FIG 2.

Proton channel activity contributes to influenza A virus M2-induced autophagosome accumulation in a dose-dependent manner. (A) TREx-293 cells carrying tetracycline-inducible amantadine-sensitive H5N1-M2 (S31) or amantadine-resistant H5N1-M2 (N31) were treated and with increasing concentrations of amantadine with or without tetracycline as indicated. Cells were collected at 24 h and subjected to Western blot analysis with the antibodies indicated. (B) HEK293 cells were transfected with plasmids expressing H5M2 (S31), H5M2 (N31), H5M2 (H37G), H5M2 (HDR/A), or H5M2 (HDR/L). Amantadine was added at 6 h after transfection. Cells were collected at 24 h and subjected to Western blot analysis with the antibodies indicated. Representative data from one of three separate experiments are shown. (C) MDCK cells were transfected with plasmids expressing H5M2 (S31), H5M2 (HDR/A), or H5M2 (HDR/L) with a pH-sensitive EGFP protein in the wells of a 96-well plate in six replicates. At 16 h after transfection, the culture medium was replaced with pH 7.4 phosphate-buffered saline containing 0.3% bovine serum albumin for detection of fluorescence intensity at neutral pH. The change in fluorescence intensity was detected at 1 min after the medium was changed to pH 5.0. The relative fluorescence was the ratio of fluorescence intensity at pH 5.0 normalized to that at pH 7.4. Statistical analyses were performed by two-tailed Mann-Whitney test. ***, P < 0.001. Representative data from one of three separate experiments are shown. The relative LC3-II/LC3-I and LC3-II/β-actin ratios were analyzed by using ImageJ (National Institutes of Health) in the same Western blot assay. M2(HDR/A) has His37Ala, Asp44Ala, and Arg45Ala mutations. M2(HDR/L) has His37Leu, Asp44 Leu, and Arg45Leu mutations.