Journal: Journal of Virology
Article Title: Relevance of Assembly-Activating Protein for Adeno-associated Virus Vector Production and Capsid Protein Stability in Mammalian and Insect Cells
Figure Lengend Snippet: Proof that AAP fosters assembly of capsids of multiple AAV serotypes. (A) Capsids of the eight AAV serotypes shown were produced by cotransfecting HEK293T cells with the corresponding wild-type or AAP-deficient (mutant) AAV helper, an AAV vector, and an adenoviral helper. Following freeze-thaw lysis at 3 days posttransfection, capsid-containing supernatants were spotted onto nitrocellulose membranes under nondenaturing conditions. In every other lane, the capsids were heat denatured via incubation at 95°C for 5 min. Intact capsids were detected with the ADK series of antibodies (ADK8 cross-reacts with AAV3) or A20 (for AAV2). For most of the shown AAV serotypes (AAV2, -3, -6, -8, and -9), lack of AAP prevented formation of assembled capsids. Arrows highlight exceptions, i.e., signals detected with the ADK antibodies for AAV1, -4, and -5 even in the absence of AAP. Note that low or no signals were detected with the VP serum or B1 antibody following heat denaturation of the same samples (arrowhead). The blots shown are representative of three independent experiments, which all gave similar results. (B) Further investigation of the three exceptions from panel A, i.e., AAV serotypes 1, 4, and 5. In lanes S, the samples were not only heated but additionally treated with SDS (see Materials and Methods for details). The arrowhead highlights the particular resistance of the AAP-independent AAV5 structures even to these harsh denaturing conditions. (C) Stabilization of free VP proteins by MG-132 treatment does not enhance capsid assembly. Capsid were produced, blotted, and detected as described for panel A. Instead of heat and/or SDS denaturation, samples in lane MG were derived from cells that were treated with the proteasome inhibitor MG-132 during vector production. At 5 and 24 h after transfection, the medium was changed to fresh medium containing MG-132, and 48 h later, supernatants from the lysed cells were analyzed. Note that AAV4 and AAV5, as well as, to a lesser extent, AAV1 (and AAV6), gave signals even in the absence of AAP, independently confirming the data in panels A and B. The blots shown are representative of two independent experiments, which gave similar results.
Article Snippet: Sixteen hours posttransfection, cells were treated with MG-132 (final concentration, 2 μM; Santa Cruz Biotechnology).
Techniques: Produced, Mutagenesis, Plasmid Preparation, Lysis, Incubation, Derivative Assay, Transfection