Eous to develop antiviral strategies that interfere with host cell factors essential for viral entry and replication. For this, systematic identification of processes that promote viral infection is necessary. Recently, five genome-wide RNAi screens for IAV infection were performed in tissue culture cells. Collectively, about 1000 genes were identified as factors that support the IAV replication cycle [5]. However, the precise role of most of these factors at different stages of the viral life cycle was not elucidated. Therefore, development of assays for the sequential steps in the infectious cycle is warranted to functionally classify hits according to the step in the entry program affected, and this in a high-throughput manner.High-Content Analysis of IAV Entry EventsFigure 1. Sequential events during host-cell entry of IAV. (a). Entry involves six steps; binding of the virus to the cell membrane (EB), internalization by endocytosis (EE), acidification in late endocytic vacuoles (EA), fusion of viral and vacuolar membranes (EF), uncoating of nucleocapsid (EU), and nuclear import of vRNPs (EI). Components of IAV are shown in the right (NA: purchase SPDB neuraminidase, M2: proton channel). (b ). Highresolution confocal images of the individual assays. (b) Binding (EB assay): (Top) AllStars negative siRNA-treated cells were Title Loaded From File incubated with IAV for 1 h in the cold. After washing, cell-bound virus particles were stained by IIF using the Pinda antibody against HA (green). The cells membrane was 1315463 visualized with WGA-AF647 (blue). (Title Loaded From File Bottom) Cells with no virus (c) Endocytosis (EE assay): (Top) Cells were incubated with IAV for 1 h in the cold. After washing, cells with bound viruses were warmed up to 37uC for 20 min to allow virus internalization. To distinguish between the endocytosed and extracellular virus particles, the HA epitopes of the virus particles accessible from the medium were masked with the Pinda antibody. The cells were then permeabilized with detergent and incubated with a mouse monoclonal antibody (HA1). After fluorescently-labeled secondary antibody treatment, the endocytosed (green) and non-internalized virus particles (red) were identified (Pinda/perm HA). Cell membrane (blue) was stained with WGA. (Bottom) After virus internalization and Title Loaded From File fixation, cells were permeabilized with detergent and similar staining procedures were followed. The endocytosed and extracellular virus particles are not distinguished and both showed same fluorescent signal (red) (perm Pinda/perm HA). (d) Acidification (EA assay): (Top) Virus particles were allowed to enter the AllStars negative siRNA-treated cells at 37uC for 1.0 h and were stained with A1 antibody to detect the acid-induced conformation of HA (green) in endocytic vacuoles near the nucleus (blue). (Bottom) Cells treated with ATP6V1B2 siRNA showed no A1 signal due to block in endosome acidification. (e) Fusion (EF assay): (Top) Virus particles were labeled with SP-DiOC18 (3) and R18, and were allowed to enter the AllStars negative siRNA-treated cells at 37uC for 1.5 h, after which the cells were fixed. Fusion of viral and vacuolar membranes of cells triggered dequenching of DiOC18(3) (green). DiOC18(3) signal colocalized with the R18 (red) signal. (Bottom) Cells treated with ATP6V1B2 siRNA showed R18 (red) signal only. (f) Uncoating (EU assay): (Top) To detect the dispersal of M1 into the cytoplasm of the cells (blue), viruses were allowed to enter the AllStars negative siRNA treated cells at 37uC for.Eous to develop antiviral strategies that interfere with host cell factors essential for viral entry and replication. For this, systematic identification of processes that promote viral infection is necessary. Recently, five genome-wide RNAi screens for IAV infection were performed in tissue culture cells. Collectively, about 1000 genes were identified as factors that support the IAV replication cycle [5]. However, the precise role of most of these factors at different stages of the viral life cycle was not elucidated. Therefore, development of assays for the sequential steps in the infectious cycle is warranted to functionally classify hits according to the step in the entry program affected, and this in a high-throughput manner.High-Content Analysis of IAV Entry EventsFigure 1. Sequential events during host-cell entry of IAV. (a). Entry involves six steps; binding of the virus to the cell membrane (EB), internalization by endocytosis (EE), acidification in late endocytic vacuoles (EA), fusion of viral and vacuolar membranes (EF), uncoating of nucleocapsid (EU), and nuclear import of vRNPs (EI). Components of IAV are shown in the right (NA: neuraminidase, M2: proton channel). (b ). Highresolution confocal images of the individual assays. (b) Binding (EB assay): (Top) AllStars negative siRNA-treated cells were incubated with IAV for 1 h in the cold. After washing, cell-bound virus particles were stained by IIF using the Pinda antibody against HA (green). The cells membrane was 1315463 visualized with WGA-AF647 (blue). (Bottom) Cells with no virus (c) Endocytosis (EE assay): (Top) Cells were incubated with IAV for 1 h in the cold. After washing, cells with bound viruses were warmed up to 37uC for 20 min to allow virus internalization. To distinguish between the endocytosed and extracellular virus particles, the HA epitopes of the virus particles accessible from the medium were masked with the Pinda antibody. The cells were then permeabilized with detergent and incubated with a mouse monoclonal antibody (HA1). After fluorescently-labeled secondary antibody treatment, the endocytosed (green) and non-internalized virus particles (red) were identified (Pinda/perm HA). Cell membrane (blue) was stained with WGA. (Bottom) After virus internalization and fixation, cells were permeabilized with detergent and similar staining procedures were followed. The endocytosed and extracellular virus particles are not distinguished and both showed same fluorescent signal (red) (perm Pinda/perm HA). (d) Acidification (EA assay): (Top) Virus particles were allowed to enter the AllStars negative siRNA-treated cells at 37uC for 1.0 h and were stained with A1 antibody to detect the acid-induced conformation of HA (green) in endocytic vacuoles near the nucleus (blue). (Bottom) Cells treated with ATP6V1B2 siRNA showed no A1 signal due to block in endosome acidification. (e) Fusion (EF assay): (Top) Virus particles were labeled with SP-DiOC18 (3) and R18, and were allowed to enter the AllStars negative siRNA-treated cells at 37uC for 1.5 h, after which the cells were fixed. Fusion of viral and vacuolar membranes of cells triggered dequenching of DiOC18(3) (green). DiOC18(3) signal colocalized with the R18 (red) signal. (Bottom) Cells treated with ATP6V1B2 siRNA showed R18 (red) signal only. (f) Uncoating (EU assay): (Top) To detect the dispersal of M1 into the cytoplasm of the cells (blue), viruses were allowed to enter the AllStars negative siRNA treated cells at 37uC for.Eous to develop antiviral strategies that interfere with host cell factors essential for viral entry and replication. For this, systematic identification of processes that promote viral infection is necessary. Recently, five genome-wide RNAi screens for IAV infection were performed in tissue culture cells. Collectively, about 1000 genes were identified as factors that support the IAV replication cycle [5]. However, the precise role of most of these factors at different stages of the viral life cycle was not elucidated. Therefore, development of assays for the sequential steps in the infectious cycle is warranted to functionally classify hits according to the step in the entry program affected, and this in a high-throughput manner.High-Content Analysis of IAV Entry EventsFigure 1. Sequential events during host-cell entry of IAV. (a). Entry involves six steps; binding of the virus to the cell membrane (EB), internalization by endocytosis (EE), acidification in late endocytic vacuoles (EA), fusion of viral and vacuolar membranes (EF), uncoating of nucleocapsid (EU), and nuclear import of vRNPs (EI). Components of IAV are shown in the right (NA: neuraminidase, M2: proton channel). (b ). Highresolution confocal images of the individual assays. (b) Binding (EB assay): (Top) AllStars negative siRNA-treated cells were incubated with IAV for 1 h in the cold. After washing, cell-bound virus particles were stained by IIF using the Pinda antibody against HA (green). The cells membrane was 1315463 visualized with WGA-AF647 (blue). (Bottom) Cells with no virus (c) Endocytosis (EE assay): (Top) Cells were incubated with IAV for 1 h in the cold. After washing, cells with bound viruses were warmed up to 37uC for 20 min to allow virus internalization. To distinguish between the endocytosed and extracellular virus particles, the HA epitopes of the virus particles accessible from the medium were masked with the Pinda antibody. The cells were then permeabilized with detergent and incubated with a mouse monoclonal antibody (HA1). After fluorescently-labeled secondary antibody treatment, the endocytosed (green) and non-internalized virus particles (red) were identified (Pinda/perm HA). Cell membrane (blue) was stained with WGA. (Bottom) After virus internalization and fixation, cells were