Journal: Cell

Article Title: A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry

doi: 10.1016/j.cell.2023.01.019

Figure Lengend Snippet: Genome characteristics of pangolin MERS-like CoV, Manis javanica HKU4-related CoV (MjHKU4r-CoV) (A) Genome structure of MjHKU4r-CoV-1. (B) Similarity plot based on the full-length genome sequences. MjHKU4r-CoV-1 was used as a query sequence, Tylonycteris bat CoV HKU4-1, Pipistrellus bat CoV HKU5-1, bat MERS-related NeoCoV, and human MERS-CoV were used as reference sequences. (C) Phylogenetic tree based on the nucleotide sequences of the complete genomes of representative Alpha coronavirus (Alpha-CoV) and Beta coronavirus (Beta-CoV). MjHKU4r-CoV-1–4 are shown in red. Shaded colors represent different subgenera of coronaviruses. The scale bars represent 0.3 substitutions per nucleotide position. The software and setting used are described in the . (D) Schematic representation of the S protein. S1 and S2 subunits are indicated, as well as four domains within S1, including the N-terminal domain (NTD), RBD, subdomain 1 (SD1), and subdomain 2 (SD2). RBM alignment is shown at the bottom left, and the 16 key residues situated at the surface between MERS-CoV RBM and human DPP4 are pink shaded. Predicted furin cleavage sites of MjHKU4r-CoV-1 and the corresponding sites in other merbecoviruses are shown at the bottom right. The key arginine sites are shown in red, and the predicted furin cleavage sites are gray shaded. See also Figures S1 , , and and Table S1 .

Article Snippet: For the detection of hDPP4 protein in Huh-7 cells, the membrane was incubated with goat anti-hDPP4 polyclonal antibody (R&D, 0.3 μg/mL) and detected with horseradish peroxidase-conjugated rabbit anti-goat IgG (Proteintech, 1:5,000).

Techniques: Sequencing, Software

Journal: Cell

Article Title: A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry

doi: 10.1016/j.cell.2023.01.019

Figure Lengend Snippet: Isolation of MjHKU4r-CoV-1 and elucidation of its cell-entry mechanism (A) Transmission electron microscopy analysis of MjHKU4r-CoV-1 virions. Scale bars, 50 nm. (B) Cytopathic effect in Caco-2 cells at 48 h.p.i. with MjHKU4r-CoV-1 or mock infected (left, scale bars, 400 μm), and immunofluorescence assay (IFA) with an antibody against the Tylonycteris bat coronavirus HKU4 NP (right, scale bars, 200 μm). Red, NP; blue, nuclei. (C) Caco-2 cells were infected with MjHKU4r-CoV-1 at a multiplicity of infection (MOI) of 1 or 0.01. Viral titers and viral RNA copies at indicated time points were determined by TCID 50 assay and qRT-PCR, respectively. (D–G) Viral infection in Huh-7 cells with or without hDPP4 expression. Wild-type (WT) or DPP4 -knockout Huh-7 (KO) cells were infected with MjHKU4r-CoV-1 or MERS-CoV for 24 h. IFA was performed (D, scale bars, 200 μm) and quantified by high content screening (E), and viral titer (F) and RNA copy number (G) were determined in the supernatant. (H–K) Viral infection in KO cells after hDPP4 re-introduction. IF staining in Huh-7 KO cells transfected with hDPP4 expression plasmid or empty vector (H, scale bars, 200 μm) quantified by high content screening (I), and viral titer (J) and RNA copies (K) were determined in the supernatant at the indicated times. Data are presented as means and standard errors of the means (SEMs) of at least triplicate measurements in (E–G and I–K). Statistical significance was assessed using a two-tailed Student’s t -test in (E and I) and two-way ANOVA in (F, G, J, and K). See also Figure S4 .

Article Snippet: For the detection of hDPP4 protein in Huh-7 cells, the membrane was incubated with goat anti-hDPP4 polyclonal antibody (R&D, 0.3 μg/mL) and detected with horseradish peroxidase-conjugated rabbit anti-goat IgG (Proteintech, 1:5,000).

