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. Author manuscript; available in PMC: 2010 Oct 22.
Published in final edited form as: Cell Host Microbe. 2009 Oct 22;6(4):367–380. doi: 10.1016/j.chom.2009.09.005

Figure 4. Influenza A virus matrix protein 2 (M2) blocks autophagosome fusion with lysosomes.

Figure 4

(A) GFP-Atg8/LC3 transfected A549 human lung epithelial cells were transfected with expression plasmids encoding either FLAG tagged M2 or NP of influenza A/WSN/33 virus. 24 hours post transfection GFP-ATG8/LC3 positive autophagosome accumulation was analyzed by fluorescence microscopy. Influenza M2 specific antibodies were used for M2 detection, and an anti-Flag antibody for NP detection. Scale bar: 80 μm. One of three experiments is shown. (B) Left: Western Blot analysis of lipidated endogenous Atg8/LC3-II accumulation in A549 cells transfected with M2 or NP. Right: Western blot analysis of M2 and NP expression in transfected cells, using anti-FLAG tag staining. One of three experiments is shown. (C) Colocalization analysis of M2 (red fluorescence) and GFP-LC3 (green fluorescence) in infected A549 cells: Serial optical sections, (0.2 um; 40 sections) were acquired and images were then deconvoluted using Huygens software. Pearson coefficients were calculated using the Imaris 6.3.0 software. White fluorescence represents colocalization. Scale bar: 10μm. One representative of 36 analyzed cells is shown. (D). Mass spectrometric analysis of density gradient fractions (1–6) of influenza A virus infected A549 cells content upon influenza infection in A549 cells. Cell organelle marker protein distribution in these fractions was determined by mass spectrometric sequencing of protein fragments. Influenza A virus M2 distribution follows the distribution of autophagosome marker protein (right panel), but not the distribution pattern of mitochondria and endoplasmic reticulum (ER) proteins. One of four experiments is shown.