FIG. 3.

Localization of Lassa virus glycoprotein GP in polarized epithelial cells. (A) MDCK-II cells were infected with LASV at an MOI of 1 from the basolateral cell surface. At 72 h p.i. cells were fixed with methanol-acetone and subsequently treated with 4% PFA for 48 h. The LASV glycoprotein subunit GP-1 (red) and the tight-junction resident protein ZO-3 (green) were immunostained. Distributions of GP and ZO-3 were monitored by using confocal laser scanning microscopy in the xy and yz directions. The xy scan is a composite of several images from the apical and middle regions of the image stack because of the irregular height of the cells. The scale bars denote 20 μm. (B) Immunodetection of GP in a stably GP-expressing nonpermeabilized MDCK-II cell line. Cells were fixed with 4% paraformaldehyde and immunostained using an α-GP-2-N antibody (red). The scale bars denote 20 μm. (C) Proteins on the cell surface of stably LASV GP- and VSV G-expressing MDCK-II cells were labeled with sulfo-NHS-biotin on either the apical or basolateral side. After cell lysis, biotinylated proteins were precipitated using streptavidin-coupled Sepharose beads and subsequently subjected to SDS-PAGE and immunoblot analysis using α-GP-2-C or α-VSV G antiserum.