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

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

For construction of the HA SSP GPC plasmid, the HA epitope was inserted between amino acids, A 10 and L 11 , in the SSP region of the fulllength glycoprotein open reading frame. The plasmid encoding only the tagged SSP was created by introduction of two consecutive stop codons at amino acids 59 and 60, the first two residues of the GP1 subunit, and immediately downstream of the SPase recognition site. Both these mutants were initially produced using the pTargeT plasmid and were shuttled into the pCAGGS backbone as stated above. SSP alignment. Amino acid sequences used to highlight the signal peptide conservation were aligned using CLC Sequence Viewer 6 and using the following arenaviruses: Western blotting. Transfected cells were incubated for 48 h, washed with 1ϫ phosphate-buffered saline (PBS) and 1 mM EDTA, collected (600 ϫ g, 4°C, 15 min), and lysed on ice with 1% NP-40 lysis buffer and complete protease inhibitors (RPI Corp.). Clarified lysates (10,000 ϫ g, 4°C, 15 min) were separated under reduced and denatured SDS-PAGE conditions and transferred to nitrocellulose membranes. For LCMV glycoprotein complex detection, the anti-GP2 (83.6) and anti-SSP (SP7) antibodies have been described previously (18, 55, 56) . We also used anti-HA (Sigma), anti-actin (Millipore), goat anti-mouse horseradish peroxidase (HRP), and goat anti-rabbit HRP (Jackson ImmunoResearch) antibodies. Blots were processed using the Amersham ECL Prime detection reagents (GE Healthcare) and were developed using the Bio-Rad ChemiDoc XRS system. Densitometry quantification was performed using ImageJ (http://imagej.nih.gov/ij/).