Techniques: Isolation, Transmission Assay, Electron Microscopy, Infection, Immunofluorescence, Quantitative RT-PCR, Expressing, Knock-Out, High Content Screening, Staining, Transfection, Plasmid Preparation, Two Tailed Test

Journal: Cell

Article Title: A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry

doi: 10.1016/j.cell.2023.01.019

Figure Lengend Snippet: MjHKU4r-CoV-1 binds to human, bat, and pangolin DPP4 proteins (A) HeLa cells overexpressing the following DPP4 orthologous: hDPP4, Tylonycteris pachypus bat DPP4 ( Tp DPP4), Pipistrellus bat DPP4 ( Pp DPP4), or Manis javanica DPP4 ( Mj DPP4) were infected with MjHKU4r-CoV-1 at a MOI of 1 and subjected to IF staining at 24 h.p.i. Shown as MjHKU4r-CoV-1 NP or DPP4 protein expression (left, scale bars, 10 μm), or the replication dynamics of MjHKU4r-CoV-1 (viral titer, middle panel; genomic RNA, right panel). For IF staining, green, DPP4; red, NP; blue, nuclei. (B) Binding affinity of MjHKU4r-CoV-1, MERS-CoV, and HKU4-CoV RBD proteins to human, bat, and pangolin DPP4 proteins as measured by Bio-layer interferometry assay. The quantification of the binding affinity is shown in Table S2 . See also Figure S5 and Table S2 .

Article Snippet: For the detection of hDPP4 protein in Huh-7 cells, the membrane was incubated with goat anti-hDPP4 polyclonal antibody (R&D, 0.3 μg/mL) and detected with horseradish peroxidase-conjugated rabbit anti-goat IgG (Proteintech, 1:5,000).

Techniques: Infection, Staining, Expressing, Binding Assay

Journal: Cell

Article Title: A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry

doi: 10.1016/j.cell.2023.01.019

Figure Lengend Snippet: Human cell tropism and human colon ex vivo infection of MjHKU4r-CoV-1, related to Figure 5 (A) NP staining (red) of the indicated human cell lines at 24 h and 72 h.p.i. with MjHKU4r-CoV-1. Blue, nuclei; scale bars, 200 μm. Replication of MjHKU4r-CoV-1 in cell culture supernatant was analyzed by TCID 50 assay. (B–D) qRT-PCR detection of the replication of MjHKU4r-CoV-1 in human ex vivo colon tissue (B) or culture supernatant (C). IF staining of human ex vivo colon at 24 h.p.i. with MjHKU4r-CoV-1 (D). Red, viral NP; green, human DPP4; blue, nuclei; scale bars, 100 μm.

Article Snippet: For the detection of hDPP4 protein in Huh-7 cells, the membrane was incubated with goat anti-hDPP4 polyclonal antibody (R&D, 0.3 μg/mL) and detected with horseradish peroxidase-conjugated rabbit anti-goat IgG (Proteintech, 1:5,000).

Techniques: Ex Vivo, Infection, Staining, Cell Culture, Quantitative RT-PCR

Journal: Cell

Article Title: A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry

doi: 10.1016/j.cell.2023.01.019

Figure Lengend Snippet: MjHKU4r-CoV-1 infection in human colon organoids and airway organoids (A–C) Replication of MjHKU4r-CoV-1 in human colon organoids (hCO), shown as viral copy numbers in cells (A) and supernatants (B) and viral titers in supernatants (C) as analyzed by qRT-PCR and TCID 50 assay, respectively. (D) Co-staining of MjHKU4r-CoV-1 NP (red) and hDPP4 (green) in human colon organoids at 72 h.p.i. (scale bars, 100 or 30 μm). Blue, nuclei. (E–G) Replication of MjHKU4r-CoV-1 in human airway organoids (hAWOs), shown as viral copy numbers in cells (E) and supernatants (F) and viral titers in supernatant (G) as analyzed by qRT-PCR and TCID 50 assay, respectively. (H) Co-staining of MjHKU4r-CoV-1 NP (red) and hDPP4 (green) in human airway organoids at 48 h.p.i. (scale bars, 50 or 10 μm). Blue, nuclei. See also Figure S6 .

Article Snippet: For the detection of hDPP4 protein in Huh-7 cells, the membrane was incubated with goat anti-hDPP4 polyclonal antibody (R&D, 0.3 μg/mL) and detected with horseradish peroxidase-conjugated rabbit anti-goat IgG (Proteintech, 1:5,000).

Techniques: Infection, Quantitative RT-PCR, Staining

Journal: Cell

Article Title: A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry

doi: 10.1016/j.cell.2023.01.019

Figure Lengend Snippet: MjHKU4r-CoV-1 is infectious in hDPP4-Tg mice (A and B) hDPP4-Tg mice were intranasally inoculated with 1 × 10 6 TCID 50 MjHKU4r-CoV-1. (A) Mean body weights (n = 7 for MjHKU4r-CoV-1 infected mice and n = 3 for mock-infected mice), (B) body weights of individual mice. (C and D) qRT-PCR detection of viral replication in the lung (C) and brain (D) of MjHKU4r-CoV-1-infected hDPP4-Tg mice at the indicated time points (n = 4 for 1, 3, and 5 d.p.i. and n = 7 for 8 d.p.i.). (E) Lung and brain sections from MjHKU4r-CoV-1-infected or mock-infected mice stained for MjHKU4r-CoV-1 NP (red) at 5 d.p.i. (lung) and 8 d.p.i. (brain) (scale bars, 100 μm). Red, viral NP; blue, nuclei. (F) Pathological changes in the lungs of MjHKU4r-CoV-1-infected hDPP4-Tg mice at 5 d.p.i. Yellow arrows indicate severely affected areas (scale bars, 1,000 or 100 μm). See also Figure S7 .

