FIG 3.

Mild acidification of viral core proteins prior to pH 5.0-mediated fusion leads to more efficient uncoating. (A) IAV uncoating upon preacidification and acid-induced fusion at the PM. Virus was pretreated for 1 h at 37°C in DMEM-based buffers adjusted to pH 7.4 and 5.8. Neutralized virus was bound to A549 cells on ice, and fusion was induced as described for the acid-bypass infection assay. Cells were incubated for 5 min (M1) and 30 min (NP) after fusion in the presence of CHX-containing stop medium, fixed, and stained for M1 and NP, respectively, by indirect immunofluorescence. (Right) Representative images. Nuclei were visualized by DRAQ5 staining. (Left) M1 and NP exposure, as a measure for viral uncoating, was determined by measurement of the total fluorescence per cell and quantified with ImageJ. Bars, 20 μm. (B) Detection of M1 and NP upon acid bypass of preacidified virus. Virus was pretreated and fused at the PM of A549 cells as described in the legend to panel A. After 5 min incubation in CHX-containing stop medium, cells were fixed and stained for M1 and NP. White arrowheads, M1-free NP-staining. Bars, 10 μm. (C) Acid-bypass infection of WSN(wt) and amantadine-sensitive WSN(AS) in the presence of 100 μM the M2 inhibitor amantadine. See Materials and Methods for details about the construction of recombinant WSN(AS). (D) Preacidification of WSN(wt) and WSN(AS) in the presence of amantadine. Virus was pretreated with amantadine for 5 min at RT prior to preacidification at pH 6.2 for 10 min at 37°C. Acid-bypass infection was performed under the same conditions used for X31. (E) Acid bypass of X31 in the presence of CCCP. As schematically shown on the right, IAV was either left untreated (condition 1), preacidified at pH 5.8 for 1 h at 37°C (condition 2), or treated with CCCP for 5 min at RT (condition 3) or preacidification was combined with CCCP treatment (condition 4). Samples were subjected to either acid-bypass infection assay or normal infection of A549 cells, as described in the legend to panel A. CCCP was further present during the 2-min fusion step but excluded from subsequent incubation in stop and infection medium. (F) IAV uncoating upon acid-induced fusion at the PM in the presence of CCCP. Preincubation with CCCP and acid bypass were performed as described in the legend to Fig. 1D. Cells were fixed for 5 min (M1) and 30 min (NP) after acid bypass and subjected to indirect immunofluorescence. Representative images with DRAQ5 nuclear staining (left) and quantification (right) are shown. Samples were analyzed as described in the legend to panel A. Bars, 20 μm. (G) Acid bypass of WSN(AS) in the presence of CCCP and amantadine. As schematically shown on the left, IAV was either left untreated (condition 1) or pretreated with amantadine (condition 2) or CCCP (condition 3) for 5 min at RT. When both drugs were combined (condition 4), virus was first treated with amantadine for 5 min to block M2 channels and then CCCP was added to the solution and the mixture was further incubated for 5 min. Drugs were present during the fusion step but excluded from subsequent incubation in stop medium. The acid-bypass infection assay was performed under the same conditions described for X31.