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

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

propagation (48) . Our microscopy results from the FALA GPC and YALL GPC mutants revealed defects in the progression through the secretory pathway. Levels of processed GP2 from the FALA GPC increased with the addition of supplemented wildtype SSP, although the presence of additional SSP failed to enhance the fusion activity of the glycoprotein. The FALA GPC and the YALL GPC, though producing the cleaved GP2 subunit, both resulted in significant ER and Golgi staining, as well as a greater than 50% reduction in both fusion activity and GP1 surface expression, indicating a defect in post-Golgi intracellular transport. The reduction in membrane localization exhibited by these mutants at 48 h posttransfection indicates an impaired maturation process, as the intracellular staining patterns observed were merely nascent glycoprotein at intermediate maturation stages, as transfection efficiencies were comparable under all conditions. Defects observed by mutating the FLLL motif indicate that this SSP functions as a sorting signal for secretory pathway trafficking. In regard to the AALL GPC and the YLAL GPC, these two mutants lacked the ability to produce a glycosylated precursor GPC. Results from the GP1/2 expression, where the glycoprotein completely lacks its native SSP, demonstrated SSP was not required for the posttranslational N-linked glycan modification. AALL GPC and YLAL GPC produced a defective, nonfunctional protein whereby the presence of in trans wild-type SSP was unable to rescue downstream functions for cleavage and transport. These two GPC variants, especially the YLAL GPC, were cytotoxic, likely due to the induction of ER stress responses, as these two mutant GPCs lacked the ability to traffic to the Golgi stacks for precursor processing. In trans SSP expression was able to moderately rescue the ALLA GPC processing, since this mutant GP did produce a glycosylated precursor, thus allowing SSP to provide some chaperone function to an otherwise able (glycosylated) precursor. Based on our fusion assay results, the levels of processed GP2 from the cotransfection of wild-type SSP with the ALLA GPC were not enough to rescue a significant amount of functional glycoprotein. For the FALA GPC and the YALL GPC mutants, the decrease in fusion activity with the in trans wild-type SSP indicated a defect in SSP-mediated transport. This fusion activity defect was likely mediated by ER stress due to unequal ratios and overabundance of SSP relative to the rest of the glycoprotein complex.