Article Snippet: For the detection of hDPP4 protein in Huh-7 cells, the membrane was incubated with goat anti-hDPP4 polyclonal antibody (R&D, 0.3 μg/mL) and detected with horseradish peroxidase-conjugated rabbit anti-goat IgG (Proteintech, 1:5,000).

Techniques: Infection, Quantitative RT-PCR, Staining

Journal: Cell

Article Title: A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry

doi: 10.1016/j.cell.2023.01.019

Figure Lengend Snippet:

Article Snippet: For the detection of hDPP4 protein in Huh-7 cells, the membrane was incubated with goat anti-hDPP4 polyclonal antibody (R&D, 0.3 μg/mL) and detected with horseradish peroxidase-conjugated rabbit anti-goat IgG (Proteintech, 1:5,000).

Techniques: Isolation, Recombinant, SYBR Green Assay, Luciferase, Reporter Assay, Sequencing, Knock-Out, Transgenic Assay, Plasmid Preparation, Software

Journal: Experimental and Therapeutic Medicine

Article Title: KDM6A suppresses hepatocellular carcinoma cell proliferation by negatively regulating the TGF-β/SMAD signaling pathway

doi: 10.3892/etm.2020.9000

Figure Lengend Snippet: KDM6A negatively regulates the TGF-β/SMAD signaling pathway. (A) The effect of KDM6A overexpression on the expression of TGF-β, p-smad2, p-smad4, Ki67 and PCNA in Huh7 and LM3 cells was (A) determined by western blotting and (B) semi-quantified. The effect of KDM6A knockdown on the expression of TGF-β, p-smad2, p-smad4, Ki67 and PCNA in YY-8103 and SNU-398 was (C) determined by western blotting and (D) semi-quantified. ** P<0.01 and *** P<0.001 vs. vector or SCR. KDM6A, lysine demethylase 6A; TGF-β, transforming growth factor-β; p, phosphorylated; PCNA, proliferating cell nuclear antigen; SCR, scrambled; sh, short hairpin RNA; ns, not significant.

Article Snippet: Subsequently, the membranes were incubated at 4˚C overnight with the following primary antibodies: Anti-KDM6A (1:1,000; cat. no. orb333886; Biorbyt Ltd.), anti-transforming growth factor (TGF)-β (1:1,000; cat. no. 3711; Cell Signaling Technology, Inc.), anti-phosphorylated (p)-smad2 (1:1,000; cat. no. 18338; Cell Signaling Technology, Inc.), anti-Smad2 (1:1,000; cat. no. 12570-1-AP; ProteinTech Group, Inc.), anti-p-smad4 (1:1,000; cat. no. 10231-8-AP; ProteinTech Group, Inc.), anti-smad4 (1:1,000; cat. no. 10231-1-AP; ProteinTech Group, Inc.), anti-proliferating cell nuclear antigen (PCNA; 1:1,000; cat. no. 10205-2-AP; ProteinTech Group, Inc.), anti-Ki67 (1:1,000; cat. no. 27309-1-AP; ProteinTech Group, Inc.), anti-GAPDH (1:1,000; cat. no. 10494-1-AP; ProteinTech Group, Inc.) and anti-Flag (1:4,000; cat. no. F7425; Sigma-Aldrich; Merck KGaA).

Techniques: Over Expression, Expressing, Western Blot, Plasmid Preparation, shRNA

Journal: Diagnostic Pathology

Article Title: Establishment of monoclonal anti-human CD26 antibodies suitable for immunostaining of formalin-fixed tissue

doi: 10.1186/1746-1596-9-30

Figure Lengend Snippet: Flow cytometry analysis with novel anti-CD26 mAbs. A. Jurkat-CD26WT cells (red lines) or Jurkat parent cells (blue lines) were incubated with the hybridoma supernatant, and subsequently stained with PE-labeled anti-mouse Ig pAb, and analyzed by flow cytometry. B. Jurkat-CD26WT cells were incubated with the hybridoma supernatant (clone 1, 5, 11, 16, 18 or 19) or purified mouse anti-CD26 mAb (5F8) or commercial mouse anti-CD26 mAb (MBL, clone 44–4), and subsequently stained with PE-labeled anti-mouse Ig pAb, and analyzed by flow cytometry. The gray areas in each histogram show the data involving the isotype control. The mean fluorescence intensity (MFI) of each staining is shown. Data shown are repeated twice (A) and five times (B) with similar results.

