Severe Acute Respiratory Syndrome Coronavirus Nonstructural Proteins 3, 4, and 6 Induce Double-Membrane Vesicles

Angelini, Megan M.; Akhlaghpour, Marzieh; Neuman, Benjamin W.; Buchmeier, Michael J.

Western blotting assays. HEK293T cells were grown in 6-well plates and lysed 24 h posttransfection using either radioimmunoprecipitation assay (RIPA) or 1% NP-40 lysis buffer with 1Ï« protease inhibitor cocktail (Research Products International Corp). Lysates were subjected to SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane for immunoblotting. Electron microscopy and phenotype quantification. Cells were grown in T-75 flasks, transfected, fixed 24 h posttransfection, and harvested with 2% EM-grade glutaraldehyde in 0.1 M sodium cacodylate buffer for at least 4 h, postfixed in 1% osmium tetroxide-0.1 M cacodylate buffer for 1 h, and stained in 2% uranyl acetate en bloc for 1 h. Samples were dehydrated in ethanol, embedded in epoxy resin, sectioned at intervals of 50 to 60 nm on a Leica UCT ultramicrotome, and picked up on Formvar and carbon-coated copper grids. Sections were stained with 2% uranyl acetate for 5 min and with Sato's lead stain for 1 min. Grids were viewed using either a Tecnai G 2 Spirit BioTWIN transmission electron microscope equipped with an Eagle 4k high-sensitivity (HS) digital camera (FEI, Hillsboro, OR) or a Phillips CM-20 camera equipped with a 2k charge-coupled device (CCD).