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Fig. 1. Structure prediction and computational modeling of PEDV OFR3 protein. Prediction of the transmembrane (TM) parts of ORF3 protein from porcine epidemic diarrhea virus (PEDV) CV777 using different secondary structure prediction programs (A). Residues highlighted in red reflect the consensus sequence and were used for molecular dynamics (MD) simulations and assembly. Representation of three PEDV ORF3 protein assemblies with the lowest potential energy on the left (B) and second (C) and third lowest models (D). View onto the bundle with tyrosines (blue) and phenylalanines (grey) highlighted (top row). Respective side views are shown in the middle panel. Top view of the bundles with Lys-55 (yellow), serines (pink) and threonines (green) highlighted.
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Fig. 2. The PEDV CV777 ORF3 protein forms an ion channel. (A) ORF3 protein was tested in PEDV infected cells by western blot. (B) Water-injected and PEDV ORF3-cRNA-injected oocytes are immunolabeled with anti-HA and monitored by confocal microscopy. (C) Typical current traces that can be detected by two-electrode voltage clamp (TEVC) in these PEDV CV777 ORF3 (wt) expressed oocytes, compared with water injected control oocytes. (D) PEDV CV777 ORF3 (wt) shows larger current in 100Â mM K+ bath solution than in barium ions containing standard bath solution. (E) PEDV ORF3 channel can complement K+ channel deficient yeast. Growth phenotype of yeast Îtrk1Îtrk2 mutants transformed with PEDV CV777 ORF3 genes and pYES2 vector, respectively.
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Fig. 3. Deletion and mutation analysis of PEDV ORF3 gene. (A) TM truncated ORF3 could not complement the growth phenotype of the potassium uptake-deficient yeast. (B) 82-98del and 151-172del mutant proteins lost channel activity in oocytes. (C) Tyr-170 in TM4 domain is important for potassium channel activity. Y170A (a single amino acid Tyr-170 replaced by alanine in TM4 domain) mutant shows only half the wild-type current, on average. Wt stands for wild-type PEDV ORF3.
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Fig. 4. The ORF3 gene of an attenuated-type PEDV encodes a truncated protein and shows less channel activity. (A) Sequence alignment of Vector NTI 9.0 with PEDV. (B) Currentâvoltage relationships of wild-type CV777 and attenuated-type PEDV ORF3 mediated ionic currents in oocytes by TEVC.
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Fig. 5. The virus production is reduced on knockdown of the ORF3 gene by siRNA. (A) Schematic diagram showing the design of siRNA for knockdown of specific PEDV ORF3 subgenomic mRNA. (B) The siRNA knockdown efficiency was checked by western blotting in PEDV ORF3 expression plasmid and siRNA cotransfected cells. (C, D) The relative virus yield is quantified by real-time RT-PCR, and also titered by TCID50 Assay (E, F). Fifty nanomolar unrelated control siRNA was used as negative control (NC), and the results from siRNA-pretreated cultures were compared with those from control transfectant (NC, defined as 100%). (âPÂ <Â 0.05, ââPÂ <Â 0.01, compared with NC).
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Figure 1. Structure prediction and computational modeling of PEDV OFR3 protein. Prediction of the transmembrane (TM) parts of ORF3 protein from porcine epidemic diarrhea virus (PEDV) CV777 using different secondary structure prediction programs (A). Residues highlighted in red reflect the consensus sequence and were used for molecular dynamics (MD) simulations and assembly. Representation of three PEDV ORF3 protein assemblies with the lowest potential energy on the left (B) and second (C) and third lowest models (D). View onto the bundle with tyrosines (blue) and phenylalanines (grey) highlighted (top row). Respective side views are shown in the middle panel. Top view of the bundles with Lysâ€55 (yellow), serines (pink) and threonines (green) highlighted.
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Figure 2. The PEDV CV777 ORF3 protein forms an ion channel. (A) ORF3 protein was tested in PEDV infected cells by western blot. (B) Waterâinjected and PEDV ORF3âcRNAâinjected oocytes are immunolabeled with antiâHA and monitored by confocal microscopy. (C) Typical current traces that can be detected by twoâelectrode voltage clamp (TEVC) in these PEDV CV777 ORF3 (wt) expressed oocytes, compared with water injected control oocytes. (D) PEDV CV777 ORF3 (wt) shows larger current in 100 mM K+ bath solution than in barium ions containing standard bath solution. (E) PEDV ORF3 channel can complement K+ channel deficient yeast. Growth phenotype of yeast Îtrk1Îtrk2 mutants transformed with PEDV CV777 ORF3 genes and pYES2 vector, respectively.
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Figure 3. Deletion and mutation analysis of PEDV ORF3 gene. (A) TM truncated ORF3 could not complement the growth phenotype of the potassium uptakeâdeficient yeast. (B) 82â98del and 151â172del mutant proteins lost channel activity in oocytes. (C) Tyrâ170 in TM4 domain is important for potassium channel activity. Y170A (a single amino acid Tyrâ170 replaced by alanine in TM4 domain) mutant shows only half the wildâtype current, on average. Wt stands for wildâtype PEDV ORF3.
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Figure 4. The ORF3 gene of an attenuatedâtype PEDV encodes a truncated protein and shows less channel activity. (A) Sequence alignment of Vector NTI 9.0 with PEDV. (B) Currentâvoltage relationships of wildâtype CV777 and attenuatedâtype PEDV ORF3 mediated ionic currents in oocytes by TEVC.
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Figure 5. The virus production is reduced on knockdown of the ORF3 gene by siRNA. (A) Schematic diagram showing the design of siRNA for knockdown of specific PEDV ORF3 subgenomic mRNA. (B) The siRNA knockdown efficiency was checked by western blotting in PEDV ORF3 expression plasmid and siRNA cotransfected cells. (C, D) The relative virus yield is quantified by realâtime RTâPCR, and also titered by TCID50 Assay (E, F). Fifty nanomolar unrelated control siRNA was used as negative control (NC), and the results from siRNAâpretreated cultures were compared with those from control transfectant (NC, defined as 100%). (â
P
< 0.05, ââ
P
< 0.01, compared with NC).
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