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 and others have shown the importance of the first signal peptide hydrophobic domain for precursor glycoprotein cleavage and pH-dependent membrane fusion (29, 34, 46) . We were able to detect the HA epitope at the cell surface by flow cytometry using live, nonpermeabilized cells, indicating that the amino-terminal portion of SSP upstream of the first hydrophobic domain was presented on the exterior surface of the plasma membrane. This suggests SSP may exhibit more than one membrane orientation, and the lack of fusion activity resulting from the inserted epitope may be interfering with late-state SSP orientation to produce a fully assembled glycoprotein complex (34, 35, 47) . This observation does not conflict with the proposed options for signal peptide hairpin-like orientation within the plasma membrane but merely provides for an alternative orientation during glycoprotein complex maturation and assembly (18, 35, 37) . In order to determine whether the carboxy-terminal region of SSP is genuinely mediating the intracellular trafficking of the glycoprotein complex and not a function of the inserted, foreign epitope, we focused on a highly conserved hydrophobic motif (FLLL) upstream of the signal peptidase recognition signal in the context of the full-length glycoprotein open reading frame. As stated previously, SSP is the most heavily conserved protein throughout the Arenaviridae family, although it is partnered with the GP2 subunit for phylogenetic analyses (7) . Results from the expression of the FLLL GPC mutants indicate that each residue within this motif plays an equal role in glycoprotein maturation cleavage. Additionally, the lack of observed fusion activity observed by the HA SSP GPC indicates a defect in one of the final stages of glycoprotein complex assembly, an observation previously noted using internally tagged Junín SSP cotransfection assays (37) . This lack of fusion activity serves as a limitation for the Previous studies reported that single point mutations within this FLLL region, specifically the phenylalanine at position 49, affect both pH-dependent membrane fusion and glycoprotein infectivity (29, 37) . Additionally, SSP residues at positions 5 and 50, the latter of which resides within this conserved FLLL motif, have been implicated to play roles in LCMV pathogenicity and virus Bound proteins from SSP immunoprecipitation (IP) using anti-HA agarose beads (B) and bound protein from GP1 immunoprecipitation (C) were separated using 12% SDS-PAGE. All membranes were probed for the GP2 (83.6) and SSP (SP7) subunits. For each blot, the "X" denotes where the nitrocellulose membranes were cut for antibody incubation prior to membrane reassembly for image acquisition.