FIG 6.

Transmission electron microscopy of AAV VP3-only capsids produced with or without AAP. HEK 293 cells were transfected with plasmids expressing VP3 proteins and AAP proteins as indicated. At 5 days posttransfection, capsids were purified by three rounds of CsCl density gradient ultracentrifugation. (A, D, G, and I) Negative-image photos and accompanying cartoons showing bands formed after the third CsCl ultracentrifugation. AAV2 VP3 with AAP2 [AAP2(+)] contained two bands at RIs of 1.3630 and 1.3645. The following ELISA indicated that the heavier of the two bands contained more capsids, and this was the band used for TEM imaging. (B and E) Assembled AAV capsids positive for anti-AAV capsid antibody in each CsCl fraction were assessed by an AAV capsid-specific ELISA. Relative optical density (OD) values at 450 nm are plotted against RIs of CsCl fractions. The OD at 450 nm obtained in the peak fraction was set to 1.0. (C, F, H, and J) Representative TEM images of samples prepared with or without AAP. The CsCl fractions showing RIs of 1.3645, 1.3640, 1.3645, 1.3640, 1.3635, 1.3640, 1.3635, and 1.3640 were used for TEM imaging of samples of AAV2 VP3 with and without AAP2 [AAP2(+) and AAP2(−), respectively], AAV5 VP3 with and without AAP5 [AAP5(+) and AAP5(−), respectively], AAV4 VP3 with and without AAP4 [AAP4(+) and AAP4(−), respectively], and AAV11 VP3 with and without AAP11 [AAP11(+) and AAP11(−), respectively]. Bar, 100 nm. (A to C) AAV2; (D to F) AAV5; (G and H) AAV4; (I and J) AAV11.