Article Snippet: Although anti-human CD26 mAbs which we have developed previously or commercially available mAbs cannot clearly detect denatured CD26 in formalin-fixed paraffin-embedded tissues, the anti-human CD26 pAb purchased from R&D Systems is able to stain CD26 with reliable clarity and intensity.

Techniques: Flow Cytometry, Incubation, Staining, Labeling, Purification, Control, Fluorescence

Journal: Diagnostic Pathology

Article Title: Establishment of monoclonal anti-human CD26 antibodies suitable for immunostaining of formalin-fixed tissue

doi: 10.1186/1746-1596-9-30

Figure Lengend Snippet: ELISA analysis with novel anti-CD26 mAbs. Non treated native soluble CD26 (sCD26) or urea treated denatured sCD26 was incubated with the hybridoma supernatant (clone 1, 5, 11, 16, 18 or 19) or purified mouse anti-CD26 mAb (5F8) or commercial mouse anti-CD26 mAb (MBL, clone 44–4) or purified goat anti-CD26 pAb (R&D Systems). The absorbance at 450 nm/570 nm was measured, and data are shown as mean ± S.E. from three independent experiments.

Article Snippet: Although anti-human CD26 mAbs which we have developed previously or commercially available mAbs cannot clearly detect denatured CD26 in formalin-fixed paraffin-embedded tissues, the anti-human CD26 pAb purchased from R&D Systems is able to stain CD26 with reliable clarity and intensity.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Purification

Journal: Diagnostic Pathology

Article Title: Establishment of monoclonal anti-human CD26 antibodies suitable for immunostaining of formalin-fixed tissue

doi: 10.1186/1746-1596-9-30

Figure Lengend Snippet: Representative results of immunostaining with novel anti-CD26 mAbs. A. The tissue specimens of liver, kidney, prostate or two cases of malignant mesothelioma were stained with 100 μl of commercial mouse anti-human CD26 mAb supernatant (MBL, clone 44–4) (i), or 10 μg/ml of purified goat anti-human CD26 pAb (R&D Systems) (ii), or newly developed hybridoma supernatant (clone 18 (iii), clone 19 (iv) or clone 3 (v)). B . Malignant mesothelioma tissue specimens were stained with commercial goat anti-human CD26 pAb (R&D Systems) (i), or purified novel mouse anti-human CD26 mAbs (clone 18 (ii) or clone 19 (iii)) at the indicated concentrations of Abs in the presence or absence of sCD26. C. The tissue specimens of hepatocellular carcinoma, renal cell carcinoma, prostate adenocarcinoma, colon adenocarcinoma or lung adenocarcinoma were stained with 100 μg/ml of commercial goat anti-human CD26 pAb (R&D Systems) (i), or purified mouse anti-human CD26 mAbs (clone 18 (ii) or clone 19 (iii)). All specimens were counterstained with hematoxylin (original magnification, 200X).

Article Snippet: Although anti-human CD26 mAbs which we have developed previously or commercially available mAbs cannot clearly detect denatured CD26 in formalin-fixed paraffin-embedded tissues, the anti-human CD26 pAb purchased from R&D Systems is able to stain CD26 with reliable clarity and intensity.

Techniques: Immunostaining, Staining, Purification

Journal: Diagnostic Pathology

Article Title: Establishment of monoclonal anti-human CD26 antibodies suitable for immunostaining of formalin-fixed tissue

doi: 10.1186/1746-1596-9-30

Figure Lengend Snippet: Analysis of crossreactivity of novel anti-CD26 mAbs with humanized anti-CD26 mAb. Jurkat-CD26WT cells were pretreated with unlabeled humanized anti-CD26 mAb (YS110) or human control IgG, and then treated with the hybridoma supernatant (clone 1, 5, 11, 16, 18 or 19) or purified mouse anti-CD26 mAb (1F7 or 5F8), and subsequently stained with PE-labeled anti-mouse Ig pAb. For staining with humanized anti-CD26 mAb, cells were stained with Alexa Fluor 647-labeled YS110 after pretreatment with unlabeled YS110. Data were analyzed by flow cytometry, and the percentage of mean fluorescence intensity (MFI) after YS110 blocking to MFI after control IgG blocking is shown. Data shown are repeated twice with similar results.

Article Snippet: Although anti-human CD26 mAbs which we have developed previously or commercially available mAbs cannot clearly detect denatured CD26 in formalin-fixed paraffin-embedded tissues, the anti-human CD26 pAb purchased from R&D Systems is able to stain CD26 with reliable clarity and intensity.

