FIG 5.

Influenza A virus M2 alters Golgi pH and reduces IBV S at the surface of EG3-expressing cells. (A) Vero cells expressing pHluorin-TGN38 and M2 were evaluated by indirect immunofluorescence microscopy. Cells were labeled with rabbit anti-GFP and mouse anti-M2, followed by Alexa 488-conjugated anti-rabbit IgG and Alexa 546-conjugated anti-mouse IgG, and Hoechst stain. Some M2 is present in the Golgi region. (B) Vero cells transiently expressing pHluorin-TGN38 with or without transient expression of IAV M2 and with or without treatment with amantadine (5 μM) were evaluated by flow cytometry. The calculated pH values from a single independent experiment are graphed (∼5,000 cells each). (C) Representative blot from Vero cells expressing IBV S with either WT IBV E or EG3, along with either the empty vector or IAV M2, after surface biotinylation. Biotinylated proteins were isolated with streptavidin-agarose beads from lysates. Both the input (IN) (10%) and surface fractions (100%) were subjected to Western blot analysis with rabbit anti-IBV S_CT followed by IRDye 680CW donkey anti-rabbit IgG. The positions of the IBV S2 species are indicated, as are the molecular weight markers in kilodaltons. (D) Quantification of the total IBV S at the cell surface from 3 experiments. The low percentage of surface S is likely due to inefficient biotinylation. One-way analysis of variance (ANOVA) was performed with GraphPad Prism. *, P < 0.05 compared to WT E plus the vector. Error bars indicate SD.