cd46 p  (Sino Biological)

Journal: Nature Communications

Article Title: CD46 facilitates entry and dissemination of human cytomegalovirus

doi: 10.1038/s41467-019-10587-1

Figure Lengend Snippet: Identification of inhibitory monoclonal antibodies and their cellular target. a Supernatants from cell-surface binding clones were subjected to a high-throughput infectivity assay (HTI) with TB40/E FLAG YFP infection of ARPE-19 cells using YFP fluorescence as readout for infection. The % infection was determined using virus incubated with media alone as 100%. b Clones demonstrating reduced infection were validated using the TB40/E FLAG YFP /ARPE-19 cells HTI with varying amounts (%) of supernatant. c Purified mAbs (10 μg ml −1 ) were analyzed using the TB40/E FLAG YFP /ARPE-19 HTI in technical triplicates. d Polypeptides recovered with mAb 2E7 and 12H8 (arrows) from ARPE-19 cells metabolically labeled with 35 S-methinionine (6 h) were resolved on a SDS-polyacrylamide gel and visualized on a radiographic film. Beads only was used as a control. e Polypeptides recovered with mAb 2E7 and 12H8 from ARPE-19 cells were subjected to immunoblot analysis using anti-CD46 antibodies. mAb W6/32 and total cell lysates (TCL) were included as controls. The polypeptides and molecular weight markers are indicated. s.d. is depicted in the experiment. * P < 0.05, ** P < 0.01 (Student’s two-tailed t test)

Article Snippet: Virus (MOI:0.5) incubated with respective mAbs (2 μg ml −1 , or otherwise noted) or soluble proteins Nrp2 (P) and CD46 (P) (Sino Biological (cat#: 10695-H08H and 12239-H08H) for 1 h 4 °C was used to infect ARPE-19, HTR-8/SVneo, or MRC5 cells (1 × 10 4 cell/well in a 96-well plate) for 2 h (37 °C) and then replaced with new media.

Techniques: Binding Assay, Clone Assay, High Throughput Screening Assay, Infection, Fluorescence, Incubation, Purification, Metabolic Labelling, Labeling, Western Blot, Molecular Weight, Two Tailed Test

Journal: Nature Communications

Article Title: CD46 facilitates entry and dissemination of human cytomegalovirus

doi: 10.1038/s41467-019-10587-1

Figure Lengend Snippet: CD46-dependent CMV entry in epithelial cells. a TB40/E wt was subjected to a mAb inhibition HTI in ARPE-19 cells with mAb 5C3 (anti-gH), mAb 2E7 (anti-CD46), mAb PY102 (non-binding control) or no-antibody control (20-0.01 μg ml −1 , in threefold dilutions). Virus infection from PY102-treated cells represents 100% infection. b Total cell lysates from uninfected, TB40/E wt and AD169 BADrUL131 infected ARPE-19 cells treated with mAbs 5C3 or 2E7 (10 and 2 μg ml −1 ), mAb PY102 (10 μg ml −1 ), and no mAb were subject to immunoblot analysis for IE1 and GAPDH. ( c ) CD46 protein model represents four consensus repeats (SCRs) where complement proteins C4b, C3b, and C4b bind, a serine/threonine/proline (STP)-rich region, an uncharacterized segment (U), a transmembrane domain (TM), and a cytoplasmic tail . Alternative splicing accounts for common isomers expressing either STP regions BC or just C and either a short cytoplasmic tail (CYT1) or long cytoplasmic tail (CYT2) . d Using flow cytometry, anti-CD46 mAbs TRA-2–10 and GB24 (2 μg ml −1 ) were analyzed on ARPE-19 cells in comparison to PY102. e ARPE-19 cells were incubated with labeled 2E7 647 (2 μg ml −1 ) and increasing concentrations (6.7–0.22 μg ml −1 , in 3-fold dilutions) of mAbs TRA-2-10 or GB24. The mean fluorescence intensity (MFI) of anti-mouse IgG Alexa647 was measured by flow cytometry with PY102 (−) and non-labeled 2E7 (+) as controls. f TB40/E wt and AD169 BADrUL131 infection of ARPE-19 cells treated with mAbs 5C3, 2E7, TRA-2-10, GB24, and PY102 (6.7–0.74 μg ml −1 , in threefold dilutions) and a no-antibody control were analyzed using an HTI. PY102-treated cells represents 100% infection. g ARPE-19 cells transfected with non-targeting siRNA (SCR) or siRNA targeting CD46 (CD46) were infected with TB40/E FLAG YFP and AD169 BADrUL131 and analyzed for infection (YFP fluorescent intensity) by flow cytometry. SCR-transfected cells represent 100% infection. h Cell lysates from ARPE-19 cells transfected with no siRNA, SCR, or CD46 targeting siRNA and infected with AD169 BADrUL131 or TB40/E wt (MOI:0.5) were subjected to anti-IE1 and GAPDH immunoblots. The polypeptides and molecular weight markers are indicated for the immunoblots. Infection experiments were performed in triplicate. s.d. is depicted in the experiment. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (Student’s two-tailed t test)

