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  • 85
    ATCC bhk 21 cells
    S.A.AR86, as well as other Sindbis-group alphaviruses, replicates in bone- and joint-associated tissues. (A) Six-week-old female CD-1 mice were infected with 10 3 PFU of s55 i.v. Three days postinfection mice were sacrificed, exsanguinated, and perfused with PBS, and their femurs and quadriceps muscles were removed by dissection and titrated for infectious virus on <t>BHK-21</t> cells. Data are shown as log PFU per gram of tissue for femur and muscle and as PFU per milliliter for serum. Each bar represents a single animal. The arrow indicates the limit of detection, and asterisks denote samples below the limit of detection. Data shown are from one of four experiments. (B) Six-week-old female CD-1 mice were infected i.v. with 10 3 PFU of s51 and sacrificed 3 days postinfection, and right femurs were removed for virus titration. Bone marrow was aspirated from the diaphyses of the femurs using 0.4 ml of PBS–1% donor calf serum per femur. Aspirates were freeze-thawed and titrated for infectious virus by plaque assay. Following marrow aspiration, the remaining femoral tissue was processed as for panel A and titrated for infectious virus by plaque assay. Titer is shown as total PFU per marrow aspirate (Asp) or femur (without aspirated marrow), with each bar representing a single animal. The arrow indicates the limit of detection, and asterisks denote samples with titers below the limit of detection. Shown is one of three comparable experiments. (C) Six-week-old female CD-1 mice were infected i.v. with 10 3 PFU of the virus s55, TR339, or TRSB. Three days postinfection mice were sacrificed and both femurs were removed for virus titration. Femurs were processed and titrated for infectious virus as for panel A. Each bar represents results from a single animal. The arrow indicates the limit of detection. N/D, not done. Shown is one of two comparable experiments.
    Bhk 21 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 85/100, based on 2876 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore bhk 21 cells
    Effect of mutations on viral replication. (A) Virion-associated reverse transcriptase (RT) activity of wild type and mutant viral particles. <t>BHK-21</t> cells were infected with 1 ml of released virus from the transfection experiment. Extracellular virions were harvested at 2, 4, and 6 d post-infection, concentrated by centrifugation, and RT activity was assessed by measuring incorporation of [ 3 H]TTP on a poly(A):(dT) 12–18 template-primer. Data are an average of three independent experiments. (B) Detection of Gag in the virion pellet. The same number of virion pellets was resolved on 12.5% sodium dodecyl sulfatepolyacrylamide gels, transferred to a membrane, and then probed with anti-Gag antibody.
    Bhk 21 Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 477 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC bhk cells
    Interaction of <t>BHK</t> cells with PMMA–PTE <t>nanoparticles</t> at a concentration of 25 ppm. The images represent the nucleus stained with DAPI (A), cellular matrix stained with nanoparticles (B), and an overlay of the two images (C), with the magnified images in the inset showing nanoparticle distribution inside the cells (D). Magnified view (D) of nucleus stained with DAPI (i), cellular matrix stained with nanoparticles (ii), cells observed under DIC mode (iii), and overlay of (i)–(iv). The cells were imaged at a magnification of 60× oil objective using a confocal microscope (Olympus FV1000).
    Bhk Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 476 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bio-Rad bhk 21 cells
    Immunofluorescence staining of protein E in <t>BHK-21</t> cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein
    Bhk 21 Cells, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 517 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher bhk 21 cells
    Intracellular localization of CD4 and chimeric CD4 proteins containing the TMD of DENV2 with different mutations by double-label immunofluorescence assay. (A) <t>BHK-21</t> cells were transfected with each of the constructs described in Fig.
    Bhk 21 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3496 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Mirus Bio bhk 21 cells
    Growth curves of chimeric viruses. A Monolayer of <t>BHK-21</t> cells was infected with wild-type and chimeric viruses at an MOI of 0.01. Media were harvested at each time point, and virus titers were determined by plaque assay in BHK-21 cells. The chimeric
    Bhk 21 Cells, supplied by Mirus Bio, used in various techniques. Bioz Stars score: 92/100, based on 104 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher cd bhk 21 production medium
    More than 2 PKs provides a replicative advantage in co-transfection competition experiments. <t>BHK-21</t> cells were co-transfected with wt , ΔPK 1234, ΔPK 234 or ΔPK 34 ptGFP replicon RNA and either a wt mCherry replicon or yeast tRNA control. Control competition of ptGFP wt , ΔPK 234 or ΔPK 34 replicons with yeast tRNA (A). Competition of wt , ΔPK 234 or ΔPK 34 ptGFP replicons with a wt mCherry replicon (B). Replication of the C11 ΔPK 1234 replicon when co-transfected with either the wt mCherry replicon or yeast tRNA control (C). Replication of the wt mCherry replicon when co-transfected with ptGFP wt , C11 ΔPK 1234, ΔPK 234 or ΔPK 34 ptGFP replicons (D). All replication is shown at 8 hours post transfection over 3 sequential passages, as measured by an Incucyte Zoom (n = 2). Initial transfection (P0), sequential passages (P1-3).
    Cd Bhk 21 Production Medium, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Corning Life Sciences bhk 21 cells
    ER Stress is activated in <t>BHK-21</t> cells transfected with 2C- or 3D-expressing plasmid. (A) A Western blotting analysis of GRP78, GRP94 and β-actin in the extracts of BHK-21 cells that were treated with 500 nM Tg for 12 h, infected with EMCV (MOI = 0.005) for 12 h or transfected with 2C- or 3D-expressing plasmid for 48 h. (B) The BHK-21 cells were transfected with pGL3-GRP78-luc, pGL3-GRP94-luc, pGL3-Carlreticulin-luc, pGL3-ATF4-luc or pGL3-CHOP-luc along with pRL-TK and pCMV-HA-2C, pCMV-HA-3D or pCMV-HA. The firefly luciferase activity was measured in cell lysates that were normalized with Renilla luciferase activities at 48 h post-transfection. Data are expressed as the means ± SD. ** p
    Bhk 21 Cells, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 92/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    S.A.AR86, as well as other Sindbis-group alphaviruses, replicates in bone- and joint-associated tissues. (A) Six-week-old female CD-1 mice were infected with 10 3 PFU of s55 i.v. Three days postinfection mice were sacrificed, exsanguinated, and perfused with PBS, and their femurs and quadriceps muscles were removed by dissection and titrated for infectious virus on BHK-21 cells. Data are shown as log PFU per gram of tissue for femur and muscle and as PFU per milliliter for serum. Each bar represents a single animal. The arrow indicates the limit of detection, and asterisks denote samples below the limit of detection. Data shown are from one of four experiments. (B) Six-week-old female CD-1 mice were infected i.v. with 10 3 PFU of s51 and sacrificed 3 days postinfection, and right femurs were removed for virus titration. Bone marrow was aspirated from the diaphyses of the femurs using 0.4 ml of PBS–1% donor calf serum per femur. Aspirates were freeze-thawed and titrated for infectious virus by plaque assay. Following marrow aspiration, the remaining femoral tissue was processed as for panel A and titrated for infectious virus by plaque assay. Titer is shown as total PFU per marrow aspirate (Asp) or femur (without aspirated marrow), with each bar representing a single animal. The arrow indicates the limit of detection, and asterisks denote samples with titers below the limit of detection. Shown is one of three comparable experiments. (C) Six-week-old female CD-1 mice were infected i.v. with 10 3 PFU of the virus s55, TR339, or TRSB. Three days postinfection mice were sacrificed and both femurs were removed for virus titration. Femurs were processed and titrated for infectious virus as for panel A. Each bar represents results from a single animal. The arrow indicates the limit of detection. N/D, not done. Shown is one of two comparable experiments.

    Journal: Journal of Virology

    Article Title: Sindbis-Group Alphavirus Replication in Periosteum and Endosteum of Long Bones in Adult Mice

    doi:

    Figure Lengend Snippet: S.A.AR86, as well as other Sindbis-group alphaviruses, replicates in bone- and joint-associated tissues. (A) Six-week-old female CD-1 mice were infected with 10 3 PFU of s55 i.v. Three days postinfection mice were sacrificed, exsanguinated, and perfused with PBS, and their femurs and quadriceps muscles were removed by dissection and titrated for infectious virus on BHK-21 cells. Data are shown as log PFU per gram of tissue for femur and muscle and as PFU per milliliter for serum. Each bar represents a single animal. The arrow indicates the limit of detection, and asterisks denote samples below the limit of detection. Data shown are from one of four experiments. (B) Six-week-old female CD-1 mice were infected i.v. with 10 3 PFU of s51 and sacrificed 3 days postinfection, and right femurs were removed for virus titration. Bone marrow was aspirated from the diaphyses of the femurs using 0.4 ml of PBS–1% donor calf serum per femur. Aspirates were freeze-thawed and titrated for infectious virus by plaque assay. Following marrow aspiration, the remaining femoral tissue was processed as for panel A and titrated for infectious virus by plaque assay. Titer is shown as total PFU per marrow aspirate (Asp) or femur (without aspirated marrow), with each bar representing a single animal. The arrow indicates the limit of detection, and asterisks denote samples with titers below the limit of detection. Shown is one of three comparable experiments. (C) Six-week-old female CD-1 mice were infected i.v. with 10 3 PFU of the virus s55, TR339, or TRSB. Three days postinfection mice were sacrificed and both femurs were removed for virus titration. Femurs were processed and titrated for infectious virus as for panel A. Each bar represents results from a single animal. The arrow indicates the limit of detection. N/D, not done. Shown is one of two comparable experiments.

    Article Snippet: The tissue homogenate was then clarified by centrifugation and assayed for infectious virus by plaque assay on BHK-21 cells (ATCC CRL 8544) as previously described ( ).

    Techniques: Mouse Assay, Infection, Dissection, Titration, Plaque Assay

    Tax does not colocalize with the ATF/CREB proteins CREB and CREM. Either uninfected BHK21 cells (A and C) or BHK21 cells infected for 18 h with SFV-Tax (B and D) were fixed and analyzed by dual immunofluorescence staining with a Tax monoclonal antibody (left panel) and specific polyclonal antibodies that recognize either CREB (A and B) or CREM (C and D) (middle panel). The panel on the right demonstrates the overlay of the red and green fluorescence.

    Journal: Molecular and Cellular Biology

    Article Title: Differential Transcriptional Activation by Human T-Cell Leukemia Virus Type 1 Tax Mutants Is Mediated by Distinct Interactions with CREB Binding Protein and p300

    doi:

    Figure Lengend Snippet: Tax does not colocalize with the ATF/CREB proteins CREB and CREM. Either uninfected BHK21 cells (A and C) or BHK21 cells infected for 18 h with SFV-Tax (B and D) were fixed and analyzed by dual immunofluorescence staining with a Tax monoclonal antibody (left panel) and specific polyclonal antibodies that recognize either CREB (A and B) or CREM (C and D) (middle panel). The panel on the right demonstrates the overlay of the red and green fluorescence.

    Article Snippet: BHK21 cells (hamster kidney, ATCC CRL 8544) were grown in Glasgow minimum essential medium (Gibco BRL, Gaithersburg, Md.) supplemented with 10% tryptose phosphate, 20 mM HEPES buffer, and 5% fetal bovine serum (FBS); HOS cells (human osteogenic sarcoma, ATCC CRL 1543) were grown in Dulbecco modified Eagle medium (Gibco BRL) supplemented with 10% FBS; and MT2 cells (human T-cell leukemia cells isolated from cord blood lymphocytes cocultured with cells from a patient with T-cell leukemia, obtained from the National Institutes of Health AIDS Research and Reagent Program) were grown in RPMI medium (Gibco BRL) supplemented with 10% FBS, 100 U of penicillin G sodium salt per ml, 100 μg of streptomycin sulfate per ml, and 2 mM glutamine.

    Techniques: Infection, Immunofluorescence, Staining, Fluorescence

    Assessment of Tax and RelA colocalization. Images from horizontal sections (Z series) were collected from a specimen of BHK21 cells infected with SFV-Tax. The cells were analyzed by dual immunofluorescence staining with antibodies to Tax and RelA. Z1 to Z4 are four of these sections, which were collected with 1-μm intervals. P is the projection of the Z series. The left panel displays the LRSC fluorescence of Tax, the middle panel displays the FITC fluorescence of RelA, and the right panel is the overlay of the red and green fluorescence staining.

    Journal: Molecular and Cellular Biology

    Article Title: Differential Transcriptional Activation by Human T-Cell Leukemia Virus Type 1 Tax Mutants Is Mediated by Distinct Interactions with CREB Binding Protein and p300

    doi:

    Figure Lengend Snippet: Assessment of Tax and RelA colocalization. Images from horizontal sections (Z series) were collected from a specimen of BHK21 cells infected with SFV-Tax. The cells were analyzed by dual immunofluorescence staining with antibodies to Tax and RelA. Z1 to Z4 are four of these sections, which were collected with 1-μm intervals. P is the projection of the Z series. The left panel displays the LRSC fluorescence of Tax, the middle panel displays the FITC fluorescence of RelA, and the right panel is the overlay of the red and green fluorescence staining.

    Article Snippet: BHK21 cells (hamster kidney, ATCC CRL 8544) were grown in Glasgow minimum essential medium (Gibco BRL, Gaithersburg, Md.) supplemented with 10% tryptose phosphate, 20 mM HEPES buffer, and 5% fetal bovine serum (FBS); HOS cells (human osteogenic sarcoma, ATCC CRL 1543) were grown in Dulbecco modified Eagle medium (Gibco BRL) supplemented with 10% FBS; and MT2 cells (human T-cell leukemia cells isolated from cord blood lymphocytes cocultured with cells from a patient with T-cell leukemia, obtained from the National Institutes of Health AIDS Research and Reagent Program) were grown in RPMI medium (Gibco BRL) supplemented with 10% FBS, 100 U of penicillin G sodium salt per ml, 100 μg of streptomycin sulfate per ml, and 2 mM glutamine.

    Techniques: Infection, Immunofluorescence, Staining, Fluorescence

    ATF-1 colocalizes with Tax in nuclear foci. BHK21 cells were infected for 18 h with control SFV (A), SFV-Tax (B), SFV-Tax F1 (C), SFV-Tax M47 (D), or SFV-Tax M148 (E). The cells were then fixed and stained by dual immunofluorescence with a rabbit polyclonal serum to Tax (left panel) and a monoclonal antibody that specifically recognizes ATF-1 (middle panel). The panel on the right demonstrates the overlay of the FITC and Texas Red fluorescence.

    Journal: Molecular and Cellular Biology

    Article Title: Differential Transcriptional Activation by Human T-Cell Leukemia Virus Type 1 Tax Mutants Is Mediated by Distinct Interactions with CREB Binding Protein and p300

    doi:

    Figure Lengend Snippet: ATF-1 colocalizes with Tax in nuclear foci. BHK21 cells were infected for 18 h with control SFV (A), SFV-Tax (B), SFV-Tax F1 (C), SFV-Tax M47 (D), or SFV-Tax M148 (E). The cells were then fixed and stained by dual immunofluorescence with a rabbit polyclonal serum to Tax (left panel) and a monoclonal antibody that specifically recognizes ATF-1 (middle panel). The panel on the right demonstrates the overlay of the FITC and Texas Red fluorescence.

    Article Snippet: BHK21 cells (hamster kidney, ATCC CRL 8544) were grown in Glasgow minimum essential medium (Gibco BRL, Gaithersburg, Md.) supplemented with 10% tryptose phosphate, 20 mM HEPES buffer, and 5% fetal bovine serum (FBS); HOS cells (human osteogenic sarcoma, ATCC CRL 1543) were grown in Dulbecco modified Eagle medium (Gibco BRL) supplemented with 10% FBS; and MT2 cells (human T-cell leukemia cells isolated from cord blood lymphocytes cocultured with cells from a patient with T-cell leukemia, obtained from the National Institutes of Health AIDS Research and Reagent Program) were grown in RPMI medium (Gibco BRL) supplemented with 10% FBS, 100 U of penicillin G sodium salt per ml, 100 μg of streptomycin sulfate per ml, and 2 mM glutamine.

    Techniques: Infection, Staining, Immunofluorescence, Fluorescence

    In vivo association of Tax with CBP and p300. BHK21 cells were infected for 18 h with the control SFV (Mock) or with SFV expressing wild-type (WT) Tax or Tax mutants F1, M47, and M148. The cells were collected, and coimmunoprecipitation assays were performed with a monoclonal antibody to Tax. Western blot analysis was then performed with the cell lysates, using antibodies to Tax (A), CBP (B), and p300 (C), or on the immunoprecipitated complexes (IP), using antibodies to CBP (D) or p300 (E).

    Journal: Molecular and Cellular Biology

    Article Title: Differential Transcriptional Activation by Human T-Cell Leukemia Virus Type 1 Tax Mutants Is Mediated by Distinct Interactions with CREB Binding Protein and p300

    doi:

    Figure Lengend Snippet: In vivo association of Tax with CBP and p300. BHK21 cells were infected for 18 h with the control SFV (Mock) or with SFV expressing wild-type (WT) Tax or Tax mutants F1, M47, and M148. The cells were collected, and coimmunoprecipitation assays were performed with a monoclonal antibody to Tax. Western blot analysis was then performed with the cell lysates, using antibodies to Tax (A), CBP (B), and p300 (C), or on the immunoprecipitated complexes (IP), using antibodies to CBP (D) or p300 (E).

    Article Snippet: BHK21 cells (hamster kidney, ATCC CRL 8544) were grown in Glasgow minimum essential medium (Gibco BRL, Gaithersburg, Md.) supplemented with 10% tryptose phosphate, 20 mM HEPES buffer, and 5% fetal bovine serum (FBS); HOS cells (human osteogenic sarcoma, ATCC CRL 1543) were grown in Dulbecco modified Eagle medium (Gibco BRL) supplemented with 10% FBS; and MT2 cells (human T-cell leukemia cells isolated from cord blood lymphocytes cocultured with cells from a patient with T-cell leukemia, obtained from the National Institutes of Health AIDS Research and Reagent Program) were grown in RPMI medium (Gibco BRL) supplemented with 10% FBS, 100 U of penicillin G sodium salt per ml, 100 μg of streptomycin sulfate per ml, and 2 mM glutamine.

    Techniques: In Vivo, Infection, Expressing, Western Blot, Immunoprecipitation

    Differential activation of NF-κB and ATF/CREB pathways by Tax mutants. (A) A schematic of the 353-aa Tax protein. The positions of the amino acid changes for the three Tax mutants F1, M148, and M47 are shown. (B) Western blot analysis was performed on extracts prepared from BHK21 cells infected with SFV-Tax for 0 h (lane 1), 6 h (lane 2), 12 h (lane 3), 18 h (lane 4), or 24 h (lane 5). BHK21 cells were infected for 18 h with either control SFV (lane 6), SFV-Tax WT (lane 7), SFV-Tax F1 (lane 8), SFV-Tax M47 (lane 9), or SFV-Tax M148 (lane 10). Cellular extracts were subjected to electrophoresis on an SDS–15% polyacrylamide gel, and Western blot analysis was performed with a monoclonal antibody directed against Tax. Molecular weights (MW) in thousands are shown on the left. (C) BHK21 cells were transfected with either 1 μg of HTLV-1 CAT or HIV-1 CAT reporter plasmids; 5 h after transfection, the cells were infected with the control SFV (WT), SFV-Tax WT, SFV-Tax F1, SFV-Tax M47, or SFV-Tax M148, and they were harvested 24 h later. The CAT activity of the cell extracts was then determined, and the fold induction by either wild-type or mutant Tax was calculated for three independent experiments with the standard deviation indicated.

    Journal: Molecular and Cellular Biology

    Article Title: Differential Transcriptional Activation by Human T-Cell Leukemia Virus Type 1 Tax Mutants Is Mediated by Distinct Interactions with CREB Binding Protein and p300

    doi:

    Figure Lengend Snippet: Differential activation of NF-κB and ATF/CREB pathways by Tax mutants. (A) A schematic of the 353-aa Tax protein. The positions of the amino acid changes for the three Tax mutants F1, M148, and M47 are shown. (B) Western blot analysis was performed on extracts prepared from BHK21 cells infected with SFV-Tax for 0 h (lane 1), 6 h (lane 2), 12 h (lane 3), 18 h (lane 4), or 24 h (lane 5). BHK21 cells were infected for 18 h with either control SFV (lane 6), SFV-Tax WT (lane 7), SFV-Tax F1 (lane 8), SFV-Tax M47 (lane 9), or SFV-Tax M148 (lane 10). Cellular extracts were subjected to electrophoresis on an SDS–15% polyacrylamide gel, and Western blot analysis was performed with a monoclonal antibody directed against Tax. Molecular weights (MW) in thousands are shown on the left. (C) BHK21 cells were transfected with either 1 μg of HTLV-1 CAT or HIV-1 CAT reporter plasmids; 5 h after transfection, the cells were infected with the control SFV (WT), SFV-Tax WT, SFV-Tax F1, SFV-Tax M47, or SFV-Tax M148, and they were harvested 24 h later. The CAT activity of the cell extracts was then determined, and the fold induction by either wild-type or mutant Tax was calculated for three independent experiments with the standard deviation indicated.

    Article Snippet: BHK21 cells (hamster kidney, ATCC CRL 8544) were grown in Glasgow minimum essential medium (Gibco BRL, Gaithersburg, Md.) supplemented with 10% tryptose phosphate, 20 mM HEPES buffer, and 5% fetal bovine serum (FBS); HOS cells (human osteogenic sarcoma, ATCC CRL 1543) were grown in Dulbecco modified Eagle medium (Gibco BRL) supplemented with 10% FBS; and MT2 cells (human T-cell leukemia cells isolated from cord blood lymphocytes cocultured with cells from a patient with T-cell leukemia, obtained from the National Institutes of Health AIDS Research and Reagent Program) were grown in RPMI medium (Gibco BRL) supplemented with 10% FBS, 100 U of penicillin G sodium salt per ml, 100 μg of streptomycin sulfate per ml, and 2 mM glutamine.

    Techniques: Activation Assay, Western Blot, Infection, Electrophoresis, Transfection, Activity Assay, Mutagenesis, Standard Deviation

    Specificity of the antibodies to CBP and p300. Western blot analysis was performed on BHK21 cell lysates with the rabbit polyclonal antibody to CBP (A) or the monoclonal antibody to p300 (B).

    Journal: Molecular and Cellular Biology

    Article Title: Differential Transcriptional Activation by Human T-Cell Leukemia Virus Type 1 Tax Mutants Is Mediated by Distinct Interactions with CREB Binding Protein and p300

    doi:

    Figure Lengend Snippet: Specificity of the antibodies to CBP and p300. Western blot analysis was performed on BHK21 cell lysates with the rabbit polyclonal antibody to CBP (A) or the monoclonal antibody to p300 (B).

    Article Snippet: BHK21 cells (hamster kidney, ATCC CRL 8544) were grown in Glasgow minimum essential medium (Gibco BRL, Gaithersburg, Md.) supplemented with 10% tryptose phosphate, 20 mM HEPES buffer, and 5% fetal bovine serum (FBS); HOS cells (human osteogenic sarcoma, ATCC CRL 1543) were grown in Dulbecco modified Eagle medium (Gibco BRL) supplemented with 10% FBS; and MT2 cells (human T-cell leukemia cells isolated from cord blood lymphocytes cocultured with cells from a patient with T-cell leukemia, obtained from the National Institutes of Health AIDS Research and Reagent Program) were grown in RPMI medium (Gibco BRL) supplemented with 10% FBS, 100 U of penicillin G sodium salt per ml, 100 μg of streptomycin sulfate per ml, and 2 mM glutamine.

    Techniques: Western Blot

    CBP and p300 are distributed in distinct nuclear speckles. Uninfected BHK21 cells were fixed and analyzed by single immunofluorescence staining with either a rabbit polyclonal antibody that recognizes CBP (A) or a monoclonal antibody to p300 (C). The anti-CBP antibody was also used in combination with the monoclonal antibody to splicing factor Sm (B) or the monoclonal antibody to p300 (D). The anti-p300 antibody was used in combination with the monoclonal antibody to Sm (E). The DNA stain TO-PRO-3 was used to stain the nuclei (A and C). The panel on the right demonstrates the overlay of the blue and green staining or the red and green staining.

    Journal: Molecular and Cellular Biology

    Article Title: Differential Transcriptional Activation by Human T-Cell Leukemia Virus Type 1 Tax Mutants Is Mediated by Distinct Interactions with CREB Binding Protein and p300

    doi:

    Figure Lengend Snippet: CBP and p300 are distributed in distinct nuclear speckles. Uninfected BHK21 cells were fixed and analyzed by single immunofluorescence staining with either a rabbit polyclonal antibody that recognizes CBP (A) or a monoclonal antibody to p300 (C). The anti-CBP antibody was also used in combination with the monoclonal antibody to splicing factor Sm (B) or the monoclonal antibody to p300 (D). The anti-p300 antibody was used in combination with the monoclonal antibody to Sm (E). The DNA stain TO-PRO-3 was used to stain the nuclei (A and C). The panel on the right demonstrates the overlay of the blue and green staining or the red and green staining.

    Article Snippet: BHK21 cells (hamster kidney, ATCC CRL 8544) were grown in Glasgow minimum essential medium (Gibco BRL, Gaithersburg, Md.) supplemented with 10% tryptose phosphate, 20 mM HEPES buffer, and 5% fetal bovine serum (FBS); HOS cells (human osteogenic sarcoma, ATCC CRL 1543) were grown in Dulbecco modified Eagle medium (Gibco BRL) supplemented with 10% FBS; and MT2 cells (human T-cell leukemia cells isolated from cord blood lymphocytes cocultured with cells from a patient with T-cell leukemia, obtained from the National Institutes of Health AIDS Research and Reagent Program) were grown in RPMI medium (Gibco BRL) supplemented with 10% FBS, 100 U of penicillin G sodium salt per ml, 100 μg of streptomycin sulfate per ml, and 2 mM glutamine.

    Techniques: Immunofluorescence, Staining

    CBP and p300 colocalize with wild-type Tax in nuclear bodies. BHK21 cells were infected for 18 h with SFV-Tax (A and B), SFV-Tax M47 (C and D), or SFV-Tax M148 (E and F). The cells were fixed and stained by dual immunofluorescence with either a monoclonal antibody to Tax in combination with a rabbit polyclonal antibody that specifically recognizes CBP (A, C, and E) or a rabbit polyclonal antibody to Tax and a monoclonal antibody to p300 (B, D, and F). The panel on the right demonstrates the overlay of the red and green fluorescence staining.

    Journal: Molecular and Cellular Biology

    Article Title: Differential Transcriptional Activation by Human T-Cell Leukemia Virus Type 1 Tax Mutants Is Mediated by Distinct Interactions with CREB Binding Protein and p300

    doi:

    Figure Lengend Snippet: CBP and p300 colocalize with wild-type Tax in nuclear bodies. BHK21 cells were infected for 18 h with SFV-Tax (A and B), SFV-Tax M47 (C and D), or SFV-Tax M148 (E and F). The cells were fixed and stained by dual immunofluorescence with either a monoclonal antibody to Tax in combination with a rabbit polyclonal antibody that specifically recognizes CBP (A, C, and E) or a rabbit polyclonal antibody to Tax and a monoclonal antibody to p300 (B, D, and F). The panel on the right demonstrates the overlay of the red and green fluorescence staining.

    Article Snippet: BHK21 cells (hamster kidney, ATCC CRL 8544) were grown in Glasgow minimum essential medium (Gibco BRL, Gaithersburg, Md.) supplemented with 10% tryptose phosphate, 20 mM HEPES buffer, and 5% fetal bovine serum (FBS); HOS cells (human osteogenic sarcoma, ATCC CRL 1543) were grown in Dulbecco modified Eagle medium (Gibco BRL) supplemented with 10% FBS; and MT2 cells (human T-cell leukemia cells isolated from cord blood lymphocytes cocultured with cells from a patient with T-cell leukemia, obtained from the National Institutes of Health AIDS Research and Reagent Program) were grown in RPMI medium (Gibco BRL) supplemented with 10% FBS, 100 U of penicillin G sodium salt per ml, 100 μg of streptomycin sulfate per ml, and 2 mM glutamine.

    Techniques: Infection, Staining, Immunofluorescence, Fluorescence

    NF-κB RelA exhibits differential colocalization with the Tax mutants M47 and M148. BHK21 cells were infected for 18 h with control SFV (A), SFV-Tax (B), SFV-Tax F1 (C), SFV-Tax M47 (D), or SFV-Tax M148 (E). The cells were fixed and stained by dual immunofluorescence with a monoclonal antibody to Tax (left panel) and a rabbit polyclonal antibody to the RelA subunit of NF-κB (middle panel). The panel on the right demonstrates the overlay of the red and green fluorescence staining.

    Journal: Molecular and Cellular Biology

    Article Title: Differential Transcriptional Activation by Human T-Cell Leukemia Virus Type 1 Tax Mutants Is Mediated by Distinct Interactions with CREB Binding Protein and p300

    doi:

    Figure Lengend Snippet: NF-κB RelA exhibits differential colocalization with the Tax mutants M47 and M148. BHK21 cells were infected for 18 h with control SFV (A), SFV-Tax (B), SFV-Tax F1 (C), SFV-Tax M47 (D), or SFV-Tax M148 (E). The cells were fixed and stained by dual immunofluorescence with a monoclonal antibody to Tax (left panel) and a rabbit polyclonal antibody to the RelA subunit of NF-κB (middle panel). The panel on the right demonstrates the overlay of the red and green fluorescence staining.

    Article Snippet: BHK21 cells (hamster kidney, ATCC CRL 8544) were grown in Glasgow minimum essential medium (Gibco BRL, Gaithersburg, Md.) supplemented with 10% tryptose phosphate, 20 mM HEPES buffer, and 5% fetal bovine serum (FBS); HOS cells (human osteogenic sarcoma, ATCC CRL 1543) were grown in Dulbecco modified Eagle medium (Gibco BRL) supplemented with 10% FBS; and MT2 cells (human T-cell leukemia cells isolated from cord blood lymphocytes cocultured with cells from a patient with T-cell leukemia, obtained from the National Institutes of Health AIDS Research and Reagent Program) were grown in RPMI medium (Gibco BRL) supplemented with 10% FBS, 100 U of penicillin G sodium salt per ml, 100 μg of streptomycin sulfate per ml, and 2 mM glutamine.

    Techniques: Infection, Staining, Immunofluorescence, Fluorescence

    Effect of mutations on viral replication. (A) Virion-associated reverse transcriptase (RT) activity of wild type and mutant viral particles. BHK-21 cells were infected with 1 ml of released virus from the transfection experiment. Extracellular virions were harvested at 2, 4, and 6 d post-infection, concentrated by centrifugation, and RT activity was assessed by measuring incorporation of [ 3 H]TTP on a poly(A):(dT) 12–18 template-primer. Data are an average of three independent experiments. (B) Detection of Gag in the virion pellet. The same number of virion pellets was resolved on 12.5% sodium dodecyl sulfatepolyacrylamide gels, transferred to a membrane, and then probed with anti-Gag antibody.

    Journal: Molecules and Cells

    Article Title: Nuclear Localization Signals in Prototype Foamy Viral Integrase for Successive Infection and Replication in Dividing Cells

    doi: 10.14348/molcells.2014.2331

    Figure Lengend Snippet: Effect of mutations on viral replication. (A) Virion-associated reverse transcriptase (RT) activity of wild type and mutant viral particles. BHK-21 cells were infected with 1 ml of released virus from the transfection experiment. Extracellular virions were harvested at 2, 4, and 6 d post-infection, concentrated by centrifugation, and RT activity was assessed by measuring incorporation of [ 3 H]TTP on a poly(A):(dT) 12–18 template-primer. Data are an average of three independent experiments. (B) Detection of Gag in the virion pellet. The same number of virion pellets was resolved on 12.5% sodium dodecyl sulfatepolyacrylamide gels, transferred to a membrane, and then probed with anti-Gag antibody.

    Article Snippet: Immunohistochemistry A 104 aliquot of BHK-21 cells was grown on glass coverslips coated with 50 μg/ml poly(D) lysine (Sigma-Aldrich) in 35 mm tissue culture plates for immunostaining detection of viral protein localization in cells.

    Techniques: Activity Assay, Mutagenesis, Infection, Transfection, Centrifugation

    Sub-cellular localization of prototype foamy viral integrase (PFV-IN) and PFV-Gag. (A) Western blotting analysis for sub-cellular localization. BHK-21 cells were infected with 1 ml of culture supernatant containing wild type and mutant viruses and harvested after the indicated incubation times. Cells were then lysed and fractionated into cytosolic and nuclear portions as described in materials and methods. An equal amount of the fractions was resolved by 12.5% SDS-PAGE. Western blot analyses were performed with anti-IN, anti-Gag and anti-caspase-3 (cytosolic control) antibodies. (B) Immunostaining analysis for PFV-IN. (C) Immunostaining analysis for PFV-Gag. BHK-21 cells were grown on coverslips and infected with 1 ml of cell-free culture supernatant for 6 h. Then, the cells were incubated for the indicated length of time, fixed and permeabilized as indicated in the “Materials and Methods”. After blocking, coverslips were successively incubated with anti-PFV-IN (1:40) and anti-Gag (1:100) for 1 h. The coverslips were incubated for 30 min with a dilution of 1:80 (PFV-IN) and 1:150 (PFV-Gag) of FITC-conjugated donkey anti-rabbit IgG secondary antibody and mounted with a drop of UltraCruz mounting medium onto a microslide. Fluorescence was detected by a fluorescence microscope.

    Journal: Molecules and Cells

    Article Title: Nuclear Localization Signals in Prototype Foamy Viral Integrase for Successive Infection and Replication in Dividing Cells

    doi: 10.14348/molcells.2014.2331

    Figure Lengend Snippet: Sub-cellular localization of prototype foamy viral integrase (PFV-IN) and PFV-Gag. (A) Western blotting analysis for sub-cellular localization. BHK-21 cells were infected with 1 ml of culture supernatant containing wild type and mutant viruses and harvested after the indicated incubation times. Cells were then lysed and fractionated into cytosolic and nuclear portions as described in materials and methods. An equal amount of the fractions was resolved by 12.5% SDS-PAGE. Western blot analyses were performed with anti-IN, anti-Gag and anti-caspase-3 (cytosolic control) antibodies. (B) Immunostaining analysis for PFV-IN. (C) Immunostaining analysis for PFV-Gag. BHK-21 cells were grown on coverslips and infected with 1 ml of cell-free culture supernatant for 6 h. Then, the cells were incubated for the indicated length of time, fixed and permeabilized as indicated in the “Materials and Methods”. After blocking, coverslips were successively incubated with anti-PFV-IN (1:40) and anti-Gag (1:100) for 1 h. The coverslips were incubated for 30 min with a dilution of 1:80 (PFV-IN) and 1:150 (PFV-Gag) of FITC-conjugated donkey anti-rabbit IgG secondary antibody and mounted with a drop of UltraCruz mounting medium onto a microslide. Fluorescence was detected by a fluorescence microscope.

