Fig 7.

AnxA6 overexpression impairs influenza virus budding. (A) Analysis of budding virions from AnxA6-overexpressing cells by TEM. A431 cells (upper panels) and the A431-AnxA6 stable cell line (lower panels) were infected with influenza A/WSN/33 virus at an MOI of 5. At 10 h p.i., cells were fixed and processed for thin sectioning and TEM. Asterisks indicate spherical virions, and arrowheads indicate elongated virions. Noninfected controls are shown on the left. (B) Semiquantitation of total numbers of virions. Virions observed in infected cells (n = 13 cells with >100 virions) of each cell type were manually counted blinded by five different investigators, and the total numbers of virions were compared. ***, P < 0.0001 by unpaired Student's t test. (C) Semiquantitation of virions according to morphology. During counting, virions were categorized into three types according to their morphology and size, as indicated. The numbers of virions for each type were determined, and their percentages were subjected to unpaired Student's t test and plotted as means ± SD (***, P < 0.0001; **, P < 0.01; *, P < 0.05). (D) Progeny virus titration of released virions. The same experiment as that in panel A was performed with cell culture supernatants collected at 9 h p.i. Virus titers were determined by plaque assay on MDCK cells. Data are shown as means ± SD for triplicates in a representative experiment. ***, P < 0.0001 by unpaired Student's t test.