Article Snippet: Virus (MOI:0.5) incubated with respective mAbs (2 μg ml −1 , or otherwise noted) or soluble proteins Nrp2 (P) and CD46 (P) (Sino Biological (cat#: 10695-H08H and 12239-H08H) for 1 h 4 °C was used to infect ARPE-19, HTR-8/SVneo, or MRC5 cells (1 × 10 4 cell/well in a 96-well plate) for 2 h (37 °C) and then replaced with new media.

Techniques: Inhibition, Binding Assay, Infection, Western Blot, Expressing, Flow Cytometry, Incubation, Labeling, Fluorescence, Transfection, Molecular Weight, Two Tailed Test

Journal: Nature Communications

Article Title: CD46 facilitates entry and dissemination of human cytomegalovirus

doi: 10.1038/s41467-019-10587-1

Figure Lengend Snippet: CD46 is an important factor for CMV infection and spread. a CD46 cell surface expression of wild-type ARPE-19 cells (Ewt), β 2 m Knock-out (KO) ARPE-19 cells (Eβ1), and three CD46-KO ARPE-19 cell clones (EC1, EC2, and EC3) was assessed by flow cytometry with mAbs against CD46 (2E7 and GB24), MHC class I (W6/32), and PY102 as a non-binding control. Following incubation with anti-mouse IgG Alexa647 , the normalized cell number was plotted based on Alexa647 fluorescence intensity. b The respective KO cells ( a ) were subjected to anti-CD46 and GAPDH immunoblots. c ARPE-19 wt and CD46- and β 2 m-KO cells infected with TB40/E wt or AD169 BADrUL131 were analyzed for infection by an HTI. Total number of infected cells was determined using anti-IE1 antibodies. d ARPE-19 wt and CD46- and β 2 m-KO cells infected with TB40/E wt, TB40/E MC UL99-eGFP , or AD169 BADrUL131 were subjected to a plaque assay, counted 14 dpi. e Supernatant from Ewt, Eβ1, and EC2 cells infected (E: epithelial cells) with AD169 BADrUL131 (MOI:0.1) from days 0–12 was added to MRC5 and analyzed for infection at 24 hpi using YFP fluorescent with a cytometer. The virus titre (infectious units (IU)/ml) was plotted for up to 12 dpi. f Cell lysates of wt HTR-8/SVneo (Twt) cells, β 2 m-KO HTR-8/SVneo (Tβ1) cells, and CD46-KO HTR-8/SVneo cell clones (TC1 and TC2, T: trophoblasts) were subjected to immunoblot using anti-CD46 and anti-GAPDH antibodies. g Cell lysates of AD169 BADrUL131 (MOI:2) infected Twt, Tβ1, TC1 and TC2 were subjected to anti-IE1 and GAPDH immunoblots. h ARPE-19 wt and Tβ1, TC1, and TC2 clones infected with AD169 BADrUL131 at varying MOIs were analyzed for infection by an HTI assay. The total number of infected cells was determined using a cytometer. i Representative cytometer images of ( h ) (overlay: Hoechst stain for nuclear stain (blue) and GFP expression (green) for virally infected cells) (MOI 2). The polypeptides and molecular weight markers are indicated for the immunoblots. Infection experiments (excluding westerns) were performed in triplicate. s.d. is depicted in the experiment. ** P < 0.01, *** P < 0.001, **** P < 0.0001 (Student’s two-tailed t test)

Article Snippet: Virus (MOI:0.5) incubated with respective mAbs (2 μg ml −1 , or otherwise noted) or soluble proteins Nrp2 (P) and CD46 (P) (Sino Biological (cat#: 10695-H08H and 12239-H08H) for 1 h 4 °C was used to infect ARPE-19, HTR-8/SVneo, or MRC5 cells (1 × 10 4 cell/well in a 96-well plate) for 2 h (37 °C) and then replaced with new media.

