Figure 5.

Novel MdRVp10 forms a cystine nose fusion peptide and traffics to the plasma membrane but is non-fusogenic. (A) Sequence alignments of the p10 FAST proteins encoded by the indicated fusogenic orthoreovirus species (Nelson Bay orthoreovirus (NBV), AF218360; ARV, AF218358; ARVN, AB914766) and two MdRVn isolates of ARV MdRVn-J18 (Md18, AFV52275) and MdRVn-Ych (MdYc, QEQ13297). The indicated hallmark p10 features are labeled as in Figure 3. The conserved cysteine residues are highlighted in yellow, completely conserved amino acids are highlighted in cyan, and residues conserved in four of five alignments are shaded green. Basic amino acids within the PB motif are in red boldface. (B) Transfected QM5 cells expressing ARV or MdRV p10 were fixed and Giemsa stained at 48h post-transfection to detect syncytium formation by bright field microscopy at 100x magnification. (C) QM5 cells expressing N-terminally FLAG-tagged versions of the indicated p10 constructs were labeled at 24 h post-transfection using membrane-impermeable Sulfo-NHS-LC-Biotin and biotinylated surface proteins were isolated using neutravidin beads and detected by Western blotting using an anti-FLAG antibody. (D) QM5 cells expressing N-terminally Myc-tagged versions of the indicated p10 constructs were labeled at 24 h post-transfection with membrane-impermeable maleimide-PEG2-biotin to detect free thiol groups on the cell surface, with or without prior treatment with 0.1 mM dithiothreitol (DTT) to reduce disulfide bonds. Biotinylated proteins were isolated using neutravidin beads and detected by Western blotting using an anti-Myc antibody.