distinguishes from the other report of uniform distribution in cytoplasm diffusely (Wongthida et al, 2017;Kaewborisuth et al, 2018). We speculate that the localization and distribution manner of PEDV ORF3 might be dependent on the virus strain-type-specific differences.…”

“…However, there are lack of reports about the influence of PEDV ORF3 on the ER stress, apoptosis and autophagy. Up to date, the investigation of ORF3 is hampered by difficulty of detecting protein expression (Kaewborisuth et al, 2018). Thus in present study, we initiated the construction of various recombinant plasmids expressing PEDV ORF3 protein fused with different tags, and found ORF3 localized in the cytoplasm in the aggregation manner.…”

“…PEDV ORF3 protein can prolong cellular S-phase, facilitate formation of vesicles and thus promote the proliferation of attenuated PEDV rather than virulent PEDV (Ye et al, 2015). Previous study demonstrated that ORF3 and S are co-localized in several cellular compartments in infected cells, and ORF3 may interact with S during PEDV assembly and consequently exert its activity at this step of virus replication (Kaewborisuth et al, 2018). On the contrary, the ORF3 accessory protein has been reported to impede reverse genetics of cellculture-adapted PEDV, because full-length ORF3, but not the truncated form, can inhibit recovery of reverse-genetics derived PEDV when expressed in trans (Wongthida et al, 2017), and the attenuated PEDV rather than virulent PEDV can grow better in ORF3-expressing Vero cells (Ye et al, 2015).…”

Porcine Epidemic Diarrhea Virus (PEDV) ORF3 Enhances Viral Proliferation by Inhibiting Apoptosis of Infected Cells

“…Interestingly, constitutive expression of the ORF3 protein exerted a positive regulatory effect on the proliferation of attenuated PEDV but not on that of virulent PEDV [17]. In a more recent study, both the wild type and a mutant ORF3 protein lacking residues 82-98 were found to co-localize with the S protein intracellularly and at the cell surface, both in infected cells and during co-expression in transfected cells [18]. Additionally, a direct interaction of the S protein with each of these ORF3 proteins was demonstrated in such transfected cells by co-immunoprecipitation, leading the authors to suggest that the ORF3 protein might be involved in virus assembly.…”

Rapid manipulation of the porcine epidemic diarrhea virus genome by CRISPR/Cas9 technology

“…Using a similar strategy, Fan et al developed an infectious cDNA clone for a Chinese PEDV variant strain, AH2012/12 (Fan et al, 2017). Li et al developed a reverse genetics system for two Chinese PEDV strains with differing virulence by ligation of cDNA fragments into BACs one by one (Li et al, some PEDV proteins, such as S protein and ORF3, in modulating PEDV pathogenicity have been examined (Beall et al, 2016;Hou et al, 2017Hou et al, , 2019Kaewborisuth et al, 2018;Wang et al, 2018). Several recombinant PEDV vaccine candidates have also been generated using reverse genetics systems (Hou et al, 2019;Kao et al, 2018;Wang et al, 2018).…”