c tail fragments  (Cytiva Europe)

Journal: bioRxiv

Article Title: Structural basis of HSV-1 DNA replication origin-binding protein UL9

doi: 10.1101/2025.10.31.685793

Figure Lengend Snippet: a Strep-tag pull-down assay demonstrating interaction of ICP8 with either full-length UL9 or UL9CTD. b GST pull-down assay analyzing binding between ICP8 and either UL9CTD or the C-tail. c Comparative ATPase activity of wild-type UL9 versus its functional mutants. ATPase activity assays were performed using the Malachite Green Phosphate Detection Kit. Data are presented as the mean with standard deviation for experiments performed in triplicate. Statistical significance was determined by a two-tailed Student’s t -test (*p < 0.05, **p < 0.01, *p < 0.001). d ICP8-mediated stimulation of UL9 helicase activity. Wild-type UL9 and two C-terminal truncation mutants (ΔC16 and ΔC8) were tested.

Article Snippet: UL9 CTD and its truncation mutants used for GST pull-down assays, along with two C-tail fragments, were cloned into the pGEX-6P-1 vector (GE Healthcare) to generate GST-tagged fusion proteins.

Techniques: Strep-tag, Pull Down Assay, Binding Assay, Activity Assay, Functional Assay, Standard Deviation, Two Tailed Test

Journal: PLoS ONE

Article Title: The Viral G Protein-Coupled Receptor ORF74 Hijacks β-Arrestins for Endocytic Trafficking in Response to Human Chemokines

doi: 10.1371/journal.pone.0124486

Figure Lengend Snippet: HEK293T cells were transiently transfected with WT-ORF74 (WT), ORF74-Rluc8 (Rluc8) or empty vector DNA (mock-transfected). (A) Cell surface expression was determined by ELISA. (B-D) Whole cell binding experiments with 100 pM 125 I-CXCL8 (B, C) or 125 I-CXCL10 (D) were performed in the presence of increasing concentrations unlabeled CXCL1 (B), CXCL8 (C) or CXCL10 (D). HEK293T cells transfected with ORF74-Rluc8 (E) or WT-ORF74 (F) were stimulated with increasing concentrations of CXCL1 (open squares), CXCL8 (filled squares) or CXCL10 (open circles) and InsP accumulation was quantified. Data are shown as the mean ± SEM of at least three independent experiments each performed in triplicate and are presented as fold over mock-transfected cells (dotted line) (A), percentage of specific 125 I-CXCL8 (B, C) or 125 I-CXCL10 binding (D) or fold over basal (E, F). Statistical differences in cell surface expression (A) were determined by a Student t test. NS = not significant.

Article Snippet: DNA fragments encoding the C-tail of ORF74 containing the different S/T to A mutations were synthesized by Eurofins (Ebersberg, Germany) and introduced into WT-ORF74-pcDEF 3 using the internal restriction site Eam1105I/AhdI.

Techniques: Transfection, Plasmid Preparation, Expressing, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: PLoS ONE

Article Title: The Viral G Protein-Coupled Receptor ORF74 Hijacks β-Arrestins for Endocytic Trafficking in Response to Human Chemokines

doi: 10.1371/journal.pone.0124486

Figure Lengend Snippet: Pharmacological characterization of ORF74-Rluc8.

Article Snippet: DNA fragments encoding the C-tail of ORF74 containing the different S/T to A mutations were synthesized by Eurofins (Ebersberg, Germany) and introduced into WT-ORF74-pcDEF 3 using the internal restriction site Eam1105I/AhdI.

Techniques: Activation Assay

Journal: PLoS ONE

Article Title: The Viral G Protein-Coupled Receptor ORF74 Hijacks β-Arrestins for Endocytic Trafficking in Response to Human Chemokines

doi: 10.1371/journal.pone.0124486

Figure Lengend Snippet: HEK293T cells co-expressing ORF74-Rluc8 and β-arrestin1-eYFP (A, C) or β-arrestin2-eYFP (B, D) were stimulated with increasing concentrations of CXCL1 (open squares), CXCL8 (filled squares) or CXCL10 (open circles) (A, B) or co-stimulated with CXCL1 and CXCL10 (C, D). β-arrestin recruitment to the receptor was measured as an increase in BRET ratio (BRETr). Data are shown as fold over basal and represent the mean of pooled data from at least three independent experiments each performed in triplicate. Error bars indicate SEM values. Significant differences between vehicle and chemokine-stimulation were determined by one-way ANOVA followed by a Bonferroni test (**** p ≤ 0.0001). NS = not significant.