permeabilized with detergent and similar staining procedures were followed. The endocytosed and extracellular virus particles are not distinguished and both showed same fluorescent signal (red) (perm Pinda/perm HA). (d) Acidification (EA assay): (Top) Virus particles were allowed to enter the AllStars negative siRNA-treated cells at 37uC for 1.0 h and were stained with A1 antibody to detect the acid-induced conformation of HA (green) in endocytic vacuoles near the nucleus (blue). (Bottom) Cells treated with ATP6V1B2 siRNA showed no A1 signal due to block in endosome acidification. (e) Fusion (EF assay): (Top) Virus particles were labeled with SP-DiOC18 (3) and R18, and were allowed to enter the AllStars negative siRNA-treated cells at 37uC for 1.5 h, after which the cells were fixed. Fusion of viral and vacuolar membranes of cells triggered dequenching of DiOC18(3) (green). DiOC18(3) signal colocalized with the R18 (red) signal. (Bottom) Cells treated with ATP6V1B2 siRNA showed R18 (red) signal only. (f) Uncoating (EU assay): (Top) To detect the dispersal of M1 into the cytoplasm of the cells (blue), viruses were allowed to enter the AllStars negative siRNA treated cells at 37uC for.Eous to develop antiviral strategies that interfere with host cell factors essential for viral entry and replication. For this, systematic identification of processes that promote viral infection is necessary. Recently, five genome-wide RNAi screens for IAV infection were performed in tissue culture cells. Collectively, about 1000 genes were identified as factors that support the IAV replication cycle [5]. However, the precise role of most of these factors at different stages of the viral life cycle was not elucidated. Therefore, development of assays for the sequential steps in the infectious cycle is warranted to functionally classify hits according to the step in the entry program affected, and this in a high-throughput manner.High-Content Analysis of IAV Entry EventsFigure 1. Sequential events during host-cell entry of IAV. (a). Entry involves six steps; binding of the virus to the cell membrane (EB), internalization by endocytosis (EE), acidification in late endocytic vacuoles (EA), fusion of viral and vacuolar membranes (EF), uncoating of nucleocapsid (EU), and nuclear import of vRNPs (EI). Components of IAV are shown in the right (NA: neuraminidase, M2: proton channel). (b ). Highresolution confocal images of the individual assays. (b) Binding (EB assay): (Top) AllStars negative siRNA-treated cells were incubated with IAV for 1 h in the cold. After washing, cell-bound virus particles were stained by IIF using the Pinda antibody against HA (green). The cells membrane was 1315463 visualized with WGA-AF647 (blue). (Bottom) Cells with no virus (c) Endocytosis (EE assay): (Top) Cells were incubated with IAV for 1 h in the cold. After washing, cells with bound viruses were warmed up to 37uC for 20 min to allow virus internalization. To distinguish between the endocytosed and extracellular virus particles, the HA epitopes of the virus particles accessible from the medium were masked with the Pinda antibody. The cells were then permeabilized with detergent and incubated with a mouse monoclonal antibody (HA1). After fluorescently-labeled secondary antibody treatment, the endocytosed (green) and non-internalized virus particles (red) were identified (Pinda/perm HA). Cell membrane (blue) was stained with WGA. (Bottom) After virus internalization and fixation, cells were permeabilized with detergent and similar staining procedures were followed. The endocytosed and extracellular virus particles are not distinguished and both showed same fluorescent signal (red) (perm Pinda/perm HA). (d) Acidification (EA assay): (Top) Virus particles were allowed to enter the AllStars negative siRNA-treated cells at 37uC for 1.0 h and were stained with A1 antibody to detect the acid-induced conformation of HA (green) in endocytic vacuoles near the nucleus (blue). (Bottom) Cells treated with ATP6V1B2 siRNA showed no A1 signal due to block in endosome acidification. (e) Fusion (EF assay): (Top) Virus particles were labeled with SP-DiOC18 (3) and R18, and were allowed to enter the AllStars negative siRNA-treated cells at 37uC for 1.5 h, after which the cells were fixed. Fusion of viral and vacuolar membranes of cells triggered dequenching of DiOC18(3) (green). DiOC18(3) signal colocalized with the R18 (red) signal. (Bottom) Cells treated with ATP6V1B2 siRNA showed R18 (red) signal only. (f) Uncoating (EU assay): (Top) To detect the dispersal of M1 into the cytoplasm of the cells (blue), viruses were allowed to enter the AllStars negative siRNA treated cells at 37uC for.