Skip to main content
. 2013 Jul 12;8(7):e68450. doi: 10.1371/journal.pone.0068450

Figure 1. Sequential events during host-cell entry of IAV.

Figure 1

(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–g). High-resolution 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 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 37°C 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 37°C 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 37°C 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 37°C for 3 h. After fixation and permeabilization, mouse monoclonal antibody HB64 was used to stain the viral M1 (green). (Bottom) Block in uncoating due to ATP6V1B2 siRNA treatment, where the virus particles (green) accumulated in the endocytic vacuoles. (g) Nuclear import (EI assay): (Top) In the AllStars negative siRNA-treated cells, virus particles were allowed to enter at 37°C for 3.5 h. Incoming NP proteins (green) were detected within the nucleus (blue) by the treatment with mouse monoclonal antibody HB65. (Bottom) Import of NP (green) was blocked in cells treated with ATP6V1B2 siRNA. Scale bar = 5 µm.