Article Snippet: DNA fragments encoding the C-tail of ORF74 containing the different S/T to A mutations were synthesized by Eurofins (Ebersberg, Germany) and introduced into WT-ORF74-pcDEF 3 using the internal restriction site Eam1105I/AhdI.

Techniques: Expressing

Journal: PLoS ONE

Article Title: The Viral G Protein-Coupled Receptor ORF74 Hijacks β-Arrestins for Endocytic Trafficking in Response to Human Chemokines

doi: 10.1371/journal.pone.0124486

Figure Lengend Snippet: (A, B) HEK293T cells were transiently transfected with WT-ORF74 (WT), ORF74-R 3.50 A (R 3.50 A) or empty vector DNA (mock-transfected). Relative receptor expression at the cell surface was determined by ELISA (A) and constitutive activation of PLC was determined by measuring InsP accumulation (B). Data are presented as fold over mock-transfected cells (dotted line). (C, D) HEK293T cells expressing ORF74-Rluc8 (WT) (filled circles) or ORF74-R 3.50 A-Rluc8 (R 3.50 A) (open squares) in combination with β-arrestin1-eYFP (C) or β-arrestin2-eYFP (D) were stimulated with increasing concentrations of CXCL1. Data are shown as fold over basal. All data are represented as the mean of pooled data from at least three independent experiments each performed in triplicate and error bars indicate SEM values. Statistical differences of cell surface expression (A) or constitutive PLC activation (B) between WT-ORF74 and ORF74-R 3.50 A were determined by a Student t test (**** p ≤ 0.0001, *** p ≤ 0.001).

Article Snippet: DNA fragments encoding the C-tail of ORF74 containing the different S/T to A mutations were synthesized by Eurofins (Ebersberg, Germany) and introduced into WT-ORF74-pcDEF 3 using the internal restriction site Eam1105I/AhdI.

Techniques: Transfection, Plasmid Preparation, Expressing, Enzyme-linked Immunosorbent Assay, Activation Assay

Journal: PLoS ONE

Article Title: The Viral G Protein-Coupled Receptor ORF74 Hijacks β-Arrestins for Endocytic Trafficking in Response to Human Chemokines

doi: 10.1371/journal.pone.0124486

Figure Lengend Snippet: (A) Schematic representation of the C-tail of ORF74, starting at the conserved VPxxY-motif in TM7. Serine and threonine residues mutated to alanine in ORF74-ST/A are shown in bold brown. The location of TM7 (delineated) and helix 8 (marked red) are based on the CCR5 crystal structure (PDB-code 4MBS) . (B-F) HEK293T cells were transiently transfected with WT-ORF74 (WT) (B-E) or ORF74-ST/A (ST/A) (B-F) or empty vector DNA (mock-transfected) (B, E). (B) Relative receptor expression at the cell surface was determined by ELISA. Binding of 125 I-CXCL10 (C) or 125 I-CXCL8 (D) to intact HEK293T cells was measured in the presence of increasing concentrations unlabeled homologous chemokines. Constitutive (E) or chemokine-induced (F) activation of PLC was determined by measuring InsP accumulation. (G) HEK293T cells expressing ORF74-Rluc8 (WT) or ORF74-ST/A-Rluc8 (ST/A) in combination with β-arrestin1-eYFP (βarr1) or β-arrestin2-eYFP (βarr2) were vehicle-stimulated (white bars) or stimulated with 300 nM CXCL1 (black bars) before measurement of BRET. Data are presented as fold over mock-transfected cells (dotted line) (B, E), percentage of specific binding (C, D) or fold over basal (F, G). All data are represented as the mean of pooled data from at least three independent experiments each performed in triplicate and error bars indicate SEM values. Statistical differences of cell surface expression (B) or constitutive PLC activation (E) between WT-ORF74 and ORF74-ST/A or between vehicle- and corresponding CXCL1-treated cells (G) were determined by a Student t test (**** p ≤ 0.0001, ** p ≤ 0.01, * p ≤ 0.05). NS = not significant.

Article Snippet: DNA fragments encoding the C-tail of ORF74 containing the different S/T to A mutations were synthesized by Eurofins (Ebersberg, Germany) and introduced into WT-ORF74-pcDEF 3 using the internal restriction site Eam1105I/AhdI.

Techniques: Transfection, Plasmid Preparation, Expressing, Enzyme-linked Immunosorbent Assay, Binding Assay, Activation Assay

Journal: PLoS ONE

Article Title: The Viral G Protein-Coupled Receptor ORF74 Hijacks β-Arrestins for Endocytic Trafficking in Response to Human Chemokines

doi: 10.1371/journal.pone.0124486

Figure Lengend Snippet: (A) Schematic representation of the C-tail of ORF74, starting at the conserved VPxxY-motif in TM7. Serine and threonine residues mutated to alanine are shown in bold brown and clustered to indicate the different ORF74-ST/A mutants (ST/A1, ST/A2 and ST/A3). The location of TM7 (delineated) and helix 8 (marked red) are based on the CCR5 crystal structure (PDB-code 4MBS) . (B) HEK293T cells were transiently transfected with ORF74-Rluc8 (WT), ORF74-ST/A1-Rluc8 (ST/A1), ORF74-ST/A2-Rluc8 (ST/A2) or ORF74-ST/A3-Rluc8 (ST/A3) or empty vector DNA (mock-transfected) and receptor cell surface expression was determined by ELISA. (C-F) HEK293T cells expressing ORF74-Rluc8 (WT) or one of the Rluc8-tagged ORF74-ST/A mutants in combination with β-arrestin1-eYFP (C, E) or β-arrestin2-eYFP (D, F) were treated with increasing concentrations CXCL1 (C, D) or were vehicle-stimulated (white bars) or stimulated with 300 nM CXCL1 (black bars) (E, F) before measurement of BRET. Data are shown as the mean of pooled data from three independent experiments each performed in triplicate. Data is presented as fold over mock-transfected cells (dotted line) (B) or fold over basal (C-F) and error bars indicate SEM values. Statistical differences between ORF74 WT and mutant cell surface expression (B) or difference between vehicle- and corresponding CXCL1-treated cells (E, F) were determined by one-way ANOVA followed by a Bonferroni test (B) or a Student t test (E, F), respectively (**** p ≤ 0.0001, ** p ≤ 0.01). NS = not significant.

Article Snippet: DNA fragments encoding the C-tail of ORF74 containing the different S/T to A mutations were synthesized by Eurofins (Ebersberg, Germany) and introduced into WT-ORF74-pcDEF 3 using the internal restriction site Eam1105I/AhdI.

Techniques: Transfection, Plasmid Preparation, Expressing, Enzyme-linked Immunosorbent Assay, Mutagenesis

Journal: PLoS ONE

Article Title: The Viral G Protein-Coupled Receptor ORF74 Hijacks β-Arrestins for Endocytic Trafficking in Response to Human Chemokines