    Article Snippet: Immunohistochemistry A 104 aliquot of BHK-21 cells was grown on glass coverslips coated with 50 μg/ml poly(D) lysine (Sigma-Aldrich) in 35 mm tissue culture plates for immunostaining detection of viral protein localization in cells.

    Techniques: Western Blot, Infection, Mutagenesis, Incubation, SDS Page, Immunostaining, Blocking Assay, Fluorescence, Microscopy

    Analyses of in vitro activities, processing, and assembly of the viral protein into the virion. (A) In vitro enzymatic activities of purified prototype foamy viral integrase (PFV-IN). Plasmids harboring both wild type and point mutated PFV-IN were used to transform E. coli BL-21. The cells were then grown, induced and recombinant PFV-IN was purified. The catalytic activities of the purified IN proteins were assessed by endonucleolytic and disintegration assays with respective substrates, completed reactions were resolved on a denaturing gel, and band separation were observed by autoradiography. (B) Detection of intracellular and extracellular viral proteins by Western blot analyses. Whole cell lysates and viral pellets were prepared from BHK-21 cells 48 and 72 h after transfection with wild type and mutant provirus expression vectors. The same amount of protein were resolved on 12.5% sodium dodecyl sulfatepolyacrylamide gels, transferred to a membrane, and then probed with anti-IN and anti-Gag antibodies. Detection of cellular protein β-actin was used as the loading control.

    Journal: Molecules and Cells

    Article Title: Nuclear Localization Signals in Prototype Foamy Viral Integrase for Successive Infection and Replication in Dividing Cells

    doi: 10.14348/molcells.2014.2331

    Figure Lengend Snippet: Analyses of in vitro activities, processing, and assembly of the viral protein into the virion. (A) In vitro enzymatic activities of purified prototype foamy viral integrase (PFV-IN). Plasmids harboring both wild type and point mutated PFV-IN were used to transform E. coli BL-21. The cells were then grown, induced and recombinant PFV-IN was purified. The catalytic activities of the purified IN proteins were assessed by endonucleolytic and disintegration assays with respective substrates, completed reactions were resolved on a denaturing gel, and band separation were observed by autoradiography. (B) Detection of intracellular and extracellular viral proteins by Western blot analyses. Whole cell lysates and viral pellets were prepared from BHK-21 cells 48 and 72 h after transfection with wild type and mutant provirus expression vectors. The same amount of protein were resolved on 12.5% sodium dodecyl sulfatepolyacrylamide gels, transferred to a membrane, and then probed with anti-IN and anti-Gag antibodies. Detection of cellular protein β-actin was used as the loading control.

    Article Snippet: Immunohistochemistry A 104 aliquot of BHK-21 cells was grown on glass coverslips coated with 50 μg/ml poly(D) lysine (Sigma-Aldrich) in 35 mm tissue culture plates for immunostaining detection of viral protein localization in cells.

    Techniques: In Vitro, Purification, Recombinant, Autoradiography, Western Blot, Transfection, Mutagenesis, Expressing

    Interaction of BHK cells with PMMA–PTE nanoparticles at a concentration of 25 ppm. The images represent the nucleus stained with DAPI (A), cellular matrix stained with nanoparticles (B), and an overlay of the two images (C), with the magnified images in the inset showing nanoparticle distribution inside the cells (D). Magnified view (D) of nucleus stained with DAPI (i), cellular matrix stained with nanoparticles (ii), cells observed under DIC mode (iii), and overlay of (i)–(iv). The cells were imaged at a magnification of 60× oil objective using a confocal microscope (Olympus FV1000).

    Journal: ACS Omega

    Article Title: Controlled Dye Aggregation in Sodium Dodecylsulfate-Stabilized Poly(methylmethacrylate) Nanoparticles as Fluorescent Imaging Probes

    doi: 10.1021/acsomega.8b00785

    Figure Lengend Snippet: Interaction of BHK cells with PMMA–PTE nanoparticles at a concentration of 25 ppm. The images represent the nucleus stained with DAPI (A), cellular matrix stained with nanoparticles (B), and an overlay of the two images (C), with the magnified images in the inset showing nanoparticle distribution inside the cells (D). Magnified view (D) of nucleus stained with DAPI (i), cellular matrix stained with nanoparticles (ii), cells observed under DIC mode (iii), and overlay of (i)–(iv). The cells were imaged at a magnification of 60× oil objective using a confocal microscope (Olympus FV1000).

    Article Snippet: Internalization of Nanoparticles into Mammalian Cells for Imaging To establish a cellular model system, BHK cells (ATCC CCL-10) were used to understand the uptake and internalization of the dye-encapsulated polymer nanoparticles.

    Techniques: Concentration Assay, Staining, Microscopy

    Extope-CFTR is internalized and recycled. (A) Cell surface pools of Extope-CFTR are internalized. BHK-21 cells stably expressing Extope-CFTR were precooled, and cell surface pools of Extope CFTR were labeled for 30 min on ice with mouse monoclonal anti-HA antibody 16B12. Cells were then washed with ice-cold PBS and reincubated for the indicated times. Bars, 10 μm. (B) Internalized pools of CFTR are rapidly recycled. Cells were labeled for 10 min with monoclonal mouse anti-HA antibody 12CA5 and then washed with PBS, pH 3.7, to remove antibody from the cell surface. Reincubatiuon in prewarmed media for different times results in reappearance of CFTR at the cell edges. Cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% saponin in PBS. Extope-CFTR labeled with 12CA5 mAb was detected with goat anti-mouse IgG conjugated to Alexa Fluor 488. Nuclei were stained with propidium iodide. (C) Recycled CFTR is redirected to the cell surface. Internalized Extope-CFTR is detectable at the cell surface of nonpermeabilized cells after 5-min reincubation in media. Extope-CFTR–expressing cells were labeled with 12CA5 and washed to remove external label as in Figure 2B. Cells were fixed immediately or after 5-min reincubation in warm media and either not permeabilized (NP) or permeabilized (P). Extope-CFTR labeled with 12CA5 mAb was immunostained by goat anti-mouse IgG conjugated to Alexa Fluor 488. (D) Quantification of CFTR recycling to the plasma membrane. The internalized pool of CFTR is almost completely redirected to the cell surface in 5 min. Experiments were performed as in Figure 2C, and mean green fluorescence per cell was determined as described in MATERIALS AND METHODS by using ImageJ 1.30v software. Each point represents the average of at least 10 cells and standard deviations are indicated.

    Journal: Molecular Biology of the Cell

    Article Title: Endocytic Trafficking Routes of Wild Type and ?F508 Cystic Fibrosis Transmembrane Conductance Regulator D⃞

    doi: 10.1091/mbc.E04-03-0176

    Figure Lengend Snippet: Extope-CFTR is internalized and recycled. (A) Cell surface pools of Extope-CFTR are internalized. BHK-21 cells stably expressing Extope-CFTR were precooled, and cell surface pools of Extope CFTR were labeled for 30 min on ice with mouse monoclonal anti-HA antibody 16B12. Cells were then washed with ice-cold PBS and reincubated for the indicated times. Bars, 10 μm. (B) Internalized pools of CFTR are rapidly recycled. Cells were labeled for 10 min with monoclonal mouse anti-HA antibody 12CA5 and then washed with PBS, pH 3.7, to remove antibody from the cell surface. Reincubatiuon in prewarmed media for different times results in reappearance of CFTR at the cell edges. Cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% saponin in PBS. Extope-CFTR labeled with 12CA5 mAb was detected with goat anti-mouse IgG conjugated to Alexa Fluor 488. Nuclei were stained with propidium iodide. (C) Recycled CFTR is redirected to the cell surface. Internalized Extope-CFTR is detectable at the cell surface of nonpermeabilized cells after 5-min reincubation in media. Extope-CFTR–expressing cells were labeled with 12CA5 and washed to remove external label as in Figure 2B. Cells were fixed immediately or after 5-min reincubation in warm media and either not permeabilized (NP) or permeabilized (P). Extope-CFTR labeled with 12CA5 mAb was immunostained by goat anti-mouse IgG conjugated to Alexa Fluor 488. (D) Quantification of CFTR recycling to the plasma membrane. The internalized pool of CFTR is almost completely redirected to the cell surface in 5 min. Experiments were performed as in Figure 2C, and mean green fluorescence per cell was determined as described in MATERIALS AND METHODS by using ImageJ 1.30v software. Each point represents the average of at least 10 cells and standard deviations are indicated.

    Article Snippet: Baby hamster kidney (BHK-21) cells were obtained from the American Type Culture Collection (Manassas, VA) and grown at 37°C in 5% CO2 .

    Techniques: Stable Transfection, Expressing, Labeling, Staining, Fluorescence, Software

    External-epitope tagged CFTR (Extope-CFTR) matures and functions like unmodified wild type CFTR. (A) Schematic of strategy used to generate Extope-CFTR. Portions of EL1 and EL4 sequences incorporated into EL2 flanking a single HA-epitope (blue) are shown in green and red, respectively. (B) Western blot of external-epitope tagged CFTR (Extope-CFTR). Extope-CFTR matures like the unmodified wild type protein. Total cell lysates (25 μg) were separated by 6% SDS-PAGE, transferred to nitrocellulose, and Extope-CFTR was detected with monoclonal mouse anti-HA antibody 16B12 or monoclonal mouse anti-CFTR antibody 596. (C) cAMP stimulated 36 Cl - efflux. Extope-CFTR functions as a chloride channel like the unmodified wild type protein. BHK-21 cells stably expressing wild type CFTR (blue diamonds) or Extope-CFTR (red squares) were loaded with Na 36 Cl, transferred to chloride-free buffer, and cAMP-dependent chloride efflux was stimulated with stimulation cocktail as described in MATERIALS AND METHODS. An arrow indicates the stimulation at time 0. Extope-CFTR–expressing cells, which were not stimulated, are shown as control (black circles). Each point represents the average of three independent samples and standard deviations are indicated. (D) Immunofluorescence detection of total and cell surface derived CFTR. Extope-CFTR was labeled on the cell surface with monoclonal mouse anti-HA antibody and cells were reincubated for 2 or 48 h. Cells were fixed, permeabilized, and internalized pools of Extope-CFTR were detected with Alexa Fluor 568 conjugated to goat anti-mouse Fab fragments (left). Total pools of Extope-CFTR were subsequently labeled with anti-CFTR antibody 570 directly conjugated to Alexa Fluor 488 (right).

    Journal: Molecular Biology of the Cell

    Article Title: Endocytic Trafficking Routes of Wild Type and ?F508 Cystic Fibrosis Transmembrane Conductance Regulator D⃞

    doi: 10.1091/mbc.E04-03-0176

    Figure Lengend Snippet: External-epitope tagged CFTR (Extope-CFTR) matures and functions like unmodified wild type CFTR. (A) Schematic of strategy used to generate Extope-CFTR. Portions of EL1 and EL4 sequences incorporated into EL2 flanking a single HA-epitope (blue) are shown in green and red, respectively. (B) Western blot of external-epitope tagged CFTR (Extope-CFTR). Extope-CFTR matures like the unmodified wild type protein. Total cell lysates (25 μg) were separated by 6% SDS-PAGE, transferred to nitrocellulose, and Extope-CFTR was detected with monoclonal mouse anti-HA antibody 16B12 or monoclonal mouse anti-CFTR antibody 596. (C) cAMP stimulated 36 Cl - efflux. Extope-CFTR functions as a chloride channel like the unmodified wild type protein. BHK-21 cells stably expressing wild type CFTR (blue diamonds) or Extope-CFTR (red squares) were loaded with Na 36 Cl, transferred to chloride-free buffer, and cAMP-dependent chloride efflux was stimulated with stimulation cocktail as described in MATERIALS AND METHODS. An arrow indicates the stimulation at time 0. Extope-CFTR–expressing cells, which were not stimulated, are shown as control (black circles). Each point represents the average of three independent samples and standard deviations are indicated. (D) Immunofluorescence detection of total and cell surface derived CFTR. Extope-CFTR was labeled on the cell surface with monoclonal mouse anti-HA antibody and cells were reincubated for 2 or 48 h. Cells were fixed, permeabilized, and internalized pools of Extope-CFTR were detected with Alexa Fluor 568 conjugated to goat anti-mouse Fab fragments (left). Total pools of Extope-CFTR were subsequently labeled with anti-CFTR antibody 570 directly conjugated to Alexa Fluor 488 (right).

    Article Snippet: Baby hamster kidney (BHK-21) cells were obtained from the American Type Culture Collection (Manassas, VA) and grown at 37°C in 5% CO2 .

    Techniques: Western Blot, SDS Page, Stable Transfection, Expressing, Immunofluorescence, Derivative Assay, Labeling

    Transient expression of HCoV-OC43 wild-type HE protein is more efficient than stably expressing BHK-21 clones to complement pBAC-HE mutants. (A) Expression of the wild-type HE protein after transfection of BHK-21 cells with either pcDNA-HE or pcDNA-HEv5, compared to pcDNA vector alone. (B) Detection of O -acetyl-esterase activity in BHK-21 cells transfected with either pcDNA-HE or pcDNA-HEv5 compared to pcDNA alone. (C) Immunofluorescence analysis (detection of the S protein) of BHK-21 cells cotransfected by either pcDNA vector alone (top panels) or by pcDNA-HE (bottom panels), together with all of the pBAC-OC43 molecular clones. (D) Production of infectious virus after transfection of BHK-21 cells by the pBAC-OC43 FL and the different pBAC-OC43-HE mutant infectious clones cotransfected with either pcDNA vector alone or pcDNA-HE. LOD, limit of detection.

    Journal: Journal of Virology

    Article Title: The Acetyl-Esterase Activity of the Hemagglutinin-Esterase Protein of Human Coronavirus OC43 Strongly Enhances the Production of Infectious Virus

    doi: 10.1128/JVI.02699-12

    Figure Lengend Snippet: Transient expression of HCoV-OC43 wild-type HE protein is more efficient than stably expressing BHK-21 clones to complement pBAC-HE mutants. (A) Expression of the wild-type HE protein after transfection of BHK-21 cells with either pcDNA-HE or pcDNA-HEv5, compared to pcDNA vector alone. (B) Detection of O -acetyl-esterase activity in BHK-21 cells transfected with either pcDNA-HE or pcDNA-HEv5 compared to pcDNA alone. (C) Immunofluorescence analysis (detection of the S protein) of BHK-21 cells cotransfected by either pcDNA vector alone (top panels) or by pcDNA-HE (bottom panels), together with all of the pBAC-OC43 molecular clones. (D) Production of infectious virus after transfection of BHK-21 cells by the pBAC-OC43 FL and the different pBAC-OC43-HE mutant infectious clones cotransfected with either pcDNA vector alone or pcDNA-HE. LOD, limit of detection.

    Article Snippet: The BHK-21 cell line (ATCC-CCL10) was also cultured in MEM-α supplemented with 10% (vol/vol) FBS and used for transfection.

    Techniques: Expressing, Stable Transfection, Clone Assay, Transfection, Plasmid Preparation, Activity Assay, Immunofluorescence, Mutagenesis

    BHK-21 cells ectopically expressing the HCoV-OC43 wild-type HE protein complement recombinant pBAC HE mutants. (A) BHK-21 subpopulations and clones analyzed by RT-PCR revealed ectopic expression of the HE. Samples were run on a 1% (wt/vol) agarose gel, and amplicons were revealed with ethidium bromide. Lane M, molecular weight markers; lanes 1 to 5, subpopulation pcDNA, HEv5-1-3, HE6E, HE6F, and HEv5-2-13, respectively; lanes 6 to 12, individual clones HE6E-G6, HEv5-H1, HEv5-C1, HEv5-E3, HE6F-A3, HE6F-E3, and HEv5-G7, respectively. (B) Quantitative real-time PCR revealed different levels of HE expression, with the two clones (HE6F-A3 and HE6F-E3) expressing the highest levels of the HE mRNA. (C) Flow cytometry analysis revealed that these two clones expressed the HE protein compared to BHK-21 cells transfected by the control vector pcDNA alone. (D) Transfection of the BHK-21 HE6F-A3 and HE6F-E3 clones by the pBAC-OC43-HE mutants produced detectable amounts of infectious HCoV-OC43 particles compared to BHK-21 cells transfected by the control vector pcDNA, from which no infectious virus was detected. LOD, limit of detection.

    Journal: Journal of Virology

    Article Title: The Acetyl-Esterase Activity of the Hemagglutinin-Esterase Protein of Human Coronavirus OC43 Strongly Enhances the Production of Infectious Virus

    doi: 10.1128/JVI.02699-12

    Figure Lengend Snippet: BHK-21 cells ectopically expressing the HCoV-OC43 wild-type HE protein complement recombinant pBAC HE mutants. (A) BHK-21 subpopulations and clones analyzed by RT-PCR revealed ectopic expression of the HE. Samples were run on a 1% (wt/vol) agarose gel, and amplicons were revealed with ethidium bromide. Lane M, molecular weight markers; lanes 1 to 5, subpopulation pcDNA, HEv5-1-3, HE6E, HE6F, and HEv5-2-13, respectively; lanes 6 to 12, individual clones HE6E-G6, HEv5-H1, HEv5-C1, HEv5-E3, HE6F-A3, HE6F-E3, and HEv5-G7, respectively. (B) Quantitative real-time PCR revealed different levels of HE expression, with the two clones (HE6F-A3 and HE6F-E3) expressing the highest levels of the HE mRNA. (C) Flow cytometry analysis revealed that these two clones expressed the HE protein compared to BHK-21 cells transfected by the control vector pcDNA alone. (D) Transfection of the BHK-21 HE6F-A3 and HE6F-E3 clones by the pBAC-OC43-HE mutants produced detectable amounts of infectious HCoV-OC43 particles compared to BHK-21 cells transfected by the control vector pcDNA, from which no infectious virus was detected. LOD, limit of detection.

