FIG 2.

PEMV CP and RTD bind aminopeptidase N. (A) Binding of WT PEMV and PEMV RNA1Δ to pea aphid BBMV. No binding was detected for the antibody (no ligand) control. (B) Binding of CP-P-GFP and GFP-RTD to pea aphid BBMV. Nonspecific binding of the GFP antibody to aphid BBMV proteins was detected in blots with CP-P-GFP or GFP as ligand. In panels A and B, arrows indicate consistent binding. Far-Western blotting conducted using pH 3 to 10 and pH 4 to 7 was replicated at least four times per ligand. (C) PEMV binding of APN using a pulldown assay. PEMV was labeled with a biotin transfer reagent to UV cross-link proteins bound to PEMV. The PEMV-APN complex was pulled down using streptavidin-agarose beads, and APN was detected by Western blotting with APN antiserum (at left). The amount of APN pulled down by PEMV averaged 4.5-fold more than the control lacking PEMV across four replicates (Student t test P = 0.025; as determined by the ImageJ program [at right]). In panels A to C, the positions of molecular mass standards are indicated on the left. (D) BIAcore SPR analysis of CP-P-GFP, GFP-RTD, GFP interactions with APN. Sensorgram showing real-time interactions between 6 μM concentrations of GFP and the GFP fusion proteins with immobilized APN-GPI(−) are shown. L1 chip surfaces were prepared with 2,000 RU of APN-GPI(−). The data shown are representative of two independent experiments.