FIG 1.

Acid-induced fusion of IAV at the PM of A549 cells. (A) Schematic overview of the acid-bypass infection assay. Virus was bound to A549 cells on ice, followed by a 2-min pH 5.0 pulse at 37°C. Acid-bypass control samples were incubated for 2 min at 37°C in infection medium. Following acid bypass, cells were incubated for 14 h at 37°C in stop medium to block endosome acidification and, thus, further viral entry via endocytosis. (B) Thin-section EM of X31 fusion at the PM of A549 cells. Virus was bound to cells on ice, directly fixed (left) or incubated for 60 to 90 s in fusion medium (pH 5.0) at 37°C (middle and right), and then fixed. Viruses with compact-appearing core elements (red asterisks) could be captured during fusion. The viral glycoproteins were clearly visible on the outer side of the PM (black arrowheads). Bar, 100 nm. (C) Fluorescence spectroscopy monitoring the fusion kinetics of R18-labeled X31 with the A549 cell surface upon acid exposure. Following binding of labeled virus to cells on ice, samples were shifted to 37°C, and then fusion was initiated by lowering the pH to the indicated value. Fluorescence dequenching of R18 was measured over time, and the reaction was terminated by addition of 0.1% (final concentration) Triton X-100, which released the maximal dequenching capacity. The percent R18 dequenching shown was normalized to the fluorescence measured in the presence of 0.1% Triton X-100. max., maximum. (D) Comparison of acid-bypass infection and normal infection via endocytosis for RNA viruses. The acid-bypass protocol was performed as described in the legend to panel A. In the case of normal infection, virus was bound to A549 cells, washed, and continuously incubated in infection medium until fixation. IAV and UUKV were scored by immunostaining, detecting newly synthesized viral nucleoproteins. SFV and VSV infection was detected on the basis of GFP expression. The percentage of infected cells was normalized to the values obtained for normal infection. (E) Schematic overview of pretreatment prior to acid-bypass infection (top). X31 was pretreated for 1 h at 37°C in DMEM-based buffers adjusted to the indicated pH values. Following binding for 1 h on ice, the normal acid-bypass protocol described in the legend to panel A was performed (red line, primary y axis). In vitro HA acidification and fusion capacity were tested for the respective pH values in separate experiments (blue lines; secondary y axis). HA acidification was assessed using a conformation-specific antibody (A1) targeting only the acidic form of HA (blue line, open circles). Fusion capacity was determined by applying a FACS-based fusion assay (blue line, closed circles). (F) Kinetics of pretreatment prior to acid-bypass infection. Virus was pretreated for the indicated incubation times in DMEM-based buffers adjusted to the indicated pH values at 37°C, followed by acid-bypass infection, as described in the legend to panel E. (G) Preacidification of WSN(wt) prior to acid-bypass infection. WSN(wt) was pretreated with the indicated DMEM-based pH buffers for 10 min at 37°C, followed by performance of the acid bypass and infection scoring protocol as described for X31.