Arenavirus Stable Signal Peptide Is the Keystone Subunit for Glycoprotein Complex Organization

Bederka, Lydia H.; Bonhomme, Cyrille J.; Ling, Emily L.; Buchmeier, Michael J.

Immunofluorescence. DBT cells were plated onto flame-sterilized glass coverslips and transfected using Lipofectamine 2000 (Life Technologies). After 48 h of incubation, cells were fixed using 3.7% paraformaldehyde (PFA) at room temperature and, when relevant, permeabilized with PBS and 0.1% Triton X-100 for 5 min. Cells were blocked using 5% bovine serum albumin (BSA) in PBS-Tween 20 for 1 h prior to overnight primary antibody incubation at 4°C. The phalloidin-fluorescein isothiocyanate (FITC) and Alexa Fluor 405-, 488-, and 594-conjugated secondary antibodies (Life Technologies) were used to detect viral or cellular epitopes. For the triple labeling microscopy, cells were incubated with phalloidin-FITC overnight, followed by mouse anti-HA (Sigma) for an additional overnight staining prior to 1 h of incubation with Alexa Fluor 405. Ultimately, cells were stained with GP1 antibody (2.11-10) directly conjugated to Alexa Fluor 594 (Life Technologies). For the SSP-HA and GP1/2 and the HA SSP GPC samples, coverslips were mounted with a mounting medium lacking DAPI (4=,6-diamidino-2-phenylindole), while other samples used DAPI-containing mounting medium (Southern Biotechnologies). The mannosidase II antibody was kindly provided by K. Moreman (University of Georgia). Confocal microscopy images were acquired using the Nikon Eclipse Ti microscope. All images were processed linearly using ImageJ and cropped for assembly using Adobe Photoshop. Colocalization measurements were acquired using the ImageJ colocalization finder plugin (http://rsb.info.nih.gov/ij/plugins/colocalizationfinder.html).