    Article Snippet: The BHK-21 cell line (ATCC-CCL10) was also cultured in MEM-α supplemented with 10% (vol/vol) FBS and used for transfection.

    Techniques: Expressing, Recombinant, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Molecular Weight, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Transfection, Plasmid Preparation, Produced

    The acetyl-esterase enzymatic activity of the HE protein of the HCoV-OC43 plays an important role in production of detectable infectious viral particles. (A) Titration of recombinant infectious HCoV-OC43 particles after transfection of BHK-21 cells at passage 0 (P0), harvested directly from transfected BHK-21 cells, and amplified between P1 and P2 on HRT-18 cells. nd, not detected. (B) IFA confirmed that the S protein was produced in transfected BHK-21 cells at equivalent levels for all of the pBAC-OC43 molecular clones and that the HE protein was produced only in BHK-21 cells transfected with pBAC-OC43 FL or pBAC-HE S40T . (C) Acetyl-esterase activity was only detected in BHK-21 cells transfected with the pBAC-OC43 FL , compared to all pBAC-OC43 HE mutants and to mock-transfected BHK-21 cells (Lipofectamine LTX).

    Journal: Journal of Virology

    Article Title: The Acetyl-Esterase Activity of the Hemagglutinin-Esterase Protein of Human Coronavirus OC43 Strongly Enhances the Production of Infectious Virus

    doi: 10.1128/JVI.02699-12

    Figure Lengend Snippet: The acetyl-esterase enzymatic activity of the HE protein of the HCoV-OC43 plays an important role in production of detectable infectious viral particles. (A) Titration of recombinant infectious HCoV-OC43 particles after transfection of BHK-21 cells at passage 0 (P0), harvested directly from transfected BHK-21 cells, and amplified between P1 and P2 on HRT-18 cells. nd, not detected. (B) IFA confirmed that the S protein was produced in transfected BHK-21 cells at equivalent levels for all of the pBAC-OC43 molecular clones and that the HE protein was produced only in BHK-21 cells transfected with pBAC-OC43 FL or pBAC-HE S40T . (C) Acetyl-esterase activity was only detected in BHK-21 cells transfected with the pBAC-OC43 FL , compared to all pBAC-OC43 HE mutants and to mock-transfected BHK-21 cells (Lipofectamine LTX).

    Article Snippet: The BHK-21 cell line (ATCC-CCL10) was also cultured in MEM-α supplemented with 10% (vol/vol) FBS and used for transfection.

    Techniques: Activity Assay, Titration, Recombinant, Transfection, Amplification, Immunofluorescence, Produced, Clone Assay

    Generation and identification of VSV-846 vaccine. (A) The schematic of pVSV-XN2-846 plasmid. The triple-antigen fusion TFP846 (Rv3615c-Mtb10.4-Rv2660c) was amplified from p846 plasmid, and the triple-antigen fusion gene was then cloned into the fifth position of the pVSV-XN2 plasmid after cleavage with XhoI and NheI. (B) The microscope images of BHK-21 cells infected with VSV-846 or non-infected mock control 6 h post-infection (200×). (C) The triple-antigen fusion TFP846 was expressed in VSV-846 infected cells. The blot was probed with anti-flag antibody. Control: Normal cells without infection.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Vesicular Stomatitis Virus-Vectored Multi-Antigen Tuberculosis Vaccine Limits Bacterial Proliferation in Mice following a Single Intranasal Dose

    doi: 10.3389/fcimb.2017.00034

    Figure Lengend Snippet: Generation and identification of VSV-846 vaccine. (A) The schematic of pVSV-XN2-846 plasmid. The triple-antigen fusion TFP846 (Rv3615c-Mtb10.4-Rv2660c) was amplified from p846 plasmid, and the triple-antigen fusion gene was then cloned into the fifth position of the pVSV-XN2 plasmid after cleavage with XhoI and NheI. (B) The microscope images of BHK-21 cells infected with VSV-846 or non-infected mock control 6 h post-infection (200×). (C) The triple-antigen fusion TFP846 was expressed in VSV-846 infected cells. The blot was probed with anti-flag antibody. Control: Normal cells without infection.

    Article Snippet: The recombinant VSV-846 virus was generated by the following procedure: In brief, baby hamster kidney cells BHK-21 (ATCC number:CCL-10) grown to 60% confluence were infected with recombinant vaccinia virus expressing T7 RNA polymerase (Fuerst et al., ) and incubated for 1 h in serum-free Dulbecco's modified Eagle's medium (DMEM).

    Techniques: Plasmid Preparation, Amplification, Clone Assay, Microscopy, Infection

    Stable transgene expression and attenuation of artLCMV in vivo. ( a – d ) Viremia of AGRAG mice infected with r3LCMV-GFP ( n =7), artLCMV-GFP ( n =7) or LCMVwt ( n =3, a , d ). Symbols show the mean±s.e.m. N =2. Blood samples obtained on d5 and d120 were processed for NP-IFF and GFP-IFF ( b ). Bars represent the mean±s.e.m. of seven mice. N =2. Representative NP-IFF and GFP-IFF analyses of d150 serum ( c , scale bar, 0.5 cm). Representative liver sections analysed for GFP + cells on d150 ( d , scale bar, 50 μm). ( e ) Growth kinetics of LCMVwt, r3LCMV-GFP and r2LCMV-S rec #1 on BHK-21 cells (compare Supplementary Fig. 2e ). Symbols represent the mean of three replicates (s.e.m. error bars hidden in the symbol size). N =2. Data in a (120 days after infection) and e (48 h after infection) were analysed by one-way ANOVA with Bonferroni post hoc test. Data in b were analysed by unpaired two-tailed Student's t -test. NS, not significant; ** P

    Journal: Nature Communications

    Article Title: Replicating viral vector platform exploits alarmin signals for potent CD8+ T cell-mediated tumour immunotherapy

    doi: 10.1038/ncomms15327

    Figure Lengend Snippet: Stable transgene expression and attenuation of artLCMV in vivo. ( a – d ) Viremia of AGRAG mice infected with r3LCMV-GFP ( n =7), artLCMV-GFP ( n =7) or LCMVwt ( n =3, a , d ). Symbols show the mean±s.e.m. N =2. Blood samples obtained on d5 and d120 were processed for NP-IFF and GFP-IFF ( b ). Bars represent the mean±s.e.m. of seven mice. N =2. Representative NP-IFF and GFP-IFF analyses of d150 serum ( c , scale bar, 0.5 cm). Representative liver sections analysed for GFP + cells on d150 ( d , scale bar, 50 μm). ( e ) Growth kinetics of LCMVwt, r3LCMV-GFP and r2LCMV-S rec #1 on BHK-21 cells (compare Supplementary Fig. 2e ). Symbols represent the mean of three replicates (s.e.m. error bars hidden in the symbol size). N =2. Data in a (120 days after infection) and e (48 h after infection) were analysed by one-way ANOVA with Bonferroni post hoc test. Data in b were analysed by unpaired two-tailed Student's t -test. NS, not significant; ** P

    Article Snippet: Cells BHK-21 cells were obtained from ECACC (Clone 13, Cat #85011433), MC57 cells (CRL-2295), EL-4 cells (TIB-39), EG7 thymoma cells (EL-4 cells expressing OVA, CRL-2113) and P815 mastocytoma cells (TIB-64) were obtained from ATCC.

    Techniques: Expressing, In Vivo, Mouse Assay, Infection, Two Tailed Test

    Genetic design and growth of artLCMV in cell culture. ( a ) Genome organization of LCMVwt, r3LCMV-GFP and artLCMV-GFP. ( b ) Non-homologous recombination unites GPC and NP ORFs of r3LCMV-GFP yielding S rec with only GFP remnants. 3′UTR and 5′UTRs of S rec pair to form a functional promoter ( Supplementary Fig. 2 ). ( c ) Hypothetical recombination event uniting GPC and NP of artLCMV-GFP at the expense of losing the 5′UTR. Duplicated 3′UTRs on such hypothetical S rec cannot form a functional promoter. ( d ) Growth kinetics of LCMVwt, artLCMV-GFP and r3LCMV-GFP on BHK-21 cells. Symbols represent the mean of three replicates (s.e.m. hidden in symbol size). PFU: plaque forming units. N =3 ( N : number of independent data sets). ( e ) artLCMV-GFP/tom and r3LCMV-GFP/tom (containing S GP -GFP and S NP -tom) contained tri-segmented and bi-segmented particles, which were discriminated and quantified by FACS analysis 12 h after infection of BHK-NP cells. Representative FACS plot for artLCMV-GFP/tom (analogous results for r3LCMV-GFP/tom, see quantification in bar graph). Bars represent the mean+s.e.m. of three replicates per group. N =3. Data in d (48 h after infection) were analysed by one-way analysis of variance (ANOVA) with Bonferroni post hoc test. *** P

    Journal: Nature Communications

    Article Title: Replicating viral vector platform exploits alarmin signals for potent CD8+ T cell-mediated tumour immunotherapy

    doi: 10.1038/ncomms15327

    Figure Lengend Snippet: Genetic design and growth of artLCMV in cell culture. ( a ) Genome organization of LCMVwt, r3LCMV-GFP and artLCMV-GFP. ( b ) Non-homologous recombination unites GPC and NP ORFs of r3LCMV-GFP yielding S rec with only GFP remnants. 3′UTR and 5′UTRs of S rec pair to form a functional promoter ( Supplementary Fig. 2 ). ( c ) Hypothetical recombination event uniting GPC and NP of artLCMV-GFP at the expense of losing the 5′UTR. Duplicated 3′UTRs on such hypothetical S rec cannot form a functional promoter. ( d ) Growth kinetics of LCMVwt, artLCMV-GFP and r3LCMV-GFP on BHK-21 cells. Symbols represent the mean of three replicates (s.e.m. hidden in symbol size). PFU: plaque forming units. N =3 ( N : number of independent data sets). ( e ) artLCMV-GFP/tom and r3LCMV-GFP/tom (containing S GP -GFP and S NP -tom) contained tri-segmented and bi-segmented particles, which were discriminated and quantified by FACS analysis 12 h after infection of BHK-NP cells. Representative FACS plot for artLCMV-GFP/tom (analogous results for r3LCMV-GFP/tom, see quantification in bar graph). Bars represent the mean+s.e.m. of three replicates per group. N =3. Data in d (48 h after infection) were analysed by one-way analysis of variance (ANOVA) with Bonferroni post hoc test. *** P

    Article Snippet: Cells BHK-21 cells were obtained from ECACC (Clone 13, Cat #85011433), MC57 cells (CRL-2295), EL-4 cells (TIB-39), EG7 thymoma cells (EL-4 cells expressing OVA, CRL-2113) and P815 mastocytoma cells (TIB-64) were obtained from ATCC.

    Techniques: Cell Culture, Homologous Recombination, Gel Permeation Chromatography, Functional Assay, FACS, Infection

    Comparison of template constructs in mammalian cells. (A) U2OS, Vero E6, and BHK-21 cells were all cotransfected with CMV-P1234 and one of CMV-Fluc-Gluc (CMV), CMV-HH-Fluc-Gluc (CMV HH), HSPolI-Fluc-Gluc (HSPolI), HSPolI-HH-Fluc-Gluc (HSPolI HH), or CGPolI-Fluc-Gluc (CGPolI). Control cells were all cotransfected with CMV-P1234 GAA and the same template-expressing plasmids. Cells were lysed at 18 h posttransfection. The Fluc (replication, left) and Gluc (transcription, right) activities generated by the active replicase were normalized to those for the controls. Each column represents an average from three independent experiments; error bars represent standard deviations. *, P

    Journal: Journal of Virology

    Article Title: Design and Use of Chikungunya Virus Replication Templates Utilizing Mammalian and Mosquito RNA Polymerase I-Mediated Transcription

    doi: 10.1128/JVI.00794-19

    Figure Lengend Snippet: Comparison of template constructs in mammalian cells. (A) U2OS, Vero E6, and BHK-21 cells were all cotransfected with CMV-P1234 and one of CMV-Fluc-Gluc (CMV), CMV-HH-Fluc-Gluc (CMV HH), HSPolI-Fluc-Gluc (HSPolI), HSPolI-HH-Fluc-Gluc (HSPolI HH), or CGPolI-Fluc-Gluc (CGPolI). Control cells were all cotransfected with CMV-P1234 GAA and the same template-expressing plasmids. Cells were lysed at 18 h posttransfection. The Fluc (replication, left) and Gluc (transcription, right) activities generated by the active replicase were normalized to those for the controls. Each column represents an average from three independent experiments; error bars represent standard deviations. *, P

    Article Snippet: BHK-21 baby hamster kidney cells (ATCC CCL-10) were grown in Glasgow’s minimal essential medium (Gibco) containing 10% FBS, 2% tryptose phosphate broth (TPB), and 200 mM HEPES, pH 7.2.

    Techniques: Construct, Expressing, Generated

    Analysis of replicon RNA-containing cells. (A) Combined green fluorescence and phase-contrast microscopic images of SCR-1 cells are shown. (B) Presence of SARS–CoV replicon and sub-replicon RNAs in replicon-carrying cells as detected by Northern blot analysis. Total RNA preparations from BHK-21 (BHK) and SCR-1 cells were analyzed as indicated. Arrows indicate full-length replicon RNA and replicon RNA-derived transcripts encoding GFP and N. (C) A flow cytometry analysis of SCR-1 cells is shown. Samples of SCR-1 cells were analyzed at passage number P10, P20, P30 and P40 under blasticidin selection and the parent BHK-21 (BHK) cells were co-analyzed. Indicated values represent the percentages of green fluorescent cells. The results are presented as histograms with green fluorescence intensity in exponential scale (horizontal axis) against cell number in linear scale (vertical axis).

    Journal: Virology

    Article Title: Derivation of a novel SARS–coronavirus replicon cell line and its application for anti-SARS drug screening

    doi: 10.1016/j.virol.2006.10.016

    Figure Lengend Snippet: Analysis of replicon RNA-containing cells. (A) Combined green fluorescence and phase-contrast microscopic images of SCR-1 cells are shown. (B) Presence of SARS–CoV replicon and sub-replicon RNAs in replicon-carrying cells as detected by Northern blot analysis. Total RNA preparations from BHK-21 (BHK) and SCR-1 cells were analyzed as indicated. Arrows indicate full-length replicon RNA and replicon RNA-derived transcripts encoding GFP and N. (C) A flow cytometry analysis of SCR-1 cells is shown. Samples of SCR-1 cells were analyzed at passage number P10, P20, P30 and P40 under blasticidin selection and the parent BHK-21 (BHK) cells were co-analyzed. Indicated values represent the percentages of green fluorescent cells. The results are presented as histograms with green fluorescence intensity in exponential scale (horizontal axis) against cell number in linear scale (vertical axis).

    Article Snippet: Cells and viruses The baby hamster kidney (BHK)-21 cell line was purchased from American Typical Culture Center (ATCC).

    Techniques: Fluorescence, Northern Blot, Derivative Assay, Flow Cytometry, Selection

    Inhibition of SARS–CoV replication. SCR-1 cells containing SARS–CoV-based replicon RNA that mediates GFP expression were used to assess the inhibitory effect of the compounds by (A) flow cytometry analysis. One representative experiment out of four is shown. Bars indicate GFP-expressing cells in flow cytometry analyses. The GFP expression of untreated cells was set at 100%. (B) Fluorescence microscopy analysis. Quadruple wells of untreated and treated SCR-1 cells are shown. The parent BHK-21 (BHK) cells were co-analyzed. (C) Quantitative real-time RT–PCR. Copy number of SARS replicon RNA in SCR-1 cells before and after inhibitor treatments are shown. The inhibitors used are indicated below. Data are the means of three independent experiments. Bar: 95% confidence intervals.

    Journal: Virology

    Article Title: Derivation of a novel SARS–coronavirus replicon cell line and its application for anti-SARS drug screening

    doi: 10.1016/j.virol.2006.10.016

    Figure Lengend Snippet: Inhibition of SARS–CoV replication. SCR-1 cells containing SARS–CoV-based replicon RNA that mediates GFP expression were used to assess the inhibitory effect of the compounds by (A) flow cytometry analysis. One representative experiment out of four is shown. Bars indicate GFP-expressing cells in flow cytometry analyses. The GFP expression of untreated cells was set at 100%. (B) Fluorescence microscopy analysis. Quadruple wells of untreated and treated SCR-1 cells are shown. The parent BHK-21 (BHK) cells were co-analyzed. (C) Quantitative real-time RT–PCR. Copy number of SARS replicon RNA in SCR-1 cells before and after inhibitor treatments are shown. The inhibitors used are indicated below. Data are the means of three independent experiments. Bar: 95% confidence intervals.

    Article Snippet: Cells and viruses The baby hamster kidney (BHK)-21 cell line was purchased from American Typical Culture Center (ATCC).

