Protease activity is VP1 and neutral pH dependent. (A) Colorimetric readout (y-axis) over time (hr, x-axis) for a casein substrate degradation by AAV5 capsids assembled from VP1, VP2 and VP3 or only VP2 and VP3. (B) Same as in (A) but for AAV5 VP1, VP2 and VP3 capsids at pH 7.4 and 5.5. (C) SDS-PAGE of AAV2 VLPs, containing the VPs shown, incubated with a casein substrate at 37 °C for 2, 4 and 6 h. Observations confirm the need to have VP1 and physiological pH for activity. (D) Superposition of AAV2 (blue) and AAV5 (gray) VP1u models. N-terminal domains of models with predicted disorder not shown.