Techniques: Control, Purification, Staining, Labeling, Flow Cytometry, Fluorescence, Blocking Assay

Journal: Diagnostic Pathology

Article Title: Establishment of monoclonal anti-human CD26 antibodies suitable for immunostaining of formalin-fixed tissue

doi: 10.1186/1746-1596-9-30

Figure Lengend Snippet: Blocking experiment of novel anti-CD26 mAb binding to CD26. Jurkat-CD26WT cells were pretreated with unlabeled mouse anti-CD26 mAbs (4G8, 1F7, 5F8, 16D4B, or 9C11) (blue lines) or mouse IgG 1 isotype control (Contl. IgG) (red lines), and subsequently stained with PE-labeled anti-mouse Ig pAb (i) or Alexa Fluor 647-labeled anti-CD26 mAbs (clone 18 (ii) or clone 19 (iii) ), and analyzed by flow cytometry. The representative histograms of CD26 expression are shown, and the gray areas in each histogram show the data involving the isotype control. Data shown are repeated twice with similar results.

Article Snippet: Although anti-human CD26 mAbs which we have developed previously or commercially available mAbs cannot clearly detect denatured CD26 in formalin-fixed paraffin-embedded tissues, the anti-human CD26 pAb purchased from R&D Systems is able to stain CD26 with reliable clarity and intensity.

Techniques: Blocking Assay, Binding Assay, Control, Staining, Labeling, Flow Cytometry, Expressing

Journal: Diagnostic Pathology

Article Title: Establishment of monoclonal anti-human CD26 antibodies suitable for immunostaining of formalin-fixed tissue

doi: 10.1186/1746-1596-9-30

Figure Lengend Snippet: Staining for CD26 expression on COS-7 cells transfected with CD26 deletion mutants by novel anti-CD26 mAbs. cDNA of deleted CD26 was cotransfected with GFP-expressing plasmid to COS-7 cells. After 24 h, the transfected cells were stained with Alexa Fluor 647-labeled anti-CD26 mAbs (YS110, clone 18 or clone 19) or isotype control, and analyzed by flow cytometry. Following gating for GFP positive cells among all acquired cells, the percentage of CD26 positive cells was analyzed. Data shown are repeated twice with similar results.

Article Snippet: Although anti-human CD26 mAbs which we have developed previously or commercially available mAbs cannot clearly detect denatured CD26 in formalin-fixed paraffin-embedded tissues, the anti-human CD26 pAb purchased from R&D Systems is able to stain CD26 with reliable clarity and intensity.

Techniques: Staining, Expressing, Transfection, Plasmid Preparation, Labeling, Control, Flow Cytometry

Journal: Journal of Virology

Article Title: Permissivity of Dipeptidyl Peptidase 4 Orthologs to Middle East Respiratory Syndrome Coronavirus Is Governed by Glycosylation and Other Complex Determinants

doi: 10.1128/JVI.00534-17

Figure Lengend Snippet: Permissivity of DPP4 orthologs to MERS-CoV. (A) Seven DPP4 orthologs were tested for their ability to support infection by rMERS-CoV-RFP. DPP4 constructs were transfected into HEK 293T cells and infected at an MOI of 5 at ∼20 h posttransfection. Cells were imaged for fluorescence at ∼24 hpi. hDPP4, human DPP4; cDPP4, camel DPP4; bDPP4, bat DPP4; mDPP4, mouse DPP4; fDPP4, ferret DPP4; haDPP4, hamster DPP4; gpDPP4, guinea pig DPP4. (B) Mean fluorescent cell counts of MERS-CoV infection utilizing various DPP4 orthologs. Cells were infected at an MOI of 0.1 and the numbers of infected cells counted at ∼72 hpi. Each DPP4 ortholog was measured in triplicate. Only hDPP4, bDPP4, and cDPP4 had levels of infection significantly higher than those seen in the absence of DPP4 (*, P < 0.05 [Student's t test]). All DPP4 orthologs had significantly lower levels of infection than hDPP4 (P < 0.05 [Student's t test]). The levels of infection seen between bDPP4 and cDPP4 were not significantly different. Error bars indicate mean values ± 1 standard deviation.

Article Snippet: Error bars indicate mean values ± 1 standard deviation. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG 4 caption a7 DPP4 and mutant variants are expressed on the surface of cells as evidenced by results of immunofluorescence assay (A) and flow cytometry (B and C). (A) Cells were transfected with each DPP4 ortholog, fixed, and probed with primary goat anti-DPP4 polyclonal antibody (R&D Systems) at 1:50 and secondary donkey anti-goat Alexa Fluor 488 (Life Technologies) at 1:500.