    Techniques: Inhibition, Expressing, Flow Cytometry, Fluorescence, Microscopy, Quantitative RT-PCR

    Dye transfer directed by the wt or mutated F and HPIV3 HN proteins. R18-labeled RBCs were bound at 4°C to BHK-21 cells coexpressing the wt or mutanted F and HN proteins. Cells were incubated for 60 min at 37°C to allow membrane fusion, images were acquired by using fluorescent microscopy. (A) Representative images of dye transfer. White arrows indicate the lipid mixing events. (B) Quantification of the events of dye transfer. The extent of dye tranfer is expressed as the average number of R18 lipid dye tranfer events of six microscopic fields. The means and standard errors are from six microscopic fields (** P

    Journal: PLoS ONE

    Article Title: Mutations in the DI-DII Linker of Human Parainfluenza Virus Type 3 Fusion Protein Result in Diminished Fusion Activity

    doi: 10.1371/journal.pone.0136474

    Figure Lengend Snippet: Dye transfer directed by the wt or mutated F and HPIV3 HN proteins. R18-labeled RBCs were bound at 4°C to BHK-21 cells coexpressing the wt or mutanted F and HN proteins. Cells were incubated for 60 min at 37°C to allow membrane fusion, images were acquired by using fluorescent microscopy. (A) Representative images of dye transfer. White arrows indicate the lipid mixing events. (B) Quantification of the events of dye transfer. The extent of dye tranfer is expressed as the average number of R18 lipid dye tranfer events of six microscopic fields. The means and standard errors are from six microscopic fields (** P

    Article Snippet: Cells and viruses BHK-21 cells, obtained from the American Type Culture Collection, were propagated in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 1% glutamine, 10% fetal calf serum (FCS) (Gibco), and 1% penicillin-streptomycin (Invitrogen).

    Techniques: Labeling, Incubation, Microscopy

    Syncytium formation in monolayers coexpressing wt or mutated F and HN proteins. After 36 h posttransfection, BHK-21 monolayers transfected with vector alone, wt F alone, wt F and wt HN, or mutant F and wt HN were fixed with methanol and stained with Giemsa stain. (A) Photomicrographs from a representative expreriment. Red arrows indicate syncytia. (B) Quantification of syncytia produced by the mutated F proteins. Values were indicated as percentages of syncytium formation detected in cells transfected with wt F and wt HN proteins and are represented as the mean ± standard deviation (SD) from three separate experiments (* P

    Journal: PLoS ONE

    Article Title: Mutations in the DI-DII Linker of Human Parainfluenza Virus Type 3 Fusion Protein Result in Diminished Fusion Activity

    doi: 10.1371/journal.pone.0136474

    Figure Lengend Snippet: Syncytium formation in monolayers coexpressing wt or mutated F and HN proteins. After 36 h posttransfection, BHK-21 monolayers transfected with vector alone, wt F alone, wt F and wt HN, or mutant F and wt HN were fixed with methanol and stained with Giemsa stain. (A) Photomicrographs from a representative expreriment. Red arrows indicate syncytia. (B) Quantification of syncytia produced by the mutated F proteins. Values were indicated as percentages of syncytium formation detected in cells transfected with wt F and wt HN proteins and are represented as the mean ± standard deviation (SD) from three separate experiments (* P

    Article Snippet: Cells and viruses BHK-21 cells, obtained from the American Type Culture Collection, were propagated in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 1% glutamine, 10% fetal calf serum (FCS) (Gibco), and 1% penicillin-streptomycin (Invitrogen).

    Techniques: Transfection, Plasmid Preparation, Mutagenesis, Staining, Giemsa Stain, Produced, Standard Deviation

    Inhibition of DENV-2 RNA replication by BP2109. (A) Schematic diagram of the DENV-2 reporter replicon, DV2Rep. Indicated are the 5′ UTR, the N-terminal 102 amino acids of the C protein (C102), the Renilla luciferase gene (Rluc), the FMDV2A cleavage site, the neomycin resistance gene (Neo), an EMCV IRES element, the C-terminal 24 amino acids of E (E24), the entire NS regions (NS1∼NS5), and the 3′ UTR. The replicon was used to quantify the inhibitory effects of BP2109 on the RNA replication of DENV-2. (B) Analysis of BP2109 using transient replicon assays. BP2109 inhibits the viral replication stages (24 to 48 h) rather than the viral translation stages (2 to 8 h). The replicon was transfected into BHK21 cells, which were then maintained in the absence or presence of 8 μM BP2109. Luciferase activity was monitored at the indicated time points posttransfection. The numbers above the BP2109-treated bars represent the percentages of luciferase signals relative to the mock-treated controls (100%), and the error bars represent the SEM from three independent experiments.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Novel Dengue Virus-Specific NS2B/NS3 Protease Inhibitor, BP2109, Discovered by a High-Throughput Screening Assay ▿Novel Dengue Virus-Specific NS2B/NS3 Protease Inhibitor, BP2109, Discovered by a High-Throughput Screening Assay ▿ †

    doi: 10.1128/AAC.00855-10

    Figure Lengend Snippet: Inhibition of DENV-2 RNA replication by BP2109. (A) Schematic diagram of the DENV-2 reporter replicon, DV2Rep. Indicated are the 5′ UTR, the N-terminal 102 amino acids of the C protein (C102), the Renilla luciferase gene (Rluc), the FMDV2A cleavage site, the neomycin resistance gene (Neo), an EMCV IRES element, the C-terminal 24 amino acids of E (E24), the entire NS regions (NS1∼NS5), and the 3′ UTR. The replicon was used to quantify the inhibitory effects of BP2109 on the RNA replication of DENV-2. (B) Analysis of BP2109 using transient replicon assays. BP2109 inhibits the viral replication stages (24 to 48 h) rather than the viral translation stages (2 to 8 h). The replicon was transfected into BHK21 cells, which were then maintained in the absence or presence of 8 μM BP2109. Luciferase activity was monitored at the indicated time points posttransfection. The numbers above the BP2109-treated bars represent the percentages of luciferase signals relative to the mock-treated controls (100%), and the error bars represent the SEM from three independent experiments.

    Article Snippet: Baby hamster kidney (BHK21) cells (ATCC CCL-10) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4.5 g/liter glucose and 5% fetal bovine serum (FBS).

    Techniques: Inhibition, Luciferase, Transfection, Activity Assay

    Inhibition of all four serotypes of DENV by BP2109. BHK21 cells were incubated with 8 μM BP2109 and then infected with four serotypes of DENV at an MOI of 0.1. The viral yield in culture medium was determined by plaque formation assays at 72 h postinfection. The mean values and SEM from three independent experiments are plotted. **, P

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Novel Dengue Virus-Specific NS2B/NS3 Protease Inhibitor, BP2109, Discovered by a High-Throughput Screening Assay ▿Novel Dengue Virus-Specific NS2B/NS3 Protease Inhibitor, BP2109, Discovered by a High-Throughput Screening Assay ▿ †

    doi: 10.1128/AAC.00855-10

    Figure Lengend Snippet: Inhibition of all four serotypes of DENV by BP2109. BHK21 cells were incubated with 8 μM BP2109 and then infected with four serotypes of DENV at an MOI of 0.1. The viral yield in culture medium was determined by plaque formation assays at 72 h postinfection. The mean values and SEM from three independent experiments are plotted. **, P

    Article Snippet: Baby hamster kidney (BHK21) cells (ATCC CCL-10) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4.5 g/liter glucose and 5% fetal bovine serum (FBS).

    Techniques: Inhibition, Incubation, Infection

    Dose-dependent inhibition of the DENV-2 yield by BP2109. The DENV-2 yield was calculated after treating BHK21 cells with increasing concentrations of BP2109 and then infecting them with DENV-2 at MOIs of 0.1 and 1.0, as described in Materials and Methods. The viral yields in culture medium at 72 h postinfection were determined by plaque formation assays. The mean values and SEM from three independent experiments are plotted.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Novel Dengue Virus-Specific NS2B/NS3 Protease Inhibitor, BP2109, Discovered by a High-Throughput Screening Assay ▿Novel Dengue Virus-Specific NS2B/NS3 Protease Inhibitor, BP2109, Discovered by a High-Throughput Screening Assay ▿ †

    doi: 10.1128/AAC.00855-10

    Figure Lengend Snippet: Dose-dependent inhibition of the DENV-2 yield by BP2109. The DENV-2 yield was calculated after treating BHK21 cells with increasing concentrations of BP2109 and then infecting them with DENV-2 at MOIs of 0.1 and 1.0, as described in Materials and Methods. The viral yields in culture medium at 72 h postinfection were determined by plaque formation assays. The mean values and SEM from three independent experiments are plotted.

    Article Snippet: Baby hamster kidney (BHK21) cells (ATCC CCL-10) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4.5 g/liter glucose and 5% fetal bovine serum (FBS).

    Techniques: Inhibition

    BP2109 selectively inhibits the NS2B/NS3 protease activity and viral yield of DENV. (A) Selective inhibition of protease activity by BP2109. Purified JEV NS2B/NS3 protease was used for an enzyme-based protease activity assay. The values represent percentages of the control (untreated) value obtained for DENV or JEV. The mean values and SEM from three independent experiments are plotted. (B) Antiviral spectrum of BP2109. BHK21 cells infected with JEV or DENV-2 at an MOI of 0.1 were maintained in the absence or presence of 12 μM BP2109. The viral yields in culture medium at 72 h postinfection were determined by plaque formation assays. The mean values and SEM from three independent experiments are plotted. (**, P

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Novel Dengue Virus-Specific NS2B/NS3 Protease Inhibitor, BP2109, Discovered by a High-Throughput Screening Assay ▿Novel Dengue Virus-Specific NS2B/NS3 Protease Inhibitor, BP2109, Discovered by a High-Throughput Screening Assay ▿ †

    doi: 10.1128/AAC.00855-10

    Figure Lengend Snippet: BP2109 selectively inhibits the NS2B/NS3 protease activity and viral yield of DENV. (A) Selective inhibition of protease activity by BP2109. Purified JEV NS2B/NS3 protease was used for an enzyme-based protease activity assay. The values represent percentages of the control (untreated) value obtained for DENV or JEV. The mean values and SEM from three independent experiments are plotted. (B) Antiviral spectrum of BP2109. BHK21 cells infected with JEV or DENV-2 at an MOI of 0.1 were maintained in the absence or presence of 12 μM BP2109. The viral yields in culture medium at 72 h postinfection were determined by plaque formation assays. The mean values and SEM from three independent experiments are plotted. (**, P

    Article Snippet: Baby hamster kidney (BHK21) cells (ATCC CCL-10) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4.5 g/liter glucose and 5% fetal bovine serum (FBS).

    Techniques: Activity Assay, Inhibition, Purification, Infection

    BP2109 reduces the levels of viral RNA in BHK21 cells infected with DENV-2. BHK21 cells infected with DENV-2 at an MOI of 0.1 or 1.0 were maintained in the absence or presence of 12 μM BP2109 for 72 h. Positive-strand DENV-2 RNA accumulations in cells were determined by using TaqMan fluorogenic quantitative RT-PCR. The RNA copy number was determined by using standards that were measured with a spectrophotometer. The mean values and SEM from three independent experiments are plotted.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Novel Dengue Virus-Specific NS2B/NS3 Protease Inhibitor, BP2109, Discovered by a High-Throughput Screening Assay ▿Novel Dengue Virus-Specific NS2B/NS3 Protease Inhibitor, BP2109, Discovered by a High-Throughput Screening Assay ▿ †

    doi: 10.1128/AAC.00855-10

    Figure Lengend Snippet: BP2109 reduces the levels of viral RNA in BHK21 cells infected with DENV-2. BHK21 cells infected with DENV-2 at an MOI of 0.1 or 1.0 were maintained in the absence or presence of 12 μM BP2109 for 72 h. Positive-strand DENV-2 RNA accumulations in cells were determined by using TaqMan fluorogenic quantitative RT-PCR. The RNA copy number was determined by using standards that were measured with a spectrophotometer. The mean values and SEM from three independent experiments are plotted.

    Article Snippet: Baby hamster kidney (BHK21) cells (ATCC CCL-10) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4.5 g/liter glucose and 5% fetal bovine serum (FBS).

    Techniques: Infection, Quantitative RT-PCR, Spectrophotometry

    Double mutations (R55K and E80K) within dengue virus NS2B resulted in BP2109 resistance of the dengue virus NS2B/NS3 protease enzyme and the dengue virus replicon. (A) BP2109 susceptibility of the WT and KK double-mutant NS2B/NS3 proteases. The curves were fitted based on the enzyme activity as percentages of the control value (untreated controls). The IC 50 s of the WT and KK mutant NS2B/NS3 proteases representing a 50% reduction in protease activity assay were calculated as 15.43 ± 2.21 μM and 158.44 ± 1.20 μM, respectively. (B) Resistance analyses of the WT replicon (DV2Rep), two K single-mutant replicons, and the KK double-mutant replicon. Two amino acid substitutions in the DENV NS2B region (R55K and E80K) were introduced into the replicon construct. The EC 50 s of the WT and KK mutant replicons were derived from Renilla luciferase activity assays. BHK21 cells were transfected with equal amounts (0.5 μg) of WT and KK mutant replicon RNAs and then treated with BP2109 at the indicated concentrations. The EC 50 values of the WT, R55K, E80K, and KK replicons were determined by calculating the luciferase activities at 72 h posttransfection; the EC 50 s were 0.17 ± 0.01 μM, 0.15 ± 0.01 μM, 10.43 ± 4.7 μM, and 12.55 ± 4.79 μM, respectively. (C) Replication kinetic curves of WT and NS2B mutant replicons. The replicons were transfected into BHK21 cells, and the luciferase activity of each clone was monitored at the indicated time points posttransfection. The mean values and SEM from three independent experiments are plotted.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Novel Dengue Virus-Specific NS2B/NS3 Protease Inhibitor, BP2109, Discovered by a High-Throughput Screening Assay ▿Novel Dengue Virus-Specific NS2B/NS3 Protease Inhibitor, BP2109, Discovered by a High-Throughput Screening Assay ▿ †

    doi: 10.1128/AAC.00855-10

    Figure Lengend Snippet: Double mutations (R55K and E80K) within dengue virus NS2B resulted in BP2109 resistance of the dengue virus NS2B/NS3 protease enzyme and the dengue virus replicon. (A) BP2109 susceptibility of the WT and KK double-mutant NS2B/NS3 proteases. The curves were fitted based on the enzyme activity as percentages of the control value (untreated controls). The IC 50 s of the WT and KK mutant NS2B/NS3 proteases representing a 50% reduction in protease activity assay were calculated as 15.43 ± 2.21 μM and 158.44 ± 1.20 μM, respectively. (B) Resistance analyses of the WT replicon (DV2Rep), two K single-mutant replicons, and the KK double-mutant replicon. Two amino acid substitutions in the DENV NS2B region (R55K and E80K) were introduced into the replicon construct. The EC 50 s of the WT and KK mutant replicons were derived from Renilla luciferase activity assays. BHK21 cells were transfected with equal amounts (0.5 μg) of WT and KK mutant replicon RNAs and then treated with BP2109 at the indicated concentrations. The EC 50 values of the WT, R55K, E80K, and KK replicons were determined by calculating the luciferase activities at 72 h posttransfection; the EC 50 s were 0.17 ± 0.01 μM, 0.15 ± 0.01 μM, 10.43 ± 4.7 μM, and 12.55 ± 4.79 μM, respectively. (C) Replication kinetic curves of WT and NS2B mutant replicons. The replicons were transfected into BHK21 cells, and the luciferase activity of each clone was monitored at the indicated time points posttransfection. The mean values and SEM from three independent experiments are plotted.

    Article Snippet: Baby hamster kidney (BHK21) cells (ATCC CCL-10) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4.5 g/liter glucose and 5% fetal bovine serum (FBS).

    Techniques: Mutagenesis, Activity Assay, Construct, Derivative Assay, Luciferase, Transfection

    Enhanced budding of late-passage BFV as determined by immunoblotting. ( A ) Cells and supernatants (bottom and top panels) were harvested at the indicated passages for immunoblotting (BFV-infected MDBK cells: p9, p45 and p108; BFV-infected BHK-21 cells: p30, p148 and p232). The Gag precursor (p59) and cleaved form (p56) are marked by arrows. ( B ) Densitometric quantification of relative Gag expression levels (bottom) and budding capacities (top) of BFV from different passages and cell lines as indicated. ImageJ was used for densitometric analysis to quantify the density of the immunoblot bands. The results from the loading controls (β-actin) were used to scale the values of the protein of interest (Gag). Densitometric data of the middle and late passages were normalized to the corresponding values of early passages (p9 and p30, respectively) showing similar intracellular Gag expression levels (bottom panel) but an increased budding capacity with continued selection (top panel).

    Journal: Viruses

    Article Title: In Vitro Evolution of Bovine Foamy Virus Variants with Enhanced Cell-Free Virus Titers and Transmission

    doi: 10.3390/v7112907

    Figure Lengend Snippet: Enhanced budding of late-passage BFV as determined by immunoblotting. ( A ) Cells and supernatants (bottom and top panels) were harvested at the indicated passages for immunoblotting (BFV-infected MDBK cells: p9, p45 and p108; BFV-infected BHK-21 cells: p30, p148 and p232). The Gag precursor (p59) and cleaved form (p56) are marked by arrows. ( B ) Densitometric quantification of relative Gag expression levels (bottom) and budding capacities (top) of BFV from different passages and cell lines as indicated. ImageJ was used for densitometric analysis to quantify the density of the immunoblot bands. The results from the loading controls (β-actin) were used to scale the values of the protein of interest (Gag). Densitometric data of the middle and late passages were normalized to the corresponding values of early passages (p9 and p30, respectively) showing similar intracellular Gag expression levels (bottom panel) but an increased budding capacity with continued selection (top panel).

    Article Snippet: The baby hamster kidney cell line BHK-21 (ATCC CCL-10) and Madin-Darby bovine kidney (MDBK) cells were grown in DMEM supplemented with 10% fetal horse serum (FHS) and 1% penicillin-streptomycin solution and used for cell-free BFV serial passaging and selection.

