FIG. 4.

Release of spike-containing virus-like particles due to solitary expression of Lassa virus glycoprotein GP. (A) Supernatants of stably LASV glycoprotein-expressing MDCK-II cells were collected and pelleted though a 20% sucrose cushion. The pellets were resuspended, fixed with 4% PFA, adsorbed on Formvar-coated copper grids, and negatively stained with 2% phosphotungstate acid before electron microscopic analysis. Scale bars denote 100 nm. (B) Size comparisons of LASV particles and GP-VLPs were performed by electron microscopy using 50 particles each. Particles were measured from the tips of glycoprotein spikes and sorted in size fractions differing by 20 nm. (C) A protease protection assay was performed with three aliquots of a VLP suspension harvested as described for panel A. One aliquot of GP-VLP suspension was left untreated (lane 1), and the second and third aliquots were incubated with proteinase K (lanes 2 and 3). The third aliquot (lane 3) was additionally treated with Triton X-100. The proteins of all three samples were subjected to SDS-PAGE and immunoblotting. LASV glycoprotein was identified by immune detection using the α-GP-2-C antibody.