Techniques: Infection, Construct, Transfection, Fluorescence, Standard Deviation

Journal: Journal of Virology

Article Title: Permissivity of Dipeptidyl Peptidase 4 Orthologs to Middle East Respiratory Syndrome Coronavirus Is Governed by Glycosylation and Other Complex Determinants

doi: 10.1128/JVI.00534-17

Figure Lengend Snippet: Sequence and structural comparison of nonpermissive DPP4 orthologs. (A) Structural comparison of threaded molecules (30) of mDPP4 (orange), fDPP4 (green), haDPP4 (blue), and gpDPP4 (purple) overlaid on hDPP4 (yellow) complexed with the MERS-CoV RBD (red) (PDB code 4L72). (B) Sequence alignment of permissive (human, camel, bat; blue) and nonpermissive (mouse, ferret, hamster, guinea pig; red) DPP4 amino acid sequences. Residue 330 is numbered relative to mDPP4. Boxes represent glycosylation sites that are either unique to nonpermissive species (black) or shared with a permissive species (gray). (C) hDPP4 (yellow) complexed with the MERS-CoV RBD (red) (PDB code 4L72). Residues aligning to the ferret (green), hamster and mouse (blue), and guinea pig (purple) glycosylation sites are highlighted. Dashed-line circles indicate the regions of the DPP4 molecule that correspond to blades IV and V.

Article Snippet: Error bars indicate mean values ± 1 standard deviation. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG 4 caption a7 DPP4 and mutant variants are expressed on the surface of cells as evidenced by results of immunofluorescence assay (A) and flow cytometry (B and C). (A) Cells were transfected with each DPP4 ortholog, fixed, and probed with primary goat anti-DPP4 polyclonal antibody (R&D Systems) at 1:50 and secondary donkey anti-goat Alexa Fluor 488 (Life Technologies) at 1:500.

Techniques: Sequencing, Comparison, Residue

Journal: Journal of Virology

Article Title: Permissivity of Dipeptidyl Peptidase 4 Orthologs to Middle East Respiratory Syndrome Coronavirus Is Governed by Glycosylation and Other Complex Determinants

doi: 10.1128/JVI.00534-17

Figure Lengend Snippet: Known and putative N-linked glycans in DPP4 orthologs a

Article Snippet: Error bars indicate mean values ± 1 standard deviation. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG 4 caption a7 DPP4 and mutant variants are expressed on the surface of cells as evidenced by results of immunofluorescence assay (A) and flow cytometry (B and C). (A) Cells were transfected with each DPP4 ortholog, fixed, and probed with primary goat anti-DPP4 polyclonal antibody (R&D Systems) at 1:50 and secondary donkey anti-goat Alexa Fluor 488 (Life Technologies) at 1:500.

Techniques: Residue

Journal: Journal of Virology

Article Title: Permissivity of Dipeptidyl Peptidase 4 Orthologs to Middle East Respiratory Syndrome Coronavirus Is Governed by Glycosylation and Other Complex Determinants

doi: 10.1128/JVI.00534-17

Figure Lengend Snippet: DPP4 ortholog glycosylation knockout mutants. (A) Neither wild-type nor glycosylation knockout (−gly) DPP4 molecules for ferret (fDPP4), hamster (haDPP4), or guinea pig (gpDPP4) support infection by MERS-CoV. (B) Successful removal of glycosylation is supported by an ∼2.5-kDa downward shift seen via Western blotting. The top blot represents DPP4, and the bottom blot represents β-actin as a control. (C) Fluorescent cell counts from MERS-CoV infection utilizing various DPP4 orthologs and their respective glycosylation knockout mutants. Cells were infected at an MOI of 0.1, and numbers of infected cells were counted at 72 hpi. The level of each DPP4 ortholog was measured in triplicate. Only hDPP4 had levels of infection significantly higher than those seen in the absence of DPP4 (*, P < 0.05 [Student's t test]). The remaining DPP4 orthologs and glycosylation knockouts had infection levels that were not significantly different from those seen in the absence of DPP4. Error bars indicate mean values ± 1 standard deviation.

Article Snippet: Error bars indicate mean values ± 1 standard deviation. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG 4 caption a7 DPP4 and mutant variants are expressed on the surface of cells as evidenced by results of immunofluorescence assay (A) and flow cytometry (B and C). (A) Cells were transfected with each DPP4 ortholog, fixed, and probed with primary goat anti-DPP4 polyclonal antibody (R&D Systems) at 1:50 and secondary donkey anti-goat Alexa Fluor 488 (Life Technologies) at 1:500.

Techniques: Knock-Out, Infection, Western Blot, Control, Standard Deviation

Journal: Journal of Virology

Article Title: Permissivity of Dipeptidyl Peptidase 4 Orthologs to Middle East Respiratory Syndrome Coronavirus Is Governed by Glycosylation and Other Complex Determinants

doi: 10.1128/JVI.00534-17

Figure Lengend Snippet: DPP4 and mutant variants are expressed on the surface of cells as evidenced by results of immunofluorescence assay (A) and flow cytometry (B and C). (A) Cells were transfected with each DPP4 ortholog, fixed, and probed with primary goat anti-DPP4 polyclonal antibody (R&D Systems) at 1:50 and secondary donkey anti-goat Alexa Fluor 488 (Life Technologies) at 1:500. Cells were imaged at a magnification of ×20 for DAPI (300 ms exposure) and DPP4 (1.5 s exposure). (B) DPP4 expression frequencies (blue outlined histogram) by DPP4 construct after subtraction of background from replicate wells stained with secondary donkey anti-goat IgG (H+L) Alexa Fluor 488 antibody only (gray-shaded histogram). Percentage values represent averages of results across two duplicate wells. Max, maximum. (C) Geometric mean fluorescence intensity of the DPP4-positive populations for each DPP4 construct.

Article Snippet: Error bars indicate mean values ± 1 standard deviation. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG 4 caption a7 DPP4 and mutant variants are expressed on the surface of cells as evidenced by results of immunofluorescence assay (A) and flow cytometry (B and C). (A) Cells were transfected with each DPP4 ortholog, fixed, and probed with primary goat anti-DPP4 polyclonal antibody (R&D Systems) at 1:50 and secondary donkey anti-goat Alexa Fluor 488 (Life Technologies) at 1:500.

Techniques: Mutagenesis, Immunofluorescence, Flow Cytometry, Transfection, Expressing, Construct, Staining, Fluorescence

Journal: Journal of Virology

Article Title: Permissivity of Dipeptidyl Peptidase 4 Orthologs to Middle East Respiratory Syndrome Coronavirus Is Governed by Glycosylation and Other Complex Determinants

doi: 10.1128/JVI.00534-17

Figure Lengend Snippet: Many amino acid changes are required to make fDPP4 and haDPP4 permissive to MERS-CoV infection. (A) Removing glycosylation alone did not confer permissivity to haDPP4. However, combining three amino acid changes on blade V (starting at residue 289) with the glycosylation knockout mutant on blade IV (N332A) resulted in high levels of MERS-CoV infection. Sequences show the alignment between hDPP4 and haDPP4, with the black boxes indicating the amino acids that were swapped from hDPP4 into haDPP4. (B) Removing glycosylation alone did not confer permissivity to fDPP4. However, introducing a set of amino acid changes on blade V (starting at residue 278) and blade IV (starting at residue 330) allowed fDPP4 to support MERS-CoV infection [fDPP4 (278) (330)]. Sequences show the alignment between hDPP4 and fDPP4, with the black boxes indicating the amino acids that were swapped from hDPP4 into fDPP4. Note that fDPP4 −gly is a negative control and includes only the single point mutation N331A. (C) Western blot analysis of fDPP4 and haDPP4 and designated variants for DPP4 and β-actin expression. Successful glycosylation knockout is indicated by a downward shift of ∼2.5 kDa. (D) Fluorescent cell counts of MERS-CoV infection utilizing DPP4 orthologs. Cells were infected at an MOI of 0.1, and numbers of red cells were counted at 72 hpi. Each DPP4 ortholog was measured in triplicate. hDPP4, fDPP4 (278) (330), and haDPP4 (289), −gly had levels of infection that were significantly greater than those seen in the absence of DPP4 (P < 0.05 [Student's t test]). fDPP4 −gly and haDPP4 −gly infection levels were not significantly different from those seen in the absence of DPP4. Error bars indicate mean values ± 1 standard deviation.

Article Snippet: Error bars indicate mean values ± 1 standard deviation. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG 4 caption a7 DPP4 and mutant variants are expressed on the surface of cells as evidenced by results of immunofluorescence assay (A) and flow cytometry (B and C). (A) Cells were transfected with each DPP4 ortholog, fixed, and probed with primary goat anti-DPP4 polyclonal antibody (R&D Systems) at 1:50 and secondary donkey anti-goat Alexa Fluor 488 (Life Technologies) at 1:500.

