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 C-terminal region is essential for GPC processing into GP1 and GP2 subunits. A native antibody directed against SSP that is applicable for immunofluorescence microscopy does not exist, as the SP7 antibody detects only SSP under denatured and reduced conditions (18, 34) . We used a C-terminal hemagglutinin-tagged SSP (SSP-HA) plasmid construct to assess the significance of the SSP for GPC maturation. This construct was previously used to define SSP membrane topology, SSP's role in pH-dependent fusion, and interactions between SSP and the Z matrix protein (35) (36) (37) (38) . Western blot analysis of HEK 293T cell lysates cotransfected with SSP-HA and the glycoprotein precursor lacking its cognate SSP (GP1/2) resulted in expression of SSP and the GPC but lacked the processed GP2 (Fig. 2) . Digestion of transfected cell lysates with the peptide--N-glycosidase F (PNGase F) enzyme to rid N-linked glycans confirmed the GPC was glycosylated (see Fig. S1 in the supplemental material). Detection of the GP2 subunit was rescued upon cotransfection with wild-type SSP, suggesting the HA epitope interfered with SSP-GPC interactions needed for appropriate precursor processing. Since the presence of the epitope at the C terminus interfered with proper glycoprotein complex expression, we inserted the HA epitope in the N-terminal SSP region of the entire glycoprotein open reading frame, downstream of the myristoylation motif (HA SSP GPC). This internally tagged glycoprotein allowed for in cis detection of and experimentation with SSP relative to the remaining glycoprotein subunits. In trans expression of SSP-HA and GP1/2 allowed for precursor cleavage and the detection of the processed GP2 subunit, although this SSP, along with the wild-type SSP, was more rapidly degraded when provided on separate plasmids (Fig. 2) .