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 mediates glycoprotein precursor processing. The glycoprotein complex is expressed as a polyprotein that undergoes two cleavage events (Fig. 1A) . SSP retains a high degree of sequence conservation across Old World and New World signal peptides ( Fig. 1B ; see also Fig . S1 in the supplemental material) (29) . This consensus alignment excludes the boa constrictor-derived arenaviruses, since this group of distantly related viruses expresses a filovirus-like glycoprotein (30) . SSP is a key component of the glycoprotein complex that is not recycled within the endoplasmic reticulum but is retained as an essential subunit within purified virions (Fig. 1C ). Previous studies have demonstrated a role of basic amino acids within transmembrane protein cytoplasmic domains for the intracellular transport and processing of proproteins into their mature forms (12, (31) (32) (33) . We introduced alanine point mutations in the final two residues within the LCMV GP2 subunit and tested the ability of these constructs to produce a cleaved GP2 subunit, since GPC cleavage into GP1 and GP2 precedes membrane transport (29) . Wild-type (WT) GPC with the RR-to-AA mutation, along with the in trans expression of wildtype SSP, resulted in the accumulation of the precursor GPC, which moderately affected levels of processed GP2, as quantified by densitometry analysis (Fig. 1D) . The GP1/2 RR-to-AA mutant lacking the native SSP did not produce the cleaved GP2 subunit. We used flow cytometry to measure the surface expression of the glycoprotein using live cells stained with a conformation-specific GP1 antibody (Fig. 1E) . Mutations within the WT GPC did not alter the amount of surface GP1 expression. The GP1/2 and the GP1/2 RRAA samples failed to produce a GP1 signal above background levels. Cotransfection of wild-type SSP with both the WT GPC RR-to-AA and with the GP1/2 RR-to-AA protein restored both GP2 processing and surface expression of GP1, suggesting the dibasic motif within the LCMV glycoprotein is not the driving force for secretory pathway trafficking, but rather this function resides within SSP.