Journal:

Article Title: Role of Nucleolin in Human Parainfluenza Virus Type 3 Infection of Human Lung Epithelial Cells

doi: 10.1128/JVI.78.15.8146-8158.2004

Figure Lengend Snippet: Identification of nucleolin as an HPIV-3 envelope binding protein. (A) VOPBA of A549 protein fractions eluted from the anion exchange column with 35S-HPIV-3 in the absence (lanes 1 to 6) and presence (lanes 7 and 8) of excess nonradioactive (cold) HPIV-3. The 35S-HPIV-3 interacting 110-kDa protein band is marked. (B) Comparison of the amino-terminal primary sequence of human nucleolin with the sequence of the 110-kDa protein. (C) 35S-HPIV-3 VOPBA in the absence (lanes 1 and 2) and presence (lanes 3 and 4) of excess nonradioactive (cold) HPIV-3 was performed by using anti-HA immunoprecipitated cell lysates obtained following transfection with HA-nucleolin (lanes 2 and 4) or an empty vector (lanes 1 and 3). (D) Western blot analysis of cell lysates (10 μg of protein) obtained from cells transfected with HA-nucleolin (lane 2) or an empty vector (lane 1) with anti-HA antibody.

Article Snippet: Human lung epithelial A549, BHK-21, and CV-1 cells were maintained in Dulbecco modified Eagle medium (GIBCO-BRL, Gaithersburg, Md.) supplemented with 10% fetal bovine serum, penicillin, streptomycin, and glutamine.

Techniques: Binding Assay, Sequencing, Immunoprecipitation, Transfection, Plasmid Preparation, Western Blot

Journal:

Article Title: Role of Nucleolin in Human Parainfluenza Virus Type 3 Infection of Human Lung Epithelial Cells

doi: 10.1128/JVI.78.15.8146-8158.2004

Figure Lengend Snippet: Cell surface expression of nucleolin. (A) Nonbiotinylated (lane 1) and biotinylated (lane 2) A549 cell lysates (100 μg of protein) were subjected to precipitation with avidin-agarose and Western blot analysis with antinucleolin antibody. A549 cell lysates (lane 3) (4 μg of protein) served as a control. (B) Biotinylated A549 cell lysates (lane 1) (200 μg protein) were subjected to precipitation with avidin-agarose and Western blot analysis with anti-β-catenin antibody. A549 cell lysates (lane 2) (5 μg of protein) served as a control. (C) Nonbiotinylated (lane 1) and biotinylated cell lysates (250 μg protein) obtained following biotinylation from either the apical (AP) (lane 2) or the basolateral (BL) (lane 3) side of a filter-grown polarized monolayer of A549 cells were subjected to precipitation with avidin-agarose and Western blot analysis with antinucleolin antibody. Cell lysates (lane 4) (12 μg of protein) from polarized A549 cells served as a control.

Article Snippet: Human lung epithelial A549, BHK-21, and CV-1 cells were maintained in Dulbecco modified Eagle medium (GIBCO-BRL, Gaithersburg, Md.) supplemented with 10% fetal bovine serum, penicillin, streptomycin, and glutamine.

Techniques: Expressing, Avidin-Biotin Assay, Western Blot

Journal:

Article Title: Role of Nucleolin in Human Parainfluenza Virus Type 3 Infection of Human Lung Epithelial Cells

doi: 10.1128/JVI.78.15.8146-8158.2004

Figure Lengend Snippet: Interaction of nucleolin with biotinylated HPIV-3. (A) Lysates obtained from A549 cells incubated with biotinylated HPIV-3 (Biotin-HPIV-3) (2 MOI) at 37°C were immunoprecipitated with either β-catenin (lane 1) or nucleolin (lane 2) antibodies. The bound proteins were then subjected to SDS-7.5% PAGE and blotting with avidin-HRP. Biotinylated HPIV-3 (lane 3) served as a control to demonstrate the biotinylated envelope proteins F and HN. (B) Lysates obtained from A549 cells incubated in the absence (lane 1) or presence (lane 2) of biotinylated HPIV-3 (Biotin-HPIV-3) (2 MOI) at 37°C were precipitated with avidin-agarose. The bound proteins were then subjected to Western blotting with antinucleolin antibody. A549 cell lysate (lane 3) served as a control. (C) Lysates obtained from A549 cells incubated in the absence (lane 1) or presence (lane 2) of biotinylated VSV (Biotin-VSV) (2 MOI) at 37°C were precipitated with avidin-agarose. The bound proteins were then subjected to Western blotting with antinucleolin antibody. A549 cell lysate (lane 3) served as a control. (D) Lysates obtained from A549 cells incubated in the absence (lane 1) or presence (lane 2) of biotinylated HPIV-3 (Biotin-HPIV-3) (2 MOI) at 4°C were precipitated with avidin-agarose. The bound proteins were then subjected to Western blotting with antinucleolin antibody. A549 cell lysate (lane 3) served as a control.

Article Snippet: Human lung epithelial A549, BHK-21, and CV-1 cells were maintained in Dulbecco modified Eagle medium (GIBCO-BRL, Gaithersburg, Md.) supplemented with 10% fetal bovine serum, penicillin, streptomycin, and glutamine.

Techniques: Incubation, Immunoprecipitation, Avidin-Biotin Assay, Western Blot

Journal:

Article Title: Role of Nucleolin in Human Parainfluenza Virus Type 3 Infection of Human Lung Epithelial Cells

doi: 10.1128/JVI.78.15.8146-8158.2004

Figure Lengend Snippet: Interaction of nucleolin with HPIV-3 F and HN proteins. (A) A549 cells transfected with either an empty vector (lane 1) or HPIV-3 FLAG-F cDNA (lane 2) were pulse labeled with [35S]methionine, and the radioactive lysate was immunoprecipitated with anti-FLAG antibody prior to SDS-7.5% PAGE and fluorography. (B) A549 cells transfected with either an empty vector (lane 1) or HPIV-3 FLAG-HN cDNA (lane 2) were pulse labeled with [35S]methionine, and the radioactive lysate was immunoprecipitated with anti-FLAG antibody prior to SDS-7.5% PAGE and fluorography. (C) Lysates (100 μg of protein) obtained from A549 cells transfected with either an empty vector (lane 1), HPIV-3 FLAG-F (lane 3) or HPIV-3 FLAG-HN (lane 4) cDNA were immunoprecipitated with anti-FLAG antibody. The proteins bound to the washed anti-FLAG-agarose beads were subjected to Western blot analysis with antinucleolin antibody. An A549 cell lysate (lane 2) (20 μg of protein) served as a control.

Article Snippet: Human lung epithelial A549, BHK-21, and CV-1 cells were maintained in Dulbecco modified Eagle medium (GIBCO-BRL, Gaithersburg, Md.) supplemented with 10% fetal bovine serum, penicillin, streptomycin, and glutamine.

Techniques: Transfection, Plasmid Preparation, Labeling, Immunoprecipitation, Western Blot

Journal:

Article Title: Role of Nucleolin in Human Parainfluenza Virus Type 3 Infection of Human Lung Epithelial Cells

doi: 10.1128/JVI.78.15.8146-8158.2004

Figure Lengend Snippet: Effect of nucleolin antibodies (Ab) on virus replication. (A) Culture supernatants collected from A549 cells mock infected or infected with HPIV-3 (0.2 MOI) in the absence or presence of nucleolin (Nuc) polyclonal (Poly) and monoclonal (Mono) antibodies or β-catenin polyclonal antibody (control) were added to CV-1 cells for a plaque assay. The plaque assay results, reflecting the viral titers, are expressed in PFU per milliliter. Each value represents the mean ± standard deviation for three determinations. (B) The average plaque assay values (in PFU per milliliter) from panel A were used to show the percentages of inhibition of infection in the presence of nucleolin antibodies. The percentage of infection, reflecting the percentage of virus release, was calculated as a ratio of the PFU-per-milliliter value obtained for cells infected with HPIV-3 in the presence of the antibodies to the value obtained for cells infected with HPIV-3 in the absence of the antibodies. The 100% level of infection represents the value (20 × 105 PFU/ml) obtained for untreated cells. (C) A549 cell lysates (10 μg of protein) obtained from mock-infected (lane 1) and HPIV-3-infected cells (36 h postinfection) in the absence (lane 2) or in the presence of β-catenin (control) (lane 3) or nucleolin (Nuc) (lane 4) antibodies were subjected to Western blot analysis with HPIV-3 anti-RNP antibody. (D) Culture supernatants collected from A549 cells mock infected or infected with VSV (0.2 MOI) in the absence or presence of nucleolin (Nuc) polyclonal (Poly) and monoclonal (Mono) antibodies were added to L929 cells for a plaque assay. The percentage of infection, reflecting the percentage of virus release, was calculated as a ratio of the PFU-per-milliliter value obtained for cells infected with VSV in the presence of the antibodies to the value obtained for cells infected with VSV in the absence of the antibodies. The 100% infection level represents the value (in PFU per milliliter) obtained for untreated cells. (E) A549 cell lysates (10 μg of protein) obtained from mock-infected (lane 1) and VSV-infected cells (36 h postinfection) in the absence (lane 2) or in the presence of β-catenin (control) (lane 3) or nucleolin (Nuc) (lane 4) antibodies were subjected to Western blot analysis with VSV anti-P antibody.

Article Snippet: Human lung epithelial A549, BHK-21, and CV-1 cells were maintained in Dulbecco modified Eagle medium (GIBCO-BRL, Gaithersburg, Md.) supplemented with 10% fetal bovine serum, penicillin, streptomycin, and glutamine.

Techniques: Infection, Plaque Assay, Standard Deviation, Inhibition, Western Blot

Journal:

Article Title: Role of Nucleolin in Human Parainfluenza Virus Type 3 Infection of Human Lung Epithelial Cells

doi: 10.1128/JVI.78.15.8146-8158.2004

Figure Lengend Snippet: Effect of purified nucleolin on virus replication. (A) Culture supernatants collected from A549 cells mock infected or infected with HPIV-3 (0.2 MOI) preincubated with either purified nucleolin (Nuc) or β-catenin (β-cat) were added to CV-1 cells for a plaque assay. The plaque assay results, reflecting the viral titers, are expressed in PFU per milliliter. Each value represents the mean ± standard deviation for three determinations. (B) The average plaque assay values (in PFU per milliliter) from panel A were used to show the percentages of inhibition of infection of HPIV-3 in the presence of purified nucleolin. The percentage of infection, reflecting the percentage of virus release, was calculated as a ratio of the PFU-per-milliliter value obtained for cells infected with HPIV-3 in the presence of purified proteins to the value obtained for cells infected with HPIV-3 in the absence of purified proteins. The 100% level of infection represents the value (23 × 105 PFU/ml) obtained for untreated cells. Similarly, culture supernatants collected from A549 cells mock infected or infected with VSV (0.2 MOI) in the absence or presence of purified nucleolin were added to L929 cells for a plaque assay. The percentage of infection, reflecting the percentage of virus release, was calculated as a ratio of the PFU-per-milliliter value obtained for cells infected with VSV in the presence of the purified protein to the value obtained for cells infected with VSV in the absence of the purified protein. The 100% level of infection represents the value (in PFU per milliliter) obtained from un-treated cells. (C) A549 cell lysates (5 μg of protein) obtained from mock-infected (lane 1) and HPIV-3-infected (36 h postinfection) cells following preincubation in the absence (lane 2) or presence of 5 nM (lane 3), 15 nM (lane 4), and 30 nM (lane 5) purified nucleolin (Nuc) were subjected to Western blot analysis with HPIV-3 anti-RNP antibody.

Article Snippet: Human lung epithelial A549, BHK-21, and CV-1 cells were maintained in Dulbecco modified Eagle medium (GIBCO-BRL, Gaithersburg, Md.) supplemented with 10% fetal bovine serum, penicillin, streptomycin, and glutamine.

Techniques: Purification, Infection, Plaque Assay, Standard Deviation, Inhibition, Western Blot

Journal:

Article Title: Role of Nucleolin in Human Parainfluenza Virus Type 3 Infection of Human Lung Epithelial Cells

doi: 10.1128/JVI.78.15.8146-8158.2004

Figure Lengend Snippet: Effect of nucleolin antibodies (Ab) and purified nucleolin (Nuc) on cellular attachment and internalization of HPIV-3. (A) The kinetics of 35S-HPIV-3 attachment to A549 cells was examined by adding different amounts of virus (0.25 to 2 MOI, or 1 × 105 to 8 × 105 cpm) to chilled A549 cells. Following attachment at 4°C for 2 h, the cells were washed extensively and the cell-associated radioactivity (in counts per minute) representing the attached virus was measured by counting the cell lysate with a liquid scintillation counter. Each value represents the mean ± standard deviation for three determinations. (B) Attachment of 35S-HPIV-3 (1 MOI, or 4 × 105 cpm) to chilled A549 cells pretreated with antibodies or the virus preincubated with the purified proteins was determined following the attachment of the virus at 4°C for 2 h. Following adsorption, the cells were washed extensively and the cell-associated radioactivity (in counts per minute) representing the attached virus was measured by counting the cell lysate with a liquid scintillation counter. The percentage of attachment was calculated as a ratio of the amount of radioactivity present in cells incubated with 35S-HPIV-3 in the presence of the antibodies or purified proteins to the amount of radioactivity present in cells incubated with 35S-HPIV-3 alone. (C) Internalization of 35S-HPIV-3 (1 MOI, or 4 × 105 cpm) into A549 cells pretreated with antibodies was determined following incubation of attached (2 h, 4°C) virus at 37°C for 0.5, 1, and 2 h. The cell-associated radioactivities (in counts per minute) representing the internalized virus at different time points were measured by counting the cell pellet with a liquid scintillation counter. Each value represents the mean ± standard deviation for three determinations. (D) Internalization of 35S-HPIV-3 (1 MOI, or 4 × 105 cpm) into A549 cells following preincubation of the virus with the purified proteins was determined following incubation of attached (2 h, 4°C) virus at 37°C for 0.5, 1, and 2 h. The cell-associated radioactivities (in counts per minute) representing the internalized virus at different time points were measured by counting the cell pellet with a liquid scintillation counter. Each value represents the mean ± standard deviation for three determinations. (E) The average internalization values (counts per minute internalized) at 2 h postinternalization at 37°C from panels C and D were used to show the percentages of inhibition of internalization in the presence of nucleolin antibody and purified nucleolin. The percentage of internalization was calculated as a ratio of the amount of radioactivity present in cells infected with 35S-HPIV-3 in the presence of the antibodies or purified proteins to the amount of radioactivity present in cells infected with 35S-HPIV-3 alone. β-cat, β-catenin.

Article Snippet: Human lung epithelial A549, BHK-21, and CV-1 cells were maintained in Dulbecco modified Eagle medium (GIBCO-BRL, Gaithersburg, Md.) supplemented with 10% fetal bovine serum, penicillin, streptomycin, and glutamine.

Techniques: Purification, Cell Attachment Assay, Radioactivity, Standard Deviation, Adsorption, Incubation, Inhibition, Infection

Journal: Viruses

Article Title: Nuclear Translocation of Crk Adaptor Proteins by the Influenza A Virus NS1 Protein

doi: 10.3390/v8040101

Figure Lengend Snippet: A functional SH3 binding motif in the non-structural protein-1 (NS1) is required for interaction with Crk-like adapter protein (CrkL) in influenza A virus (IAV)-infected cells. ( A ) The consensus sequence of class II SH3 binding motif, and its presence ( + ) or absence ( − ) in the C-terminal region (residues 212–217 shown) of NS1 proteins of the recombinant IAV strains used in this study. In SH3 binding consensus x indicates any residue, ɸ a hydrophopic residue, and + a positively charged amino acid, which for Crk-family SH3 domains is preferable a lysine residue; ( B,C ) Co-immunoprecipation of NS1 proteins with CrkL from lysates of A549 cells infected with recombinant A/WSN-based IAV strains expressing wild-type or mutant NS1 proteins derived from A/Mallard ( B ) or A/WSN ( C ) for 24 h at a multiplicity of infections (MOI) 2. Note that these NS1 proteins naturally differ in their SH3 binding capacity, and the mutations introduced in them thus have opposite effects. NS1 and nucleoprotein ( NP ) blots from whole cell extracts ( WCE ) before anti-CrkL immunoprecipitation are shown to control equal infection of the cells by the different viruses.

Article Snippet: The human lung epithelial (A549) and the human hepatocellular carcinoma (Huh-7) cell lines were maintained in Dulbecco′s Modified Eagle Medium (DMEM) (Sigma Aldrich, St. Louis, MO, USA) supplemented with 4500 mg/L of glucose, 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA), 0.05 mg/mL penicillin, 0.05 mg/mL streptomycin (Sigma Aldrich), and 1 mM L-glutamine (Sigma Aldrich) at 37 °C in 5% CO 2 .

Techniques: Functional Assay, Binding Assay, Infection, Sequencing, Recombinant, Expressing, Mutagenesis, Derivative Assay, Immunoprecipitation

Journal: Viruses

Article Title: Nuclear Translocation of Crk Adaptor Proteins by the Influenza A Virus NS1 Protein

doi: 10.3390/v8040101

Figure Lengend Snippet: Infection of cells with IAV expressing SH3 binding-competent NS1 causes nuclear relocalization of CrkL. ( A ) Immunofluorescence staining of NS1 and CrkL in A549 cells that were mock-infected (upper panel) or infected with A/WSN-NS1 Mallard(wt) (middle panel) or A/WSN-NS1 Mallard(K217E) (bottom panel) for 20 h at a MOI 0.5. The nuclei were visualized by staining with Hoechst. The mean intensity of CrkL fluorescence in the nuclei was quantified from 50 cells infected with A/WSN-NS1 Mallard(wt) or A/WSN-NS1 Mallard(K217E) that also stained positive for positive for NS1, and was normalized to the mean fluorescence intensity of CrkL immunostaining of 50 mock-infected cells. The standard error is presented in the figure. The statistical significance of the differences was determined by Student′s t-test (* p < 0.001); ( B ) A549 cells were infected with A/WSN-NS1 Mallard(wt) for different time points at a MOI 0.5. The localization of CrkL was scored from 100 cells as a cytoplasmic, an intermediate, or a nuclear pattern.

Article Snippet: The human lung epithelial (A549) and the human hepatocellular carcinoma (Huh-7) cell lines were maintained in Dulbecco′s Modified Eagle Medium (DMEM) (Sigma Aldrich, St. Louis, MO, USA) supplemented with 4500 mg/L of glucose, 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA), 0.05 mg/mL penicillin, 0.05 mg/mL streptomycin (Sigma Aldrich), and 1 mM L-glutamine (Sigma Aldrich) at 37 °C in 5% CO 2 .

Techniques: Infection, Expressing, Binding Assay, Immunofluorescence, Staining, Fluorescence, Immunostaining

Journal: Viruses

Article Title: Nuclear Translocation of Crk Adaptor Proteins by the Influenza A Virus NS1 Protein

doi: 10.3390/v8040101

Figure Lengend Snippet: Nuclear translocation of CrkI, CrkII, and CrkL by SH3 binding-competent NS1 proteins demonstrated by subcellular fractionation. ( A ) Western blot analysis of cytoplasmic ( C ) and nuclear extracts ( N ) prepared from A549 cells that were mock-infected ( MOCK ) or infected for 24 h with recombinant A/WSN containing either the wild-type ( WT ) or the K217E mutant NS1 from A/Mallard virus at an MOI 2. In addition to antibodies against the Crk-family proteins and NS1, the blotted A549 fractions were also probed with antibodies against Histone H3 and α-tubulin to confirm successful separation of nuclear and cytoplasmic fractions. In addition, unfractionated whole cell extracts ( WCE ) of the infected cells were Western blotted with anti-NS1 and anti-NP antibodies to confirm uniform infection of cells by the different viruses; ( B ) Same as ( A ) except that the cells were infected with recombinant A/WSN virus carrying wild-type ( WT ) or the T215P mutant NS1 from A/WSN.

Article Snippet: The human lung epithelial (A549) and the human hepatocellular carcinoma (Huh-7) cell lines were maintained in Dulbecco′s Modified Eagle Medium (DMEM) (Sigma Aldrich, St. Louis, MO, USA) supplemented with 4500 mg/L of glucose, 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA), 0.05 mg/mL penicillin, 0.05 mg/mL streptomycin (Sigma Aldrich), and 1 mM L-glutamine (Sigma Aldrich) at 37 °C in 5% CO 2 .

Techniques: Translocation Assay, Binding Assay, Fractionation, Western Blot, Infection, Recombinant, Mutagenesis

Journal: Viruses

Article Title: Nuclear Translocation of Crk Adaptor Proteins by the Influenza A Virus NS1 Protein

doi: 10.3390/v8040101

Figure Lengend Snippet: Nuclear targeting of Crk by NS1 causes a change in the nuclear protein tyrosine phosphorylation pattern of IAV-infected cells. A549 cells were infected with recombinant viruses as indicated for 24 h at a MOI 2, and treated with pervanadate for 10 min before cytoplasmic ( C ) and nuclear ( N ) extracts were prepared. The extracts were probed with an anti-phosphotyrosine antibody. As in , shown are also H3 and α-tubulin blots to verify the quality of the subcellular fractionation, as well as blotting of whole cell extracts (WCE) with antibodies for NS1 and NP to verify equal NS1 expression of NS1 and uniform infection of cells with the different viruses.

Article Snippet: The human lung epithelial (A549) and the human hepatocellular carcinoma (Huh-7) cell lines were maintained in Dulbecco′s Modified Eagle Medium (DMEM) (Sigma Aldrich, St. Louis, MO, USA) supplemented with 4500 mg/L of glucose, 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA), 0.05 mg/mL penicillin, 0.05 mg/mL streptomycin (Sigma Aldrich), and 1 mM L-glutamine (Sigma Aldrich) at 37 °C in 5% CO 2 .

Techniques: Infection, Recombinant, Fractionation, Expressing