Human metapneumovirus Induces Reorganization of the Actin Cytoskeleton for Direct Cell-to-Cell Spread
Fig 1
HMPV infection in BEAS-2B cells results in the formation of branched filamentous networks and intercellular extensions.
(A) HMPV infected BEAS-2B cells at 18 or 24 h.p.i., were fixed and processed for immunofluorescence staining. White arrows indicate intercellular extensions, red arrowheads indicate branched viral filaments and white arrowheads indicate viral filaments. Scale bars = 10 μm. (B) HMPV infected BEAS-2B cells at 48 h.p.i. were fixed, processed for immunofluorescence and stained with antibodies for HMPV N or P. Scale bar = 50 μm. (C) BEAS-2B cells were inoculated with PIV5 and at 48 h.p.i. cells were fixed, processed for immunofluorescence and stained with antibody against the F protein of PIV5. Arrow indicates an intercellular extension in a PIV5-infected cell and inset shows filaments. Scale bar = 10 μm. (D) BEAS-2B cells were inoculated with HMPV at M.O.I. of 3, and at 24 h.p.i; cells were fixed and stained with the plasma membrane marker WGA and immunostained for HMPV N. Scale bar = 10 μm. (E) Table showing range and average diameter of filaments and intercellular extensions. Images were taken for a total of 100 HMPV-infected cells and imageJ analysis tool was used to determine the diameter of branched filaments and intercellular extensions. (F) Cells were inoculated with HMPV, and 24h later, cells were processed for Stochastic Optical Reconstruction Microscopy (STORM) and stained with an anti-N antibody (green) and an anti-M antibody (red). Inset shows filaments with a central core of N protein surrounded by matrix protein. Scale bar = 1 μm. (G) BEAS-2B cells were inoculated with rgHMPV at an M.O.I. of 2, and at different times post infection, cells or culture media were collected and virus titers determined. Graph shows mean ± SD for three independent experiments.