Fig 2. pX mediates ESCRT-dependent extracellular release of assembled EPN-pX nanocages and noncytopathic p16 virus.

(A) (top) EPN-pX, in which pX is fused to the C-terminus of the nanocage protein with an intervening Myc tag. (bottom) pX sequence in EPN-pX and related mutants. The pentamer assembly domain (PAD) [21] and YxxL motif are indicated. Δ1, Δ2 and Δ3 deletions were described previously [23]. (B) Representative electron micrograph showing multiple EPN-pX nanocages (white arrows) within a membrane-bound vesicle (black arrow) in extracellular fluids of transfected 293T cells. Scale bar = 20 nm. The inset shows nanocages in extracellular fluids treated with CHAPS. Scale bar = 30 nm. (C) pX and Myc immunoblots of anti-pX immunoprecipitates of extracellular fluids, with and without NP-40 treatment, versus lysate from EPN-pX transfected cells. (D) Myc immunoblot of released EPN protein recovered following centrifugation through a 20% sucrose cushion, with soluble and insoluble intracellular EPN proteins expressed by cells transfected with EPN-pX and related constructs shown below the solid horizontal line. Cells were co-transfected with vectors expressing eGFP (control, lanes 1–6) or the E228Q VPS4A mutant (lanes 7–12). (E) Quantitation of nanocage protein release normalized to intracellular protein expression. Results are means ±S.E.M. from 3 independent experiments. ****p<0.0001 by ANOVA with Sidak’s multiple comparison test. (F) Protein-fragment complementation assay with (left) VP1pX and (right) pX GLuc2 baits with and without YxxL/A substitutions and GLuc1 prey fused to ESCRT components. Results are means ± S.D. from two independent experiments, each with 3 technical replicates. ***p<0.001 by two-way ANOVA with Sidak’s multiple comparison test. (G) Extracellular HAV RNA determined by RT-PCR [genome equivalents (GE) per mL supernatant fluids] and intracellular HAV RNA [GE per μg total RNA], and calculated extracellular/intracellular RNA ratios, 12 days after transfection of cells with p16 or p16-YxxL/A RNA; GE = genome equivalents. Data are from n = 6 cultures from two transfections, and are representative of multiple independent experiments. **p<0.01 calculated by Mann-Whitney test.