    Techniques: Infection, Expressing, Selection

    Enhanced budding of late-passage BFV determined by TEM. TEM was performed to directly examine particle release of BFV from different passages. ( A ) BFV-infected MDBK and BHK-21 cells from the indicated passages (BFV-infected MDBK cells: MP6, MP37, and MP180; BFV-infected BHK-21 cells: BP10, BP62, and BP230) were washed three times, fixed for 10 min at room temperature, and processed as described previously [ 38 ]. Some views with BFV capsids indicated by red boxes are magnified. Black bars represent 4 µm. ( B ) Quantification of BFV budding from different passages. Different TEM images were used to quantify budding. Only cells with intracellular, clearly visible BFV capsids were scored for the presence of budding events at the cell surface.

    Journal: Viruses

    Article Title: In Vitro Evolution of Bovine Foamy Virus Variants with Enhanced Cell-Free Virus Titers and Transmission

    doi: 10.3390/v7112907

    Figure Lengend Snippet: Enhanced budding of late-passage BFV determined by TEM. TEM was performed to directly examine particle release of BFV from different passages. ( A ) BFV-infected MDBK and BHK-21 cells from the indicated passages (BFV-infected MDBK cells: MP6, MP37, and MP180; BFV-infected BHK-21 cells: BP10, BP62, and BP230) were washed three times, fixed for 10 min at room temperature, and processed as described previously [ 38 ]. Some views with BFV capsids indicated by red boxes are magnified. Black bars represent 4 µm. ( B ) Quantification of BFV budding from different passages. Different TEM images were used to quantify budding. Only cells with intracellular, clearly visible BFV capsids were scored for the presence of budding events at the cell surface.

    Article Snippet: The baby hamster kidney cell line BHK-21 (ATCC CCL-10) and Madin-Darby bovine kidney (MDBK) cells were grown in DMEM supplemented with 10% fetal horse serum (FHS) and 1% penicillin-streptomycin solution and used for cell-free BFV serial passaging and selection.

    Techniques: Transmission Electron Microscopy, Infection

    Kinetics of adaptation of cell-free infectious BFV in MDBK and BHK-21 cultures. Cell-free infectivity of selected BFV was monitored during serial passaging. (A-B) Culture supernatants from serially passaged BFV-infected ( A ) MDBK and ( B ) BHK-21 cells were filtered and titrated on MICL and BICL cells, respectively. Titers are expressed as focus-forming units per ml (FFU/ml). (C,D) Percentages of BFV Gag-positive cells as determined by IIF for early passages (P) of BFV from ( C ) MDBK and ( D ) BHK-21 cells were determined at different time points. BFV-infected MDBK cells: P7 and P13; BFV-infected BHK-21 cells: P5 and P14.

    Journal: Viruses

    Article Title: In Vitro Evolution of Bovine Foamy Virus Variants with Enhanced Cell-Free Virus Titers and Transmission

    doi: 10.3390/v7112907

    Figure Lengend Snippet: Kinetics of adaptation of cell-free infectious BFV in MDBK and BHK-21 cultures. Cell-free infectivity of selected BFV was monitored during serial passaging. (A-B) Culture supernatants from serially passaged BFV-infected ( A ) MDBK and ( B ) BHK-21 cells were filtered and titrated on MICL and BICL cells, respectively. Titers are expressed as focus-forming units per ml (FFU/ml). (C,D) Percentages of BFV Gag-positive cells as determined by IIF for early passages (P) of BFV from ( C ) MDBK and ( D ) BHK-21 cells were determined at different time points. BFV-infected MDBK cells: P7 and P13; BFV-infected BHK-21 cells: P5 and P14.

    Article Snippet: The baby hamster kidney cell line BHK-21 (ATCC CCL-10) and Madin-Darby bovine kidney (MDBK) cells were grown in DMEM supplemented with 10% fetal horse serum (FHS) and 1% penicillin-streptomycin solution and used for cell-free BFV serial passaging and selection.

    Techniques: Infection, Passaging

    Syncytia, “filopodia-like” structure in BFV, and loss of syncytia formation by serially passaged HT–BFV as determined by indirect immunofluorescence (IIF) microscopy. BFV-infected KTR cells were fixed with ice-cold methanol/EGTA and incubated with a rabbit antiserum against BFV Gag MA. Alexa-594 (red) or Alexa-488 (green)-coupled anti-rabbit IgG was used as secondary antibody. Nuclei were counter-stained with Hoechst 33342 (blue). ( A ) Syncytia of original BFV Riems-infected KTR cells by IIF against Gag. The bars are 20 µm in length. ( B ) “Filopodia-like” structures in original BFV Riems-infected KTR cells detected via IIF. The bar is 50 µm in length. ( C ) BFV-infected KTR cells using the original BFV–Riems isolate (left hand panel) and serially passaged BFV (HT–BFV from passage 30 in BHK-21 cells, right hand panel) were analyzed by IIF against Gag MA. BFV Gag MA (red) and nuclear Hoechst 33342 (blue) staining allowed clear detection of loss of syncytia formation upon serial passaging. The bar is 50 µm in length.

    Journal: Viruses

    Article Title: In Vitro Evolution of Bovine Foamy Virus Variants with Enhanced Cell-Free Virus Titers and Transmission

    doi: 10.3390/v7112907

    Figure Lengend Snippet: Syncytia, “filopodia-like” structure in BFV, and loss of syncytia formation by serially passaged HT–BFV as determined by indirect immunofluorescence (IIF) microscopy. BFV-infected KTR cells were fixed with ice-cold methanol/EGTA and incubated with a rabbit antiserum against BFV Gag MA. Alexa-594 (red) or Alexa-488 (green)-coupled anti-rabbit IgG was used as secondary antibody. Nuclei were counter-stained with Hoechst 33342 (blue). ( A ) Syncytia of original BFV Riems-infected KTR cells by IIF against Gag. The bars are 20 µm in length. ( B ) “Filopodia-like” structures in original BFV Riems-infected KTR cells detected via IIF. The bar is 50 µm in length. ( C ) BFV-infected KTR cells using the original BFV–Riems isolate (left hand panel) and serially passaged BFV (HT–BFV from passage 30 in BHK-21 cells, right hand panel) were analyzed by IIF against Gag MA. BFV Gag MA (red) and nuclear Hoechst 33342 (blue) staining allowed clear detection of loss of syncytia formation upon serial passaging. The bar is 50 µm in length.

    Article Snippet: The baby hamster kidney cell line BHK-21 (ATCC CCL-10) and Madin-Darby bovine kidney (MDBK) cells were grown in DMEM supplemented with 10% fetal horse serum (FHS) and 1% penicillin-streptomycin solution and used for cell-free BFV serial passaging and selection.

    Techniques: Immunofluorescence, Microscopy, Infection, Incubation, Staining, Passaging

    D471G substitution affects the ability of LCMV-NP to promote replication and gene expression of an LCMV MG. BHK-21 cells were co-transfected in triplicate with 0.5 μg of pPolI GFP-Pur/Gluc, together with 0.6 μg of pC-L, 0.3 μg of pC wt or mutant NPs HA-tagged, and 0.1μg of the pSV40-Cluc expression vector to normalize transfection efficiencies At 48 hpt, MG driven GFP expression (A) and luciferase activity in TCS (B) were determined. Cell lysates were used to determine expression levels of wt and mutant NPs by WB using an anti-HA antibody (C). GAPDH was used as a loading control. Empty pC was used as a negative control. Percentages of relative luciferase units (% RLUs) were normalized with respect to the activity of the wt NP, after normalization of transfection efficiencies based on Cluc luminescence values. Numbers at the bottom of each WB lane represent the quantification of band intensities normalized to wt NP lane as described in material and methods.

    Journal: Viruses

    Article Title: D471G Mutation in LCMV-NP Affects its Ability to Self-associate and Results in a Dominant Negative Effect in Viral RNA Synthesis

    doi: 10.3390/v4102137

    Figure Lengend Snippet: D471G substitution affects the ability of LCMV-NP to promote replication and gene expression of an LCMV MG. BHK-21 cells were co-transfected in triplicate with 0.5 μg of pPolI GFP-Pur/Gluc, together with 0.6 μg of pC-L, 0.3 μg of pC wt or mutant NPs HA-tagged, and 0.1μg of the pSV40-Cluc expression vector to normalize transfection efficiencies At 48 hpt, MG driven GFP expression (A) and luciferase activity in TCS (B) were determined. Cell lysates were used to determine expression levels of wt and mutant NPs by WB using an anti-HA antibody (C). GAPDH was used as a loading control. Empty pC was used as a negative control. Percentages of relative luciferase units (% RLUs) were normalized with respect to the activity of the wt NP, after normalization of transfection efficiencies based on Cluc luminescence values. Numbers at the bottom of each WB lane represent the quantification of band intensities normalized to wt NP lane as described in material and methods.

    Article Snippet: Cells and Viruses Baby hamster kidney (BHK-21) cells (ATCC CCL-10) and human embryonic kidney (293T) cells (ATCC CRL-11268) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), L-glutamine (2mM), penicillin (100 units/ml) and streptomycin (100μg/ml), in a 5 % CO2 atmosphere at 37°C [ , ].

    Techniques: Expressing, Transfection, Mutagenesis, Plasmid Preparation, Luciferase, Activity Assay, Western Blot, Negative Control

    Effects of D471 substitution on LCMV-NP functions. (A) NP-NP interaction: Human 293T cells were co-transfected as described in Figure 1 B with 2 μg of the indicated pC wt or mutant NP-VP16 fusion proteins, together with 2 μg of NP-GAL4 expression plasmids. At 72 hpt, cell extracts were prepared to determine the strength of the interaction. VP16 expression plasmid was used as negative control. Reporter gene activation (FFL) is shown as percentage of wt interaction (pC-NP-VP16 and pC-NP-GAL4) after normalization of transfection efficiencies based on levels of Renilla luciferase activity driven by plasmid pRL SV40. Cell lysates were used to detect expression of wt and mutant NPs by WB using an anti-VP16 polyclonal antibody. GAPDH was used as a loading control. (B) NP-Z interaction: Human 293T cells were co-transfected as described in Figure 2 B with 2 μg of the indicated pC wt or mutant NP-VP16, together with 2 μg of GAL4-Z expression plasmids. At 48 hpt, cell extracts were prepared to determine the strength of the interaction and protein expression. Reporter gene activation (FFL) is shown as percentage of wt interaction (pC-NP-VP16 and pC-GAL4-Z) after normalization of transfection efficiencies based on Renilla luciferase values. Cell lysates were used to detect expression of wt and mutant NPs by WB using an anti-VP16 polyclonal antibody. GAPDH was used as a loading control. (C) Replication and transcription activity: BHK-21 cells were co-transfected with the LCMV MG as described in Figure 3 together with expression plasmids for the viral polymerase (L) and wt or indicated mutant NPs, and pSV40-Cluc expression vector to normalize transfection efficiencies. At 48 hpt, TCS were collected for luciferase assay and cell lysates were prepared for protein detection. Empty pC was used as a negative control. RLUs (%) were calculated based on the replication and transcription activity mediated by wt NP, after normalization by Cluc luminescence values. Expression levels of wt and mutant NPs were determined by WB using an anti-HA antibody. GAPDH was used as a loading control. (D) Inhibition of induction of IFN-I: Human 293T cells were co-transfected as described in Figure 4 with 0.1 μg of the indicated pC-NP HA-tagged expression vectors together with the IFNβ reporter plasmids. At 16 hpt, cells were infected with SeV (moi=3) to induce activation of the IFNβ promoter, and 24 hours later cell lysates were prepared for luciferase assay and detection of protein expression. Luciferase values were normalized with respect to those obtained in cells transfected with Empty pC and infected with SeV, after adjusting for renilla luminescence values. Expression of wt and mutant NPs were determined by WB using an anti-HA antibody. GAPDH was used as a loading control.

    Journal: Viruses

    Article Title: D471G Mutation in LCMV-NP Affects its Ability to Self-associate and Results in a Dominant Negative Effect in Viral RNA Synthesis

    doi: 10.3390/v4102137

    Figure Lengend Snippet: Effects of D471 substitution on LCMV-NP functions. (A) NP-NP interaction: Human 293T cells were co-transfected as described in Figure 1 B with 2 μg of the indicated pC wt or mutant NP-VP16 fusion proteins, together with 2 μg of NP-GAL4 expression plasmids. At 72 hpt, cell extracts were prepared to determine the strength of the interaction. VP16 expression plasmid was used as negative control. Reporter gene activation (FFL) is shown as percentage of wt interaction (pC-NP-VP16 and pC-NP-GAL4) after normalization of transfection efficiencies based on levels of Renilla luciferase activity driven by plasmid pRL SV40. Cell lysates were used to detect expression of wt and mutant NPs by WB using an anti-VP16 polyclonal antibody. GAPDH was used as a loading control. (B) NP-Z interaction: Human 293T cells were co-transfected as described in Figure 2 B with 2 μg of the indicated pC wt or mutant NP-VP16, together with 2 μg of GAL4-Z expression plasmids. At 48 hpt, cell extracts were prepared to determine the strength of the interaction and protein expression. Reporter gene activation (FFL) is shown as percentage of wt interaction (pC-NP-VP16 and pC-GAL4-Z) after normalization of transfection efficiencies based on Renilla luciferase values. Cell lysates were used to detect expression of wt and mutant NPs by WB using an anti-VP16 polyclonal antibody. GAPDH was used as a loading control. (C) Replication and transcription activity: BHK-21 cells were co-transfected with the LCMV MG as described in Figure 3 together with expression plasmids for the viral polymerase (L) and wt or indicated mutant NPs, and pSV40-Cluc expression vector to normalize transfection efficiencies. At 48 hpt, TCS were collected for luciferase assay and cell lysates were prepared for protein detection. Empty pC was used as a negative control. RLUs (%) were calculated based on the replication and transcription activity mediated by wt NP, after normalization by Cluc luminescence values. Expression levels of wt and mutant NPs were determined by WB using an anti-HA antibody. GAPDH was used as a loading control. (D) Inhibition of induction of IFN-I: Human 293T cells were co-transfected as described in Figure 4 with 0.1 μg of the indicated pC-NP HA-tagged expression vectors together with the IFNβ reporter plasmids. At 16 hpt, cells were infected with SeV (moi=3) to induce activation of the IFNβ promoter, and 24 hours later cell lysates were prepared for luciferase assay and detection of protein expression. Luciferase values were normalized with respect to those obtained in cells transfected with Empty pC and infected with SeV, after adjusting for renilla luminescence values. Expression of wt and mutant NPs were determined by WB using an anti-HA antibody. GAPDH was used as a loading control.

    Article Snippet: Cells and Viruses Baby hamster kidney (BHK-21) cells (ATCC CCL-10) and human embryonic kidney (293T) cells (ATCC CRL-11268) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), L-glutamine (2mM), penicillin (100 units/ml) and streptomycin (100μg/ml), in a 5 % CO2 atmosphere at 37°C [ , ].

    Techniques: Transfection, Mutagenesis, Expressing, Plasmid Preparation, Negative Control, Activation Assay, Luciferase, Activity Assay, Western Blot, Inhibition, Infection

    Dominant negative effect of LCMV-NP D471G on viral replication and transcription. BHK-21 cells were co-transfected in triplicate with 0.5 μg of pPolI GFP-Pur/Gluc, 0.6 μg of pC-L, and the indicated amounts (ng) of pC-LCMV-NP HA-tagged (NP); together with empty pC (Empty) or pC LCMV-NP D471G HA-tagged (D471G), and 0.1μg of the pSV40-Cluc expression vector to normalize transfection efficiencies. At 48 hpt, MG activity was determined by GFP expression (A) and luciferase activity from TCS (B). Reporter gene activation is shown as induction over an empty pC vector-transfected control.

    Journal: Viruses

    Article Title: D471G Mutation in LCMV-NP Affects its Ability to Self-associate and Results in a Dominant Negative Effect in Viral RNA Synthesis

    doi: 10.3390/v4102137

    Figure Lengend Snippet: Dominant negative effect of LCMV-NP D471G on viral replication and transcription. BHK-21 cells were co-transfected in triplicate with 0.5 μg of pPolI GFP-Pur/Gluc, 0.6 μg of pC-L, and the indicated amounts (ng) of pC-LCMV-NP HA-tagged (NP); together with empty pC (Empty) or pC LCMV-NP D471G HA-tagged (D471G), and 0.1μg of the pSV40-Cluc expression vector to normalize transfection efficiencies. At 48 hpt, MG activity was determined by GFP expression (A) and luciferase activity from TCS (B). Reporter gene activation is shown as induction over an empty pC vector-transfected control.

    Article Snippet: Cells and Viruses Baby hamster kidney (BHK-21) cells (ATCC CCL-10) and human embryonic kidney (293T) cells (ATCC CRL-11268) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), L-glutamine (2mM), penicillin (100 units/ml) and streptomycin (100μg/ml), in a 5 % CO2 atmosphere at 37°C [ , ].

    Techniques: Dominant Negative Mutation, Transfection, Expressing, Plasmid Preparation, Activity Assay, Luciferase, Activation Assay

    Dominant negative effect of NP D471G on LCMV infection. (A) Characterization of stable cell lines expressing HA-tagged wt mutant or D471G NPs. Parental and NP-HA expressing BHK-21 cell lines were examined by immunofluorescence with an anti-HA (α-HA) antibody (NP staining). Cellular nuclei were stained with DAPI. Representative merged images are illustrated. Cells lysates were prepared and 100 μg of total cellular protein content were analyzed by WB using an anti-HA antibody (B). GAPDH was used as a loading control. Numbers at the bottom of each WB lane represent the quantification of band intensities normalized to wt NP lane, as described in material and methods. Kinetics of LCMV (C) and VSV-GFP (D) propagation: Parental and NP-HA expressing BHK-21 cell lines were infected with LCMV (moi = 0.01) or VSV-GFP (moi = 0.001) in triplicates. TCS at the indicated hpi were titrated using a focus forming unit assay on Vero cells as described in materials and methods. Dashed lines indicate the limit of detection for the assay.

