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

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

We introduced a panel of double point mutations within this FLLL motif in order to determine whether any one amino acid was key for a processed and mature GP complex. The AALL GPC was defective in its ability to allow posttranslational modifications and revealed a dominant negative maturation phenotype, since the addition of wild-type SSP did not rescue glycosylated glycoprotein expression or processing ( Fig. 4A ; see Fig. S2 in the supplemental material). The ALLA GPC supported precursor GP posttranslational modifications but was defective in producing a processed GP2 subunit. The ALLA GPC produced a cleavage GP2 subunit with the in trans addition of wild-type SSP (Fig. 4B ). The FALA GPC retaining the conserved phenylalanine encoded by all known rodent arenaviruses and resulted in a reduced level of GP2 cleavage from the precursor GPC, while levels of GP2 were increased with the cotransfection of the wild-type SSP. Densitometry analysis of the FALA GPC and wild-type SSP cotransfection, from three independent replicates, resulted in an overall increase in the amount of GPC processed to yield the GP2 subunit, while the representative Western blot in Fig. 4A does not highlight this slight rescue in GP2 processing (Fig. 4B ). The YALL GPC resulted in the highest percentage of processed glycoprotein of all SSP mutants we analyzed, though not at the level of the wild-type GP2 cleavage. Interestingly, the cotransfection of the wild-type SSP with YALL GPC resulted in the reduced expression of the total glycoprotein complex. The final variant we examined, YLAL GPC, was toxic to transfected cells and consistently yielded low levels of detectable glycoprotein. Overall, double point mutations within this conserved SSP motif indicate that this entire motif, and not one residue in particular, plays a dominant role in downstream processes that mediate processing of the precursor into mature glycoprotein subunits.