Techniques: Infection, Expressing, Knock-Out, Clone Assay, Flow Cytometry, Binding Assay, Incubation, Fluorescence, Western Blot, Plaque Assay, Cytometry, Staining, Molecular Weight, Two Tailed Test

Journal: Nature Communications

Article Title: CD46 facilitates entry and dissemination of human cytomegalovirus

doi: 10.1038/s41467-019-10587-1

Figure Lengend Snippet: CD46 is involved in a post-binding entry step. a TB40/E wt infection of ARPE-19 cells untreated or treated at −0.5, 0.5, and 5hpi with mAbs targeting HCMV gH (5C3) and CD46 (2E7), heparin, a non-binding antibody control (PY102), or a no-antibody control (10 μg ml −1 ) was analyzed with a HTI. PY102-treated cells was used as 100% infection. b TB40/E wt infection of ARPE-19 cells and ( c ) AD169 BADrUL131 infection of HTR-8/SVneo cells incubated with mAbs 5C3, 2E7, and anti-Nrp2 (αNrp2), or soluble CD46 and Nrp2 proteins (P) (20–0.24 μg ml −1 , in threefold dilutions) were subjected to the HTI assay. The % infection was determined using incubation with PY102 as 100% infection. Experiments were performed in triplicate. s.d. is depicted in the experiment. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (Student’s two-tailed t test)

Article Snippet: Virus (MOI:0.5) incubated with respective mAbs (2 μg ml −1 , or otherwise noted) or soluble proteins Nrp2 (P) and CD46 (P) (Sino Biological (cat#: 10695-H08H and 12239-H08H) for 1 h 4 °C was used to infect ARPE-19, HTR-8/SVneo, or MRC5 cells (1 × 10 4 cell/well in a 96-well plate) for 2 h (37 °C) and then replaced with new media.

Techniques: Binding Assay, Infection, Incubation, Two Tailed Test

Journal: Oncotarget

Article Title: Cap-dependent translational control of oncolytic measles virus infection in malignant mesothelioma

doi: 10.18632/oncotarget.18656

Figure Lengend Snippet: Relative CD46 expression levels on surfaces of a panel of non-transformed and MM cells expressed in mean fluorescence intensity (A) . Representative histograms of CD46 receptor are displayed from non-transformed and mesothelioma cells. Cells were stained with anti-CD46 PE antibody (grey histogram) or an isotype control (open histogram) and analyzed by flow cytometry (B) . Nectin-4 expression levels on the surfaces of non-transformed and MM cells conveyed in mean fluorescence intensity (C) . Histograms of nectin-4 on cells stained with anti-nectin-4 PE antibody (grey histogram) or an isotype control (open histogram) and measured by flow cytometry (D) . Microscopy, both Light (LM-top) and fluorescence (FM-bottom) of H513 cells infected with MV-Edm producing green fluorescent protein (MV-GFP) following exposure to blocking antibodies against CD46, nectin-4 or the combination (E) . Results for fluorescence measurement following H513 cell treatment with blocking antibodies for CD46 and nectin-4 prior to MV-GFP infection. Isotype IgG treatment and H513 cells untreated with MV-GFP were used as positive and negative controls, respectively (F) .

Article Snippet: The following day cells were treated with isotype IgG (R & D Systems, catalog number AB-108-C), or nectin-4 IgG (R & D Systems, catalog number AF2659) or CD46 IgG (Santa Cruz, Dallas TX, catalog number sc-5267-clone m177) or both CD46 and nectin-4 antibodies.

Techniques: Expressing, Transformation Assay, Fluorescence, Staining, Control, Flow Cytometry, Microscopy, Infection, Blocking Assay