Figure 7

Trypsin protection assay on LASV Z+GPC+NP VLP. LASV VLP expressing Z, GPC, and NP proteins were subjected to trypsin protection assays to assess the enveloped nature of pseudoparticles and compartmentalization of viral proteins. LASV VLP incorporated unprocessed 75 kDa GPC precursor (7A, 7B, lane 1), and monomeric 42 kDa GP1 (7A, lane 1), and 38 kDa GP2 (7B, lane 1). LASV VLP also incorporated trimerized, non-reduceable 126 kDa GP1 isoforms (7A, lane 1), and 114 kDa GP2 trimers to a lesser extent (7B, lane 1). For trypsin protection assays ten μg of LASV VLP were either left untreated (lane 1), treated with 3 mg/mL soybean trypsin inhibitor (lane 2), 1% Triton X-100 (lane 3), 100 μg/mL trypsin (lane 4), 1% Triton X-100 and 100 μg/mL trypsin (lane 5), or 100 μg/mL trypsin in the presence of 3 mg/mL soybean trypsin inhibitor (lane 6). Trypsin alone completely digested trimerized GP1 (7A, lane 4) and GP2 (7B, lane 4), while partially degrading GPC precursor, but having little effect on monomeric glycoproteins. Trypsin treatment of intact VLP did not significantly affect the levels of NP (7C, lane 4), and Z (7D, lane 4) proteins. Treatment of VLP with Triton X-100 in the presence of trypsin resulted in the complete digestion of NP (7C, lane 5) and Z (7D, lane 5), while only partially degrading monomeric GP1 (7A, lane 5) and GP2 (7B, lane 5) proteins. Treatment of VLP with trypsin in the presence of soybean trypsin inhibitor completely prevented digestion of any form of all viral proteins (7A - 7D, lane 6).