The signal peptide of GP5 from different PRRSV type 2 strains is cleaved.

<p>(A), N-terminal sequences (residues 1–60) of representative PRRSV type 2 strains (VR-2332: prototype strain, MLV: modified live vaccine “RespPRRS” (containing just one exchange – R13Q – in the signal peptide/ectodomain region relative to VR-2332), JXA-1: Chinese virulent strain, Neb-1: US-American virulent strain) with potential glycosylation sites (grey), predicted signal peptide (black), and propensity of signal peptide cleavage (D score according to SignalP 4.0, <a href="http://www.cbs.dtu.dk/services/SignalP/" target="_blank">www.cbs.dtu.dk/services/SignalP/</a>). Note that the difference in signal peptide cleavage at site 1 and 2 was small as shown in detail in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065548#pone.0065548.s001" target="_blank">Table S1</a>. (B/C), MARC-145 cells were transfected with GP5–HA with the signal peptide/ectodomain sequence as depicted in (A), or with empty plasmid (Ø), subsequently lysed and assessed by SDS-PAGE and Western blot (anti-HA tag) before (B) and after (C) PNGase F digestion to remove glycans. Labels as in Fig. 2; the thin black arrow denotes additional glycosylation partially achieved in JXA-1; the number of glycans in the mature proteins is indicated on the bottom. The black arrowhead in (C) indicates the position of deglycosylated GP5–HA without signal peptide.