    Journal: Viruses

    Article Title: D471G Mutation in LCMV-NP Affects its Ability to Self-associate and Results in a Dominant Negative Effect in Viral RNA Synthesis

    doi: 10.3390/v4102137

    Figure Lengend Snippet: Dominant negative effect of NP D471G on LCMV infection. (A) Characterization of stable cell lines expressing HA-tagged wt mutant or D471G NPs. Parental and NP-HA expressing BHK-21 cell lines were examined by immunofluorescence with an anti-HA (α-HA) antibody (NP staining). Cellular nuclei were stained with DAPI. Representative merged images are illustrated. Cells lysates were prepared and 100 μg of total cellular protein content were analyzed by WB using an anti-HA antibody (B). GAPDH was used as a loading control. Numbers at the bottom of each WB lane represent the quantification of band intensities normalized to wt NP lane, as described in material and methods. Kinetics of LCMV (C) and VSV-GFP (D) propagation: Parental and NP-HA expressing BHK-21 cell lines were infected with LCMV (moi = 0.01) or VSV-GFP (moi = 0.001) in triplicates. TCS at the indicated hpi were titrated using a focus forming unit assay on Vero cells as described in materials and methods. Dashed lines indicate the limit of detection for the assay.

    Article Snippet: Cells and Viruses Baby hamster kidney (BHK-21) cells (ATCC CCL-10) and human embryonic kidney (293T) cells (ATCC CRL-11268) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), L-glutamine (2mM), penicillin (100 units/ml) and streptomycin (100μg/ml), in a 5 % CO2 atmosphere at 37°C [ , ].

    Techniques: Dominant Negative Mutation, Infection, Stable Transfection, Expressing, Mutagenesis, Immunofluorescence, Staining, Western Blot

    Comparison of biological characteristics between recovered GZ01 and the parental strain. (A) Identification of the KpnI-marker in recovered ZIKV. Viral RNA was isolated from supernatants of GZ01 and rGZ01 and ZIKV cDNA fragments were amplified by RT-PCR and digested by the endonuclease KpnI. The KpnI-digested PCR products [KpnI (+)] and undigested controls [KpnI (−)] were analyzed by electrophoresis in a 1.5% agarose–TAE gel. Lane M, DNA marker DL 2,000. (B) Plaque morphologies of rGZ01 and parental GZ01. Mock, uninfected BHK-21 cells. (C) BHK-21 cells were infected with an MOI of 0.1 of rGZ01 and GZ01, respectively, and viral E protein expression was monitored by IFA at different time points after infection. Uninfected BHK-21 cells were subjected to IFA and the results were shown in parallel (mock). (D and E) Comparison of the growth kinetics of rGZ01 and GZ01. (D) Infectious viruses in the supernatants of MOI 0.1-infected BHK-21 cells were determined by plaque assay. (E) C6/36 cells were infected with rGZ01 and GZ01 at an MOI of 0.1, culture supernatants were collected at different time points postinfection, and vRNA levels were determined by qRT-PCR. (F) Neurovirulence of rGZ01 and GZ01 in neonatal BALB/c mice. One-day-old BALB/c suckling mice ( n = 7) were incubated with 10 PFU of rGZ01 or GZ01, respectively, and the survival statistics were monitored daily.

    Journal: Journal of Virology

    Article Title: Characterization of cis-Acting RNA Elements of Zika Virus by Using a Self-Splicing Ribozyme-Dependent Infectious Clone

    doi: 10.1128/JVI.00484-17

    Figure Lengend Snippet: Comparison of biological characteristics between recovered GZ01 and the parental strain. (A) Identification of the KpnI-marker in recovered ZIKV. Viral RNA was isolated from supernatants of GZ01 and rGZ01 and ZIKV cDNA fragments were amplified by RT-PCR and digested by the endonuclease KpnI. The KpnI-digested PCR products [KpnI (+)] and undigested controls [KpnI (−)] were analyzed by electrophoresis in a 1.5% agarose–TAE gel. Lane M, DNA marker DL 2,000. (B) Plaque morphologies of rGZ01 and parental GZ01. Mock, uninfected BHK-21 cells. (C) BHK-21 cells were infected with an MOI of 0.1 of rGZ01 and GZ01, respectively, and viral E protein expression was monitored by IFA at different time points after infection. Uninfected BHK-21 cells were subjected to IFA and the results were shown in parallel (mock). (D and E) Comparison of the growth kinetics of rGZ01 and GZ01. (D) Infectious viruses in the supernatants of MOI 0.1-infected BHK-21 cells were determined by plaque assay. (E) C6/36 cells were infected with rGZ01 and GZ01 at an MOI of 0.1, culture supernatants were collected at different time points postinfection, and vRNA levels were determined by qRT-PCR. (F) Neurovirulence of rGZ01 and GZ01 in neonatal BALB/c mice. One-day-old BALB/c suckling mice ( n = 7) were incubated with 10 PFU of rGZ01 or GZ01, respectively, and the survival statistics were monitored daily.

    Article Snippet: The baby hamster kidney fibroblast cell line BHK-21 (ATCC CCL10) was cultured in Dulbecco modified Eagle medium (DMEM; Gibco, Thermo Fisher Scientific) containing 5% fetal bovine serum (FBS; Biowest) and 1% penicillin-streptomycin (PS; Thermo Fisher Scientific).

    Techniques: Marker, Isolation, Amplification, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Electrophoresis, Infection, Expressing, Immunofluorescence, Plaque Assay, Quantitative RT-PCR, Mouse Assay, Incubation

    shRNAs targeting viral gene and inhibiting replication of FMDV in BHK-21 cells. BHK-21 cells stable expressing shRNA were cultured in 12-well plates in four replicates, each well inoculated with 100 TCID 50 of the FMDV ASIA1/YS/CHA/05 strain. 48 hrs after FMDV inoculation, the cells and virus were collected, and mRNA expression of viral genes was determined by RT-PCR, β-actin as control (A), and FMDV viral replication was determined by TCID 50 (B). The results from three independent experiments in quadruplicate were presented as 1 minus a percentage of average TCID 50 in treated cells to that in LacZ shRNA expressed control cells± one standard deviation.

    Journal: PLoS ONE

    Article Title: Identification of Short Hairpin RNA Targeting Foot-And-Mouth Disease Virus with Transgenic Bovine Fetal Epithelium Cells

    doi: 10.1371/journal.pone.0042356

    Figure Lengend Snippet: shRNAs targeting viral gene and inhibiting replication of FMDV in BHK-21 cells. BHK-21 cells stable expressing shRNA were cultured in 12-well plates in four replicates, each well inoculated with 100 TCID 50 of the FMDV ASIA1/YS/CHA/05 strain. 48 hrs after FMDV inoculation, the cells and virus were collected, and mRNA expression of viral genes was determined by RT-PCR, β-actin as control (A), and FMDV viral replication was determined by TCID 50 (B). The results from three independent experiments in quadruplicate were presented as 1 minus a percentage of average TCID 50 in treated cells to that in LacZ shRNA expressed control cells± one standard deviation.

    Article Snippet: Cells BHK-21 and 293T cells were obtained from the American Type Culture Collection and grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

    Techniques: Expressing, shRNA, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

    Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein

    Journal: Journal of Virology

    Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus

    doi: 10.1128/JVI.79.18.11813-11823.2005

    Figure Lengend Snippet: Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein

    Article Snippet: BHK-21 cells were grown in Eagle's minimal essential medium supplemented with 5% fetal calf serum, 1% glutamine, and 0.5% neomycin at 37°C (or, in some experiments, 28°C) in 5% CO2 .

    Techniques: Immunofluorescence, Staining, Transfection, In Vitro, Clone Assay

    Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein

    Journal: Journal of Virology

    Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus

    doi: 10.1128/JVI.79.18.11813-11823.2005

    Figure Lengend Snippet: Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein

    Article Snippet: BHK-21 cells were grown in Eagle's minimal essential medium supplemented with 5% fetal calf serum, 1% glutamine, and 0.5% neomycin at 37°C (or, in some experiments, 28°C) in 5% CO2 .

    Techniques: Transfection, Mutagenesis

    Intracellular localization of CD4 and chimeric CD4 proteins containing the TMD of DENV2 with different mutations by double-label immunofluorescence assay. (A) BHK-21 cells were transfected with each of the constructs described in Fig.

    Journal: Journal of Virology

    Article Title: The Length of and Nonhydrophobic Residues in the Transmembrane Domain of Dengue Virus Envelope Protein Are Critical for Its Retention and Assembly in the Endoplasmic Reticulum ▿

    doi: 10.1128/JVI.01963-09

    Figure Lengend Snippet: Intracellular localization of CD4 and chimeric CD4 proteins containing the TMD of DENV2 with different mutations by double-label immunofluorescence assay. (A) BHK-21 cells were transfected with each of the constructs described in Fig.

    Article Snippet: Plasmid DNA was transfected to 293T cells prepared in a six-well plate by the calcium phosphate method or to BHK-21 cells by Lipofectamine 2000 (Invitrogen, Carsblad, CA).

    Techniques: Immunofluorescence, Transfection, Construct

    Growth curves of chimeric viruses. A Monolayer of BHK-21 cells was infected with wild-type and chimeric viruses at an MOI of 0.01. Media were harvested at each time point, and virus titers were determined by plaque assay in BHK-21 cells. The chimeric

    Journal: Journal of Virology

    Article Title: A Critical Determinant of Neurological Disease Associated with Highly Pathogenic Tick-Borne Flavivirus in Mice

    doi: 10.1128/JVI.00421-14

    Figure Lengend Snippet: Growth curves of chimeric viruses. A Monolayer of BHK-21 cells was infected with wild-type and chimeric viruses at an MOI of 0.01. Media were harvested at each time point, and virus titers were determined by plaque assay in BHK-21 cells. The chimeric

    Article Snippet: BHK-21 cells were infected with each recombinant virus at a multiplicity of infection (MOI) of 0.01.

    Techniques: Infection, Plaque Assay

    Growth properties of chimeric viruses containing NS5 amino acid substitutions. (A) Monolayers of BHK-21 or NA cells were infected with wild-type and chimeric viruses at an MOI of 0.01. Media were harvested at each time point, and virus titers were determined

    Journal: Journal of Virology

    Article Title: A Critical Determinant of Neurological Disease Associated with Highly Pathogenic Tick-Borne Flavivirus in Mice

    doi: 10.1128/JVI.00421-14

    Figure Lengend Snippet: Growth properties of chimeric viruses containing NS5 amino acid substitutions. (A) Monolayers of BHK-21 or NA cells were infected with wild-type and chimeric viruses at an MOI of 0.01. Media were harvested at each time point, and virus titers were determined

    Article Snippet: BHK-21 cells were infected with each recombinant virus at a multiplicity of infection (MOI) of 0.01.

    Techniques: Infection

    More than 2 PKs provides a replicative advantage in co-transfection competition experiments. BHK-21 cells were co-transfected with wt , ΔPK 1234, ΔPK 234 or ΔPK 34 ptGFP replicon RNA and either a wt mCherry replicon or yeast tRNA control. Control competition of ptGFP wt , ΔPK 234 or ΔPK 34 replicons with yeast tRNA (A). Competition of wt , ΔPK 234 or ΔPK 34 ptGFP replicons with a wt mCherry replicon (B). Replication of the C11 ΔPK 1234 replicon when co-transfected with either the wt mCherry replicon or yeast tRNA control (C). Replication of the wt mCherry replicon when co-transfected with ptGFP wt , C11 ΔPK 1234, ΔPK 234 or ΔPK 34 ptGFP replicons (D). All replication is shown at 8 hours post transfection over 3 sequential passages, as measured by an Incucyte Zoom (n = 2). Initial transfection (P0), sequential passages (P1-3).

    Journal: bioRxiv

    Article Title: The RNA pseudoknots in foot-and-mouth disease virus are dispensable for genome replication but essential for the production of infectious virus

    doi: 10.1101/2020.01.10.901801

    Figure Lengend Snippet: More than 2 PKs provides a replicative advantage in co-transfection competition experiments. BHK-21 cells were co-transfected with wt , ΔPK 1234, ΔPK 234 or ΔPK 34 ptGFP replicon RNA and either a wt mCherry replicon or yeast tRNA control. Control competition of ptGFP wt , ΔPK 234 or ΔPK 34 replicons with yeast tRNA (A). Competition of wt , ΔPK 234 or ΔPK 34 ptGFP replicons with a wt mCherry replicon (B). Replication of the C11 ΔPK 1234 replicon when co-transfected with either the wt mCherry replicon or yeast tRNA control (C). Replication of the wt mCherry replicon when co-transfected with ptGFP wt , C11 ΔPK 1234, ΔPK 234 or ΔPK 34 ptGFP replicons (D). All replication is shown at 8 hours post transfection over 3 sequential passages, as measured by an Incucyte Zoom (n = 2). Initial transfection (P0), sequential passages (P1-3).

    Article Snippet: For complementation assays, BHK-21 cells seeded into 24-well plates were allowed to adhere for 16 hours before transfection with 1 µg of replicon RNA using Lipofectin.

    Techniques: Cotransfection, Transfection

    The poly-C-tract is dispensable and only one PK is required for wt replication. Wt , 3D-GNN and a replicon with a truncated poly-C-tract (C11) were transfected into BHK-21 and MDBK cell lines, replication was monitored using the Incucyte Zoom (A). A replicon with entire poly-C-tract removed (C0) was transfected alongside wt , 3D-GNN and C11 replicons into BHK-21 cells (B). Replicons with sequentially deleted PKs (ΔPK 34, ΔPK 234 and C11 ΔPK 1234) were assayed for replication in BHK-21 cells (C) and MDBK cells (D). Replication of replicon with PK 4 as the sole remaining PK (C11 PK 4), transfected into MDBK and BHK-21 cells (E). All replication assays were measured by counting the number of GFP positive cells per well using the Incucyte Zoom with data shown at 8 hours post-transection. Error bars shown are calculated by SEM, n = 3. Significance is shown compared to the wt (A, B) or the wt and 3D-GNN (C, D, E) * P

    Journal: bioRxiv

    Article Title: The RNA pseudoknots in foot-and-mouth disease virus are dispensable for genome replication but essential for the production of infectious virus

    doi: 10.1101/2020.01.10.901801

    Figure Lengend Snippet: The poly-C-tract is dispensable and only one PK is required for wt replication. Wt , 3D-GNN and a replicon with a truncated poly-C-tract (C11) were transfected into BHK-21 and MDBK cell lines, replication was monitored using the Incucyte Zoom (A). A replicon with entire poly-C-tract removed (C0) was transfected alongside wt , 3D-GNN and C11 replicons into BHK-21 cells (B). Replicons with sequentially deleted PKs (ΔPK 34, ΔPK 234 and C11 ΔPK 1234) were assayed for replication in BHK-21 cells (C) and MDBK cells (D). Replication of replicon with PK 4 as the sole remaining PK (C11 PK 4), transfected into MDBK and BHK-21 cells (E). All replication assays were measured by counting the number of GFP positive cells per well using the Incucyte Zoom with data shown at 8 hours post-transection. Error bars shown are calculated by SEM, n = 3. Significance is shown compared to the wt (A, B) or the wt and 3D-GNN (C, D, E) * P

    Article Snippet: For complementation assays, BHK-21 cells seeded into 24-well plates were allowed to adhere for 16 hours before transfection with 1 µg of replicon RNA using Lipofectin.

    Techniques: Transfection

    Disrupting the PK structure and reversing the orientation of a PK reduces replication. Cartoon representations of disrupting and restoring mutations made to PK 1, where nucleotides in the bulge of the stem loop and interacting region downstream were mutated to disrupt structure formation ‘PK disrupt’, or mutated to maintain bulge and downstream interaction but with different nucleotides ‘PK restore’ (A i ). Replication of PK disrupt and restore mutants were measured by transfection of RNA into BHK-21 cells and shown here at 8 hours post-transfection alongside wt , 3D-GNN and C11 ΔPK 1234 controls. Significance is shown comparing the replication of C11 PK disrupt and C11 PK restore (A ii ). Visual representation of the reversing of the nucleotide sequence of PK1 creating the C11 PK Rvs construct (B i ). Replication of PK Rvs at 8 hours post transfection of BHK-21 cells (B ii ). Significance shown is compared to wt replicon. Error bars are calculated by SEM, n = 3, * P

    Journal: bioRxiv

    Article Title: The RNA pseudoknots in foot-and-mouth disease virus are dispensable for genome replication but essential for the production of infectious virus

    doi: 10.1101/2020.01.10.901801

    Figure Lengend Snippet: Disrupting the PK structure and reversing the orientation of a PK reduces replication. Cartoon representations of disrupting and restoring mutations made to PK 1, where nucleotides in the bulge of the stem loop and interacting region downstream were mutated to disrupt structure formation ‘PK disrupt’, or mutated to maintain bulge and downstream interaction but with different nucleotides ‘PK restore’ (A i ). Replication of PK disrupt and restore mutants were measured by transfection of RNA into BHK-21 cells and shown here at 8 hours post-transfection alongside wt , 3D-GNN and C11 ΔPK 1234 controls. Significance is shown comparing the replication of C11 PK disrupt and C11 PK restore (A ii ). Visual representation of the reversing of the nucleotide sequence of PK1 creating the C11 PK Rvs construct (B i ). Replication of PK Rvs at 8 hours post transfection of BHK-21 cells (B ii ). Significance shown is compared to wt replicon. Error bars are calculated by SEM, n = 3, * P

    Article Snippet: For complementation assays, BHK-21 cells seeded into 24-well plates were allowed to adhere for 16 hours before transfection with 1 µg of replicon RNA using Lipofectin.