Techniques: Infection, Residue, Knock-Out, Mutagenesis, Negative Control, Western Blot, Expressing, Standard Deviation

Journal: Journal of Virology

Article Title: Permissivity of Dipeptidyl Peptidase 4 Orthologs to Middle East Respiratory Syndrome Coronavirus Is Governed by Glycosylation and Other Complex Determinants

doi: 10.1128/JVI.00534-17

Figure Lengend Snippet: Residues identified to be important for MERS-CoV permissivity a

Article Snippet: Error bars indicate mean values ± 1 standard deviation. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG 4 caption a7 DPP4 and mutant variants are expressed on the surface of cells as evidenced by results of immunofluorescence assay (A) and flow cytometry (B and C). (A) Cells were transfected with each DPP4 ortholog, fixed, and probed with primary goat anti-DPP4 polyclonal antibody (R&D Systems) at 1:50 and secondary donkey anti-goat Alexa Fluor 488 (Life Technologies) at 1:500.

Techniques: Residue

Journal: Journal of Virology

Article Title: Permissivity of Dipeptidyl Peptidase 4 Orthologs to Middle East Respiratory Syndrome Coronavirus Is Governed by Glycosylation and Other Complex Determinants

doi: 10.1128/JVI.00534-17

Figure Lengend Snippet: Bat and guinea pig DPP4 share the same glycosylation site downstream of the site identified to be important in mDPP4 (Fig. 2B). (A) Removal of the glycosylation site from bDPP4 showed no decrease in infection, while removal of glycosylation from gpDPP4 resulted in no increase in infection. (B) Western blot analysis of bDPP4 and gpDPP4 and their respective glycosylation knockout mutants for DPP4 and β-actin expression. Successful glycosylation knockout is indicated by a downward shift of ∼2.5 kDa. (C) DPP4 and mutant variants are expressed on the surface of cells, visible by immunofluorescence. Cells were transfected with each DPP4 ortholog, fixed, and probed with primary goat anti-DPP4 polyclonal antibody (R&D Systems) at 1:50 and secondary donkey anti-goat Alexa Fluor 488 (Life Technologies) at 1:500. Cells were imaged at a magnification of ×20 for DAPI (300-ms exposure) and DPP4 (1.5-s exposure). (D) Fluorescent cell counts of MERS-CoV infection utilizing various DPP4 orthologs. Cells were infected at an MOI of 0.1, and numbers of infected cells were counted at 72 hpi. Each DPP4 ortholog was measured in triplicate. hDPP4, bDPP4, and bDPP4 −gly had levels of infection that are significantly higher than those seen in the absence of DPP4 (P < 0.05 [Student's t test]). gpDPP4 and gpDPP4 −gly infection levels were not significantly different from those seen in the absence of DPP4. Error bars indicate mean values ± 1 standard deviation.

Article Snippet: Error bars indicate mean values ± 1 standard deviation. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG 4 caption a7 DPP4 and mutant variants are expressed on the surface of cells as evidenced by results of immunofluorescence assay (A) and flow cytometry (B and C). (A) Cells were transfected with each DPP4 ortholog, fixed, and probed with primary goat anti-DPP4 polyclonal antibody (R&D Systems) at 1:50 and secondary donkey anti-goat Alexa Fluor 488 (Life Technologies) at 1:500.

Techniques: Infection, Western Blot, Knock-Out, Expressing, Mutagenesis, Immunofluorescence, Transfection, Standard Deviation

Journal: Journal of Virology

Article Title: Permissivity of Dipeptidyl Peptidase 4 Orthologs to Middle East Respiratory Syndrome Coronavirus Is Governed by Glycosylation and Other Complex Determinants

doi: 10.1128/JVI.00534-17

Figure Lengend Snippet: DPP4 protein phylogenetic tree based on amino acid sequences. Shaded colors indicate the group each species falls in. Blue, reptiles and amphibians; green, avian species; orange, other mammals; red, Chiroptera (bats); purple, ungulates; gray, rodents; pink: primates. Colored circles to the right of the species names indicate whether the sequence has a glycosylation site upstream of (first column), at the same site as (second column), or downstream of (third column) the NXT glycosylation site in mDPP4 (residues 332 to 334). Numbers inside the circle designate how many amino acids upstream (or downstream) the N of the NXT or NXS glycosylation site is. For the second column, a circle indicates that there was a glycosylation site aligning to the site present in mDPP4. Squares in the rightmost column indicate permissive (green) or nonpermissive (red) species, as determined from either in vivo or in vitro studies. Numbers indicate bootstrap support values >50.

Article Snippet: Error bars indicate mean values ± 1 standard deviation. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG 4 caption a7 DPP4 and mutant variants are expressed on the surface of cells as evidenced by results of immunofluorescence assay (A) and flow cytometry (B and C). (A) Cells were transfected with each DPP4 ortholog, fixed, and probed with primary goat anti-DPP4 polyclonal antibody (R&D Systems) at 1:50 and secondary donkey anti-goat Alexa Fluor 488 (Life Technologies) at 1:500.

Techniques: Sequencing, In Vivo, In Vitro