PEDV ORF3 Independently Regulates IκB Kinase β-Mediated NF-κB and IFN-β Promoter Activities

The Open Reading Frame 3 (ORF3), an accessory protein of porcine epidemic diarrhea virus (PEDV), has been shown to interact with a myriad of cellular proteins, among which include the IκB kinase β (IKBKB). Here, specific IKBKB domains responsible for ORF3–IKBKB interaction were identified. Dysregulation of NF-κB and Type I interferon (IFN) in the presence of ORF3 was also demonstrated. We showed that while ORF3 was capable of up-regulating IKBKB-meditated NF-κB promoter activity, it surprisingly down-regulated the activation of IKBKB-meditated IFN-β promoter and expression of IFN-β mRNA. When overexpressed, ORF3 could suppress Poly I:C mediated type I IFN production and induction. Finally, we demonstrated that IKBKB- and RIG-I-mediated type I IFN induction by ORF3 resulted in different outcomes. Our study is the first to demonstrate the potential and complex roles of ORF3 in the involvement of aberrant immune signaling as well as in the virus–host interaction

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

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

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

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 (PEDV) ORF3 Interactome Reveals Inhibition of Virus Replication by Cellular VPS36 Protein

The accessory protein ORF3 of porcine epidemic diarrhea virus (PEDV) has been proposed to play a key role in virus replication. However, our understanding of its function regarding virus and host interaction is still limited. In this study, we employed immunoprecipitation and mass spectrometry to screen for cellular interacting partners of ORF3. Gene ontology analysis of the host interactome highlighted the involvement of ORF3 in endosomal and immune signaling pathways. Among the identified ORF3-interacting proteins, the vacuolar protein-sorting-associated protein 36 (VPS36) was assessed for its role in PEDV replication. VPS36 was found to interact with ORF3 regardless of its GLUE domain. As a result of VPS36–ORF3 interaction, PEDV replication was substantially suppressed in cells overexpressing VPS36. Interestingly, the ORF3 protein expression was diminished in VPS36-overexpressing cells, an effect that could not be restored by treatment of lysosomal inhibitors. In addition, disruption of endogenously-expressed VPS36 by siRNA could partially augment PEDV replication. Taken together, our study provides mechanistic insights into the contribution of ORF3 in PEDV replication

A Single V672F Substitution in the Spike Protein of Field-Isolated PEDV Promotes Cell–Cell Fusion and Replication in VeroE6 Cells

While porcine epidemic diarrhea virus (PEDV) infects and replicates in enterocytes lining villi of neonatal piglets with high efficiency, naturally isolated variants typically grow poorly in established cell lines, unless adapted by multiple passages. Cells infected with most cell-adapted PEDVs usually displayed large syncytia, a process triggered by the spike protein (S). To identify amino acids responsible for S-mediated syncytium formation, we constructed and characterized chimeric S proteins of the cell-adapted variant, YN144, in which the receptor binding domain (RBD) and S1/S2 cleavage site were replaced with those of a poorly culturable field isolate (G2). We demonstrated that the RBD, not the S1/S2 cleavage site, is critical for syncytium formation mediated by chimeric S proteins. Further mutational analyses revealed that a single mutation at the amino acid residue position 672 (V672F) could enable the chimeric S with the entire RBD derived from the G2 strain to trigger large syncytia. Moreover, recombinant PEDV viruses bearing S of the G2 strain with the single V672F substitution could induce extensive syncytium formation and replicate efficiently in VeroE6 cells stably expressing porcine aminopeptidase N (VeroE6-APN). Interestingly, we also demonstrated that while the V672F mutation is critical for the syncytium formation in VeroE6-APN cells, it exerts a minimal effect in Huh-7 cells, thereby suggesting the difference in receptor preference of PEDV among host cells

A Novel Plasmid DNA-Based Foot and Mouth Disease Virus Minigenome for Intracytoplasmic mRNA Production

Picornaviruses are non-enveloped, single-stranded RNA viruses that cause highly contagious diseases, such as polio and hand, foot-and-mouth disease (HFMD) in human, and foot-and-mouth disease (FMD) in animals. Reverse genetics and minigenome of picornaviruses mainly depend on in vitro transcription and RNA transfection; however, this approach is inefficient due to the rapid degradation of RNA template. Although DNA-based reverse genetics systems driven by mammalian RNA polymerase I and/or II promoters display the advantage of rescuing the engineered FMDV, the enzymatic functions are restricted in the nuclear compartment. To overcome these limitations, we successfully established a novel DNA-based vector, namely pKLS3, an FMDV minigenome containing the minimum cis-acting elements of FMDV essential for intracytoplasmic transcription and translation of a foreign gene. A combination of pKLS3 minigenome and the helper plasmids yielded the efficient production of uncapped-green florescent protein (GFP) mRNA visualized in the transfected cells. We have demonstrated the application of the pKLS3 for cell-based antiviral drug screening. Not only is the DNA-based FMDV minigenome system useful for the FMDV research and development but it could be implemented for generating other picornavirus minigenomes. Additionally, the prospective applications of this viral minigenome system as a vector for DNA and mRNA vaccines are also discussed

Additional file 3: Fig. S3. of G45R mutation in the nonstructural protein 1 of A/Puerto Rico/8/1934 (H1N1) enhances viral replication independent of dsRNA-binding activity and type I interferon biology

Western blot analysis of STAT1, pSTAT1 and NS1 protein. A549 cells were infected with rX31 encoding WT and G45R/NS1 viruses at an MOI of 2. At 8 h post infection, cell lysates were clarified by centrifugation at 13,000 rpm for 15 min. The lysates were mixed in Laemmli sample buffer (Bio-Rad) and denatured at 95 °C for 5 mins prior to perform SDS-PAGE gel electrophoresis. Immunoblots were probed for STAT1 (purified rabbit anti-Stat1 N-terminus, BD Transduction Laboratories), pSTAT1 (purified mouse anti-Stat1 pY701 BD Transduction Laboratories), NS1 (polyclonal rabbit anti-NS1; Thermoscientific) and actin (purified mouse anti-actin Ab-5; BD Transduction Laboratories). Antibodies were detected by incubation with goat anti-rabbit (GE healthcare) or goat anti-mouse HRP-linked antibody (Cell signaling). The immunoblots were visualized by using ChemiDoc XRS imager (Bio-Rad). Band intensity of proteins was quantified by using Image Lab version 5.0 (Bio-Rad). (TIFF 2319 kb

Additional file 4: Fig. S4. of G45R mutation in the nonstructural protein 1 of A/Puerto Rico/8/1934 (H1N1) enhances viral replication independent of dsRNA-binding activity and type I interferon biology

Plaque assay of the virus inoculums. The virus inoculums (WT and G45R/NS1) at MOI of 0.01 were serially diluted before they were infected onto MDCK cells to confirm that each inoculum had equal amount of the virus. Plaque assay of the viruses at 10−5 dilution is shown. The average virus titers of both WT and G45R/NS1 inoculums are 5× 106 pfu/ml. (TIFF 1781 kb