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

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

SSP is vital for glycoprotein complex association. We performed immunoprecipitation (IP) assays to investigate the interaction between SSP and the rest of the GP complex, since mutations within SSP result in fusion activity deficiencies. Previous studies using GP2-targeted IP showed an interaction between the GP2 subunit and SSP (10, 34) . We subjected a panel of point mutations within the SSP FLLL motif, as well as within the FALA GPC and YALL GPC double point mutants, to immunoprecipitation assays using both the HA and the confirmation-specific, neutralizing GP1 subunit 2.11-15 antibodies (Fig. 6 ). Cells transfected with the wild-type GPC, the cotransfected SSP and GP1/2, and the HA SSP GPC resulted in properly processed GP2 subunit detection (Fig. 6A, lanes 2, 4, and 6 ). The GP1/2 missing its native SSP, as well as the cotransfected SSP-HA plus GP1/2, did not result in processed GP2 (Fig. 6A, lanes 3 and 5) . In order to directly assess SSP interactions by IP, we introduced FLLL motif point mutations within the context of the HA SSP GPC open reading frame. The HA SSP GPC 49A point mutant did not result in processed GP2 (Fig. 6A, lane 7) . The HA SSP GPC 49Y, 50A, and 52A point mutants, as well as FALA GPC and YALL GPC double point mutants, all allowed GPC processing and GP2 detection (Fig. 6A, lanes 8 to 12) .