doi: 10.1371/journal.pone.0124486

Figure Lengend Snippet: (A) Sequence alignment of V 2 R, CCR5, CXCR2, and ORF74 with a focus on serine (S) and threonine (T) residues (bold, brown) and aspartate (D) and glutamate (E) residues (bold). Underlined S/T residues are experimentally determined to be phosphorylated [ – ] or are phosphorylated in the active β-arrestin1 crystal structure . The location of TM7 (delineated) and helix 8 (marked red) are based on the CCR5 crystal structure (PDB-code 4MBS) (35). Residues of the V 2 R-peptide solved in the active β-arrestin1 crystal structure are marked light green (the grey residues were not solved in the crystal structure), and the final 19 C-terminal residues of ORF74 that were used to build the model are marked light blue. (B) 3D model of the C-tail of ORF74 (blue) based on the crystal structure of the C-tail of V 2 R (green) in β-arrestin1 (gold) (PDB-code 4JQI). Spheres indicate the Cα-atoms of S/T residues of V 2 R and ORF74. A detailed view of S335/S338 (C) and T341/T342 (D) interacting with β-arrestin1 highlighting interactions between phosphorylated S/T residues in ORF74 with several key residues in β-arrestin1 (i.e. K10, K11, K107, R165, K294). Dashed lines indicate H-bonds or ionic interactions. β-arrestin1-carbon, ORF74-carbon, nitrogen, oxygen, and phosphate atoms are colored gold, slate, blue, red, and orange respectively. Water molecules are depicted as red spheres.

Article Snippet: DNA fragments encoding the C-tail of ORF74 containing the different S/T to A mutations were synthesized by Eurofins (Ebersberg, Germany) and introduced into WT-ORF74-pcDEF 3 using the internal restriction site Eam1105I/AhdI.

Techniques: Sequencing

Journal: PLoS ONE

Article Title: The Viral G Protein-Coupled Receptor ORF74 Hijacks β-Arrestins for Endocytic Trafficking in Response to Human Chemokines

doi: 10.1371/journal.pone.0124486

Figure Lengend Snippet: HEK293T cells were transiently transfected with ORF74-Rluc8 (WT) (A-D) or ORF74-ST/A2-Rluc8 (ST/A2) (E-H) in combination with Venus-K-Ras (plasma membrane marker) (A, E), Venus-Rab5a (early endosome marker) (B, F), Venus-Rab7a (late endosome/lysosome marker) (C, G) or Venus-Rab11 (recycling endosome marker) (D, H) and stimulated with CXCL1, CXCL8 or CXCL10 for indicated time and BRET was measured. Data are shown as the mean of pooled data from three independent experiments each performed in triplicate. Data is presented as fold over vehicle-stimulated cells (dotted line) and error bars indicate SEM values. Statistical differences between the area under the curve of vehicle- and corresponding CXCL1-, CXCL8- or CXCL10-treated cells (baseline = 1) were determined by one-way ANOVA followed by a Bonferroni test (**** p ≤ 0.0001, *** p≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05). NS = not significant.

Article Snippet: DNA fragments encoding the C-tail of ORF74 containing the different S/T to A mutations were synthesized by Eurofins (Ebersberg, Germany) and introduced into WT-ORF74-pcDEF 3 using the internal restriction site Eam1105I/AhdI.

Techniques: Transfection, Clinical Proteomics, Membrane, Marker

Journal: PLoS ONE

Article Title: The Viral G Protein-Coupled Receptor ORF74 Hijacks β-Arrestins for Endocytic Trafficking in Response to Human Chemokines

doi: 10.1371/journal.pone.0124486

Figure Lengend Snippet: HEK293T cells were transiently transfected with ORF74-Rluc8 and Venus-K-Ras (plasma membrane marker) (A, C) or Venus-Rab5a (early endosome marker) (B, D) in combination with control (Contr) or β-arrestin1/2 (βarr1/2) siRNA. (A, B) Downregulation of β-arrestin1/2 levels was determined by immunoblotting. STAT3 levels were determined as loading control. (C, D) Cells were stimulated with CXCL1 for indicated time and BRET was measured. Data are shown as the mean of pooled data from three independent experiments each performed in triplicate. Data is presented as fold over vehicle-stimulated cells (dotted line) and error bars indicate SEM values. Statistical differences between the area under the curve of cells treated with control or β-arrestin1/2 siRNA (baseline = 1) were determined by a Student t test (** p ≤ 0.01).

Article Snippet: DNA fragments encoding the C-tail of ORF74 containing the different S/T to A mutations were synthesized by Eurofins (Ebersberg, Germany) and introduced into WT-ORF74-pcDEF 3 using the internal restriction site Eam1105I/AhdI.

Techniques: Transfection, Clinical Proteomics, Membrane, Marker, Control, Western Blot