    Techniques: Transfection, Sequencing, Construct

    Recovered virus showed a delay in rate of CPE and a small plaque phenotype. BHK-21 cells were infected with wt , ΔPK 34 and ΔPK 234 virus at a MOI of 0.01, alongside a mock infected control and cell confluency (shown as phase object confluence %) monitored every half an hour for 62 hours using an Incucyte Zoom (A). Representative plaque assay of BHK-21 cells infected with wt , ΔPK 34 and ΔPK 234 viruses. Recovered virus from passage 5 of blind passaging was infected onto BHK-21 cells, cells were fixed and stained 48 hours post infection (B). Virus plaques were imaged and size of plaques measured using Image J, all plaques per well were counted, additional wells were used until a minimum plaque count of 40 was reached. Significance is shown when compared to the wt , box and whisker plots were made using the Tukey method.

    Journal: bioRxiv

    Article Title: The RNA pseudoknots in foot-and-mouth disease virus are dispensable for genome replication but essential for the production of infectious virus

    doi: 10.1101/2020.01.10.901801

    Figure Lengend Snippet: Recovered virus showed a delay in rate of CPE and a small plaque phenotype. BHK-21 cells were infected with wt , ΔPK 34 and ΔPK 234 virus at a MOI of 0.01, alongside a mock infected control and cell confluency (shown as phase object confluence %) monitored every half an hour for 62 hours using an Incucyte Zoom (A). Representative plaque assay of BHK-21 cells infected with wt , ΔPK 34 and ΔPK 234 viruses. Recovered virus from passage 5 of blind passaging was infected onto BHK-21 cells, cells were fixed and stained 48 hours post infection (B). Virus plaques were imaged and size of plaques measured using Image J, all plaques per well were counted, additional wells were used until a minimum plaque count of 40 was reached. Significance is shown when compared to the wt , box and whisker plots were made using the Tukey method.

    Article Snippet: For complementation assays, BHK-21 cells seeded into 24-well plates were allowed to adhere for 16 hours before transfection with 1 µg of replicon RNA using Lipofectin.

    Techniques: Infection, Plaque Assay, Passaging, Staining, Whisker Assay

    PKs are not essential for viral genome replication. BHK-21 cells were transfected with RNA transcribed from infectious clones of wt , ΔPK 34, ΔPK 234 and C11 ΔPK 1234. Non-transfected cells and a wt transfection treated with 3 mM GuHCl were used as negative controls. Cells were harvested, fixed and labelled using an anti-3A antibody and fluorescent anti-mouse secondary before separation by flow cytometry. Gates were used to select for live cell and single cell populations, and virus positive/negative populations were identified based on levels of Alexa-488 fluorescence. Where distinct virus positive and negative populations exist, gates were drawn to separate these in order to determine the mean fluorescent intensity (MFI) of the virus positive cells. Where no clear separate populations exist ( wt with GuHCl, mock treatment and untreated cells) gates could not be drawn and therefore the total MFI has been reported (denoted by #). Representative images have been shown here for the live cell gate from the wt virus transfection (A), the single cell gate from the wt virus transfection (B) and the relative fluorescence of the cells with mock antibody treatment (C), wt virus (D), C11 ΔPK 1234 virus (E) and wt virus transfection with 3 mM GuHCl (F). The experiment was performed in triplicate and the MFI values for each condition were calculated (G). The error bars represent the SEM. Significance is shown compared to the wt plus 3 mM GuHCl control (**** P

    Journal: bioRxiv

    Article Title: The RNA pseudoknots in foot-and-mouth disease virus are dispensable for genome replication but essential for the production of infectious virus

    doi: 10.1101/2020.01.10.901801

    Figure Lengend Snippet: PKs are not essential for viral genome replication. BHK-21 cells were transfected with RNA transcribed from infectious clones of wt , ΔPK 34, ΔPK 234 and C11 ΔPK 1234. Non-transfected cells and a wt transfection treated with 3 mM GuHCl were used as negative controls. Cells were harvested, fixed and labelled using an anti-3A antibody and fluorescent anti-mouse secondary before separation by flow cytometry. Gates were used to select for live cell and single cell populations, and virus positive/negative populations were identified based on levels of Alexa-488 fluorescence. Where distinct virus positive and negative populations exist, gates were drawn to separate these in order to determine the mean fluorescent intensity (MFI) of the virus positive cells. Where no clear separate populations exist ( wt with GuHCl, mock treatment and untreated cells) gates could not be drawn and therefore the total MFI has been reported (denoted by #). Representative images have been shown here for the live cell gate from the wt virus transfection (A), the single cell gate from the wt virus transfection (B) and the relative fluorescence of the cells with mock antibody treatment (C), wt virus (D), C11 ΔPK 1234 virus (E) and wt virus transfection with 3 mM GuHCl (F). The experiment was performed in triplicate and the MFI values for each condition were calculated (G). The error bars represent the SEM. Significance is shown compared to the wt plus 3 mM GuHCl control (**** P

    Article Snippet: For complementation assays, BHK-21 cells seeded into 24-well plates were allowed to adhere for 16 hours before transfection with 1 µg of replicon RNA using Lipofectin.

    Techniques: Transfection, Clone Assay, Flow Cytometry, Fluorescence

    ER Stress is activated in BHK-21 cells transfected with 2C- or 3D-expressing plasmid. (A) A Western blotting analysis of GRP78, GRP94 and β-actin in the extracts of BHK-21 cells that were treated with 500 nM Tg for 12 h, infected with EMCV (MOI = 0.005) for 12 h or transfected with 2C- or 3D-expressing plasmid for 48 h. (B) The BHK-21 cells were transfected with pGL3-GRP78-luc, pGL3-GRP94-luc, pGL3-Carlreticulin-luc, pGL3-ATF4-luc or pGL3-CHOP-luc along with pRL-TK and pCMV-HA-2C, pCMV-HA-3D or pCMV-HA. The firefly luciferase activity was measured in cell lysates that were normalized with Renilla luciferase activities at 48 h post-transfection. Data are expressed as the means ± SD. ** p

    Journal: Virology Journal

    Article Title: Nonstructural proteins 2C and 3D are involved in autophagy as induced by the encephalomyocarditis virus

    doi: 10.1186/1743-422X-11-156

    Figure Lengend Snippet: ER Stress is activated in BHK-21 cells transfected with 2C- or 3D-expressing plasmid. (A) A Western blotting analysis of GRP78, GRP94 and β-actin in the extracts of BHK-21 cells that were treated with 500 nM Tg for 12 h, infected with EMCV (MOI = 0.005) for 12 h or transfected with 2C- or 3D-expressing plasmid for 48 h. (B) The BHK-21 cells were transfected with pGL3-GRP78-luc, pGL3-GRP94-luc, pGL3-Carlreticulin-luc, pGL3-ATF4-luc or pGL3-CHOP-luc along with pRL-TK and pCMV-HA-2C, pCMV-HA-3D or pCMV-HA. The firefly luciferase activity was measured in cell lysates that were normalized with Renilla luciferase activities at 48 h post-transfection. Data are expressed as the means ± SD. ** p

    Article Snippet: Transmission electron microscopy (TEM) Sub-confluent monolayers of BHK-21 cells grown on 6-well culture plates (Corning Inc.) were transfected with pCMV-HA-2C, pCMV-HA-3D, pCMV-HA-VP1 or pCMV-HA and mock-transfected cells for 48 h. The cell samples were then processed as previously described [ ].

    Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, Infection, Luciferase, Activity Assay

    The regulation of the UPR pathway in the EMCV-infected or with expressed EMCV 2C or 3D BHK 21 cells. (A) BHK-21 cells were harvested after infecting with EMCV (MOI = 0.005) for 12 h or transfecting with pCMV-HA-2C, pCMV-HA-3D or pCMV-HA (as a control) for 48 h, and subjected to Western blotting analysis for PERK, p-PERK, eIF2α, p-eIF2α and β-actin with the indicated antibodies. (B) The analysis scheme for XBP1 mRNA splicing. The relative locations of the 26-nt intron and the PstI restriction site are shown. The sizes of PCR-amplified fragments from spliced XBP1 (XBP1s) and unspliced XBP1 (XBP1u) with or without PstI cleavage are also listed. (C) RT-PCR analysis. The BHK-21 cells were treated with Tu (1 μg/ml) for 12 h, infected with EMCV (MOI = 0.005) for 12 h or transfected with the pCMV-HA-2C, pCMV-HA-3D or pCMV-HA (as a control) for 48 h. Total cellular RNAs were prepared, and RT-PCR was performed by using specific primers to determine the XBP1 splicing (XBP1s) level. To detect XBP1 splicing (XBP1s), the PCR products were digested with PstI and electrophoresed. The XBP1u (291/307 bp) and XBP1s (572 bp) bands are indicated. (D) A Western blotting analysis of ATF6α and β-actin in the cell extracts of BHK-21 cells that were infected with EMCV (MOI = 0.005) for 12 h or transfected with 2C- or 3D-expressing plasmid for 48 h.

    Journal: Virology Journal

    Article Title: Nonstructural proteins 2C and 3D are involved in autophagy as induced by the encephalomyocarditis virus

    doi: 10.1186/1743-422X-11-156

    Figure Lengend Snippet: The regulation of the UPR pathway in the EMCV-infected or with expressed EMCV 2C or 3D BHK 21 cells. (A) BHK-21 cells were harvested after infecting with EMCV (MOI = 0.005) for 12 h or transfecting with pCMV-HA-2C, pCMV-HA-3D or pCMV-HA (as a control) for 48 h, and subjected to Western blotting analysis for PERK, p-PERK, eIF2α, p-eIF2α and β-actin with the indicated antibodies. (B) The analysis scheme for XBP1 mRNA splicing. The relative locations of the 26-nt intron and the PstI restriction site are shown. The sizes of PCR-amplified fragments from spliced XBP1 (XBP1s) and unspliced XBP1 (XBP1u) with or without PstI cleavage are also listed. (C) RT-PCR analysis. The BHK-21 cells were treated with Tu (1 μg/ml) for 12 h, infected with EMCV (MOI = 0.005) for 12 h or transfected with the pCMV-HA-2C, pCMV-HA-3D or pCMV-HA (as a control) for 48 h. Total cellular RNAs were prepared, and RT-PCR was performed by using specific primers to determine the XBP1 splicing (XBP1s) level. To detect XBP1 splicing (XBP1s), the PCR products were digested with PstI and electrophoresed. The XBP1u (291/307 bp) and XBP1s (572 bp) bands are indicated. (D) A Western blotting analysis of ATF6α and β-actin in the cell extracts of BHK-21 cells that were infected with EMCV (MOI = 0.005) for 12 h or transfected with 2C- or 3D-expressing plasmid for 48 h.

    Article Snippet: Transmission electron microscopy (TEM) Sub-confluent monolayers of BHK-21 cells grown on 6-well culture plates (Corning Inc.) were transfected with pCMV-HA-2C, pCMV-HA-3D, pCMV-HA-VP1 or pCMV-HA and mock-transfected cells for 48 h. The cell samples were then processed as previously described [ ].

    Techniques: Infection, Western Blot, Polymerase Chain Reaction, Amplification, Reverse Transcription Polymerase Chain Reaction, Transfection, Expressing, Plasmid Preparation

    Immunofluorescence puncta in the cytoplasm of BHK-21 cells that were co-transfected with each HA-tagged EMCV protein-expressing plasmid and pEGFP-LC3. (A) Confocal immunofluorescence analysis was performed on BHK-21 cells transfected with the pEGFP-LC3 plasmid at 18 h after transfection, and the cells were then infected with the EMCV BJC3 strain for 12 h (MOI = 0.005). A GFP-LC3 signal (green) and EMCV VP1 protein staining (red) are shown. (B-N) The BHK 21 cells were co-transfected with each HA-tagged EMCV protein-expressing plasmid or GST protein-expressing plasmid and pEGFP-LC3. At 48 h after transfection, the cells were fixed and processed by immunostaining with a mouse monoclonal antibody against HA and goat anti-mouse secondary antibodies conjugated to TRITC. The HA (red) and GFP-LC3 (green) proteins were examined by immunoconfocal microscopy. The white arrows show the positive puncta of LC3 proteins or the colocalization of the LC3 proteins and EMCV proteins. The scale bars in panels (A-N) represent 10 μm.

    Journal: Virology Journal

    Article Title: Nonstructural proteins 2C and 3D are involved in autophagy as induced by the encephalomyocarditis virus

    doi: 10.1186/1743-422X-11-156

    Figure Lengend Snippet: Immunofluorescence puncta in the cytoplasm of BHK-21 cells that were co-transfected with each HA-tagged EMCV protein-expressing plasmid and pEGFP-LC3. (A) Confocal immunofluorescence analysis was performed on BHK-21 cells transfected with the pEGFP-LC3 plasmid at 18 h after transfection, and the cells were then infected with the EMCV BJC3 strain for 12 h (MOI = 0.005). A GFP-LC3 signal (green) and EMCV VP1 protein staining (red) are shown. (B-N) The BHK 21 cells were co-transfected with each HA-tagged EMCV protein-expressing plasmid or GST protein-expressing plasmid and pEGFP-LC3. At 48 h after transfection, the cells were fixed and processed by immunostaining with a mouse monoclonal antibody against HA and goat anti-mouse secondary antibodies conjugated to TRITC. The HA (red) and GFP-LC3 (green) proteins were examined by immunoconfocal microscopy. The white arrows show the positive puncta of LC3 proteins or the colocalization of the LC3 proteins and EMCV proteins. The scale bars in panels (A-N) represent 10 μm.

    Article Snippet: Transmission electron microscopy (TEM) Sub-confluent monolayers of BHK-21 cells grown on 6-well culture plates (Corning Inc.) were transfected with pCMV-HA-2C, pCMV-HA-3D, pCMV-HA-VP1 or pCMV-HA and mock-transfected cells for 48 h. The cell samples were then processed as previously described [ ].

    Techniques: Immunofluorescence, Transfection, Expressing, Plasmid Preparation, Infection, Staining, Immunostaining, Microscopy

    Nonstructural proteins 2C and 3D increased autophagic activity and the formation of autophagosomes. (A) Western blotting analyses of LC3, p62 and β-actin in BHK-21 cells that were transfected individually with an HA-tagged EMCV protein-expressing plasmid. HA, the BHK-21 cells transfected with pCMV-HA; Mock, the untransfected BHK-21 cells. The band intensities of LC3-II, p62 and β-actin were quantified, and the relative ratios of LC3-II/β-actin and p62/β-actin are shown in the lower blots. (B) The BHK-21 cells were transfected with pCMV-HA-2C, pCMV-HA-3D, pCMV-HA-VP1 or pCMV-HA and mock (untransfected BHK-21 cells) for 48 h, and they were fixed, processed, and imaged by transmission electron microscopy (TEM). The morphologically characteristic double-membrane vesicles are indicated by black arrows in the relevant areas. Magnification, 10,000×; scale bars, 2 μm. (C) A quantification of the number of autophagosome-like vesicles per cell profile in BHK-21 cells that were transfected with 2C-, 3D-expressing plasmid, pCMV-HA-VP1 or pCMV-HA and mock (untransfected BHK-21 cells) Data are the means ± SD (error bars) for 8 cells per experimental condition from three independent experiments. ***p

    Journal: Virology Journal

    Article Title: Nonstructural proteins 2C and 3D are involved in autophagy as induced by the encephalomyocarditis virus

    doi: 10.1186/1743-422X-11-156

    Figure Lengend Snippet: Nonstructural proteins 2C and 3D increased autophagic activity and the formation of autophagosomes. (A) Western blotting analyses of LC3, p62 and β-actin in BHK-21 cells that were transfected individually with an HA-tagged EMCV protein-expressing plasmid. HA, the BHK-21 cells transfected with pCMV-HA; Mock, the untransfected BHK-21 cells. The band intensities of LC3-II, p62 and β-actin were quantified, and the relative ratios of LC3-II/β-actin and p62/β-actin are shown in the lower blots. (B) The BHK-21 cells were transfected with pCMV-HA-2C, pCMV-HA-3D, pCMV-HA-VP1 or pCMV-HA and mock (untransfected BHK-21 cells) for 48 h, and they were fixed, processed, and imaged by transmission electron microscopy (TEM). The morphologically characteristic double-membrane vesicles are indicated by black arrows in the relevant areas. Magnification, 10,000×; scale bars, 2 μm. (C) A quantification of the number of autophagosome-like vesicles per cell profile in BHK-21 cells that were transfected with 2C-, 3D-expressing plasmid, pCMV-HA-VP1 or pCMV-HA and mock (untransfected BHK-21 cells) Data are the means ± SD (error bars) for 8 cells per experimental condition from three independent experiments. ***p

    Article Snippet: Transmission electron microscopy (TEM) Sub-confluent monolayers of BHK-21 cells grown on 6-well culture plates (Corning Inc.) were transfected with pCMV-HA-2C, pCMV-HA-3D, pCMV-HA-VP1 or pCMV-HA and mock-transfected cells for 48 h. The cell samples were then processed as previously described [ ].

    Techniques: Activity Assay, Western Blot, Transfection, Expressing, Plasmid Preparation, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy