Journal: PLoS ONE

Article Title: Integrin-Mediated Signaling Induced by Simian Virus 40 Leads to Transient Uncoupling of Cortical Actin and the Plasma Membrane

doi: 10.1371/journal.pone.0055799

Figure Lengend Snippet: (A) Cell surface N-glycoproteins that are significantly altered in cell surface abundance upon exposure to SV40 are visualized with a network view. The glycoproteins whose abundance was either increased (yellow) or decreased (blue) during exposure to SV40 (and which either did not change during exposure to VSV, or changed in the opposite direction compared to SV40) are depicted as nodes in the network. The different shades represent different degrees of relative abandunce (log2 values). The remaining nodes in the network are the hits from the RNAi screen (see ), which either increased (green) or decreased (red) SV40 infection upon siRNA knockdown. For the common hits in the CSC and RNAi screens, the node border represents the RNAi phenotype (ITGA6, ITGB6 and CD47 were CSC-hits but gave no RNAi phenotype when tested). The grey connecting lines between nodes illustrate protein interactions, which were assessed using the STRING database with a combined score of at least 0.9 , and were visualized using Cytoscape ( www.cytoscape.org ) and the Cerebral plugin .

Article Snippet: Antibodies were from the following sources: rabbit anti-p-ERM: Cell Signaling Technology; mouse anti-p-AKT (S473) and rabbit anti-p-AKT (S473): Upstate, clone 11E6 and Cell Signaling Technology, respectively; anti-p-p85 (Y458): Cell Signaling Technology; mouse integrin α2β1: abcam, clone P1E6; rabbit anti-SV40 VP1: abcam, ab53977; mouse anti-transferrin receptor: Zymed/Invitrogen, 13–6800; rabbit anti-beta tubulin: abcam, ab6046; mouse anti-RhoA: Santa Cruz, clone 26C4; Antisera against viral large T-antigen were provided by Dr. G. Brandner (University of Freiburg, Germany).

Techniques: Infection, Knockdown

Journal: PLoS ONE

Article Title: Integrin-Mediated Signaling Induced by Simian Virus 40 Leads to Transient Uncoupling of Cortical Actin and the Plasma Membrane

doi: 10.1371/journal.pone.0055799

Figure Lengend Snippet: (A) A targeted siRNA screen reveals several structural and signaling components of cell adhesion to regulate the SV40 infectious route. A set of four siRNAs against 263 genes was applied in A431 human epithelial cells and virus infection was assessed by the presence of nuclear large T-antigen. Low-resolution imaging and image processing with the CellProfiler analysis software were subsequently performed. A Support Vector Machine (SVM)-based classification method was then used to determine percentage of infection upon siRNA treatment. The table shows the genes that reduced (red shades) or enhanced (green shades) SV40 infection with different strength when knocked down. The values in the boxes represent the number of different siRNAs that gave a similar phenotype. (B) Epistasis analysis between Cav1, GRAF1, and Ezrin. A431 cells were treated with siRNA against each one of these genes or combinations of two. Two or three siRNAs were used per gene. Cells were subsequently treated with SV40 and infection levels were assessed by the presence of nuclear T-antigen. The graph shows values pooled from the individual infection indices. p-values: 1.3×10 −4 (Ezrin-siRNA, Cav1-siRNA), 0.39 (Ezrin-siRNA, GRAF1-siRNA). (C) Blocking integrin α2 function with an antibody reduces SV40 infection, similar to siRNA-mediated knock down. A431 cells were pre-incubated with 0.02 µg/µL of blocking antibody 20 min prior to infection (p-values 1×10 −4– 7×10 −4 ). (D) siRNA against integrins α2 and β1 reduces binding of SV40 at the surface of A431 cells. Binding was performed at cold for 2 h and binding capacity was determined by immunoblotting for the presence of the major capsid protein VP1 in cell extracts. Signal intensity was quantified by the ImageJ software and standard deviation corresponds to two independent experiments. (E) SV40 binds onto the surface of various cell lines with different intensity; GM1-deficient cells retain the ability to bind SV40. Quantification of signal intensity from two independent experiments was performed as in (D). (F) SV40-like particles (VLPs) can bind cells that lack its native receptor GM1 in a dose-dependent manner. (G) SV40 can bind cells that lack its native receptor GM1 via integrins. GM95 cells were treated with siRNA against integrin α2 and SV40 binding was determined by the abundance of VP1 protein, as described in (D). (H) Integrins can serve as binding sites for SV40. Integrin α2β1 was immunoprecipitated from A431 cells pretreated for 2 h with SV40 in the cold, and the VP1 protein was detected in the immunocomplex by immunoblotting (red box). Transferrin receptor was used as a negative control.

Article Snippet: Antibodies were from the following sources: rabbit anti-p-ERM: Cell Signaling Technology; mouse anti-p-AKT (S473) and rabbit anti-p-AKT (S473): Upstate, clone 11E6 and Cell Signaling Technology, respectively; anti-p-p85 (Y458): Cell Signaling Technology; mouse integrin α2β1: abcam, clone P1E6; rabbit anti-SV40 VP1: abcam, ab53977; mouse anti-transferrin receptor: Zymed/Invitrogen, 13–6800; rabbit anti-beta tubulin: abcam, ab6046; mouse anti-RhoA: Santa Cruz, clone 26C4; Antisera against viral large T-antigen were provided by Dr. G. Brandner (University of Freiburg, Germany).

Techniques: Virus, Infection, Imaging, Software, Plasmid Preparation, Blocking Assay, Knockdown, Incubation, Binding Assay, Western Blot, Standard Deviation, Immunoprecipitation, Negative Control

Journal: PLoS ONE

Article Title: Integrin-Mediated Signaling Induced by Simian Virus 40 Leads to Transient Uncoupling of Cortical Actin and the Plasma Membrane

doi: 10.1371/journal.pone.0055799

Figure Lengend Snippet: (A) AKT is activated in a wortmannin-sensitive manner in A431 cells following SV40 treatment. A431 cells were treated with wortmannin for 1 h prior to SV40 treatment. Phosphorylated AKT at Ser473 was tested by immunoblotting. (B, C) Phosphorylated AKT is recruited to the plasma membrane of A431 cells in a cholesterol-dependent manner. p-AKT (Ser473) was detected by immunofluorescence in fixed cells (B) or immunoblotting (C), before or 30 min after SV40 treatment. Cholesterol was extracted from the plasma membrane using methyl-β-cyclodextrin (mβCD) for 45 min. Cholesterol re-administration was succeeded with cholesterol coupled to mβCD for 3 h. Scale bars in (B) represent 15 µm. (D) Incubating A431 cells with an antibody against integrin α2β1 results in activation of AKT signaling. Incubation was carried out for 15 min before the addition of SV40 for another 10 min. Cholesterol extraction prior to antibody application was performed as described in (B). (E, F) siRNA-mediated knockdown of human Integrin Linked Kinase (ILK) (E) and integrins α2 and β1 (F) leads to defective activation of AKT in A431 cells, 10 min following SV40 treatment. (G) AKT is phosphorylated in GM95 cells following SV40 treatment. Quantification of all western blots was done with the ImageJ software; values represent averages of two independent experiments ± standard deviation.

Article Snippet: Antibodies were from the following sources: rabbit anti-p-ERM: Cell Signaling Technology; mouse anti-p-AKT (S473) and rabbit anti-p-AKT (S473): Upstate, clone 11E6 and Cell Signaling Technology, respectively; anti-p-p85 (Y458): Cell Signaling Technology; mouse integrin α2β1: abcam, clone P1E6; rabbit anti-SV40 VP1: abcam, ab53977; mouse anti-transferrin receptor: Zymed/Invitrogen, 13–6800; rabbit anti-beta tubulin: abcam, ab6046; mouse anti-RhoA: Santa Cruz, clone 26C4; Antisera against viral large T-antigen were provided by Dr. G. Brandner (University of Freiburg, Germany).

Techniques: Western Blot, Clinical Proteomics, Membrane, Immunofluorescence, Activation Assay, Incubation, Extraction, Knockdown, Software, Standard Deviation

Journal: PLoS ONE

Article Title: Integrin-Mediated Signaling Induced by Simian Virus 40 Leads to Transient Uncoupling of Cortical Actin and the Plasma Membrane

doi: 10.1371/journal.pone.0055799

Figure Lengend Snippet: (A) Cortical actin alterations occur in A431 cells following incubation with SV40 for 10 min, as visualized with phalloidin staining in fixed cells. (B) Phosphorylated ERM proteins become inactive shortly after SV40 treatment. A431 cells were treated with SV40 for the indicated time points, fixed and immunostained with an antibody against phosphorylated T567 of ERM proteins. p-ERM disappears as soon as 5 min after incubation with SV40 and it resumes after 1 h. (C) A phosphomimetic mutant form of Ezrin, T567D, fails to fall off the membrane following SV40 treatment. A431 cells were transfected with EzrinT567D-GFP, treated with SV40 for 15 min, and subsequently fixed and tested for the GFP signal localization. (D) Expression of both a phosphomimetic and an inactive Ezrin mutant form negatively correlates with SV40 infection. Large populations of A431 cells were transfected with EzrinT567D, EzrinT567A or wild-type Ezrin GFP constructs and subjected to SV40. Cells that were both transfected (GFP signal) and infected (T-antigen signal) were scored and compared with the expected number emerging from a random occurrence of the two signals. Negative log2 ratio values represent an anti-correlation, which demonstrates inhibition of infection by the transfected construct (p-value 6.8×10 −3 and 0.05 for the T567D and T567A scores, respectively). (E, F) Inhibition of PDK1 function leads to increased levels of p-ERM. The PDK1 inhibitor was applied onto A431 cells for 1.5 h before the addition of SV40 for another 15 min. Levels of p-ERM were assessed using either immunofluorescence in fixed cells (E) or immunoblotting (F). Quantification of two different blots was performed using the ImageJ software. (G) Inhibition of PDK1 function blocks increased levels of infection as caused by Ezrin knockdown. A431 cells were subjected to siRNA against Ezrin or control siRNA, and subsequently treated with the PDK1 inhibitor 1.5 h prior to SV40 infection, or left untreated. Percentage of infection was calculated from a large number of cells. p-values: 1×10 −4 (PDK1 inhibitor), 5.4×10 −3 (Ezrin siRNA), 5.8×10 −4 (Ezrin siRNA, PDK1 inhibitor).

Article Snippet: Antibodies were from the following sources: rabbit anti-p-ERM: Cell Signaling Technology; mouse anti-p-AKT (S473) and rabbit anti-p-AKT (S473): Upstate, clone 11E6 and Cell Signaling Technology, respectively; anti-p-p85 (Y458): Cell Signaling Technology; mouse integrin α2β1: abcam, clone P1E6; rabbit anti-SV40 VP1: abcam, ab53977; mouse anti-transferrin receptor: Zymed/Invitrogen, 13–6800; rabbit anti-beta tubulin: abcam, ab6046; mouse anti-RhoA: Santa Cruz, clone 26C4; Antisera against viral large T-antigen were provided by Dr. G. Brandner (University of Freiburg, Germany).

Techniques: Incubation, Staining, Mutagenesis, Membrane, Transfection, Expressing, Infection, Construct, Inhibition, Immunofluorescence, Western Blot, Software, Knockdown, Control

Journal: PLoS ONE

Article Title: Integrin-Mediated Signaling Induced by Simian Virus 40 Leads to Transient Uncoupling of Cortical Actin and the Plasma Membrane

doi: 10.1371/journal.pone.0055799

Figure Lengend Snippet: (A) Constitutively active RhoA leads to increased levels of phosphorylated Ezrin that persist upon SV40 treatment. Inactive RhoA abrogates basal levels of p-ERM. A431 cells were transfected with RhoA-G14V-GFP or RhoA-T19N-GFP before SV40 was applied for 15 min and fixed cells were stained with a p-ERM antibody. (B) Expression of an inactive RhoA mutant form positively correlates with SV40 infection. Large populations of A431 cells were transfected with RhoA-G14V, RhoA-T19N and wild-type RhoA GFP constructs and subjected to SV40. Cells that were both transfected (GFP signal) and infected (T-antigen signal) were scored and compared with the expected number emerging from a random occurrence of the two signals. Positive log2 ratio values represent a positive correlation, demonstrating a stimulation of infection, whereas negative values denote anti-correlation, demonstrating an inhibition of infection (p-value 4.2×10 −4 ). (C) Inhibition of GRAF1 function leads to increased levels of p-ERM. siRNA was applied onto A431 cells followed by addition of SV40 for 15 min. Levels of p-ERM were assessed using immunofluorescence in fixed cells. (D) RhoA is inactivated 10 min after SV40 treatment. Inhibition of PI3K and PDK1 with wortmannin or the PDK1 inhibitor, respectively, abolished the SV40-induced reduction in RhoA activity. Cells that had undergone the indicated treatment were subjected to RhoA-GTP immunoprecipitation, which was subsequently detected with a RhoA antibody using immunoblotting. The graph shows the quantification of the RhoA-GTP signal in SV40 exposed cells, as expressed in % reduction compared to the non-treated cells, and normalized against total RhoA and tubulin (quantification based on two different experiments). (E) PDK1 and GRAF1 act both upstream of RhoA to signal to ERM proteins. A431 cells were subjected to the following conditions before being scored for the presence or absence of p-ERM signal using immunofluorescence: transfection with RhoA-WT-GFP, RhoA-G14V-GFP or RhoA-T19N-GFP constucts, incubation with the PDK1 inhibitor for 1.5 h, siRNA treatment against GRAF1, or a combination of RhoA-T19N-GFP expression and the PDK1 inhibitor or GRAF1 siRNA. Acquired confocal images were processed with ImageJ to quantify the number of p-ERM-expressing cells. Inhibition of PDK1 function or silencing of GRAF1 led to partial or no restoration of the fraction of p-ERM-positive RhoA-T19N-expressing cells (asterisks), respectively. Values shown are the average of 2–4 independent experiments ± standard deviation. (F) Representative image used to extract the values shown in (E). The white line outlines manually segmented cells, whereas green and red depict RhoA-T19N-GFP transfected cells and p-ERM-positive cells, respectively. Dapi-stained nuclei are shown in blue.

Article Snippet: Antibodies were from the following sources: rabbit anti-p-ERM: Cell Signaling Technology; mouse anti-p-AKT (S473) and rabbit anti-p-AKT (S473): Upstate, clone 11E6 and Cell Signaling Technology, respectively; anti-p-p85 (Y458): Cell Signaling Technology; mouse integrin α2β1: abcam, clone P1E6; rabbit anti-SV40 VP1: abcam, ab53977; mouse anti-transferrin receptor: Zymed/Invitrogen, 13–6800; rabbit anti-beta tubulin: abcam, ab6046; mouse anti-RhoA: Santa Cruz, clone 26C4; Antisera against viral large T-antigen were provided by Dr. G. Brandner (University of Freiburg, Germany).

Techniques: Transfection, Staining, Expressing, Mutagenesis, Infection, Construct, Inhibition, Immunofluorescence, Activity Assay, Immunoprecipitation, Western Blot, Incubation, Standard Deviation

Journal: PLoS ONE

Article Title: Integrin-Mediated Signaling Induced by Simian Virus 40 Leads to Transient Uncoupling of Cortical Actin and the Plasma Membrane

doi: 10.1371/journal.pone.0055799

Figure Lengend Snippet: (A) The various players participating in the integrin-mediated signaling through host SV40 engagement. The respective time intervals during which these signaling events occur are also depicted.

Article Snippet: Antibodies were from the following sources: rabbit anti-p-ERM: Cell Signaling Technology; mouse anti-p-AKT (S473) and rabbit anti-p-AKT (S473): Upstate, clone 11E6 and Cell Signaling Technology, respectively; anti-p-p85 (Y458): Cell Signaling Technology; mouse integrin α2β1: abcam, clone P1E6; rabbit anti-SV40 VP1: abcam, ab53977; mouse anti-transferrin receptor: Zymed/Invitrogen, 13–6800; rabbit anti-beta tubulin: abcam, ab6046; mouse anti-RhoA: Santa Cruz, clone 26C4; Antisera against viral large T-antigen were provided by Dr. G. Brandner (University of Freiburg, Germany).

Techniques: