Proteolytic mapping of the adeno-associated virus capsid.
The three-dimensional structures of the viral capsid of three AAV serotypes have previously been determined by X-ray crystallography or cryoelectron microscopy. These studies of AAV and similar studies of autonomous parvoviruses have yielded important structural information about the virions in a low-energy conformation. However, there is little information on the structural properties of AAV virions in solution under physiological conditions. We demonstrate that proteolytic digestion of AAV2 virions with trypsin results in cleavage at a specific site on the capsid surface while the capsid remains intact. The products of digestion were mapped using unique antibodies, protein sequencing, mass spectroscopy, and 3D structure modeling to a region on a surface loop that is common to all three AAV2 structural proteins. Empty AAV2 capsids could be distinguished from full (DNA-containing) capsids, having an increased susceptibility of VP2 to trypsin and being digested more rapidly by chymotrypsin. Proteolytic analysis utilizing trypsin or chymotrypsin was also capable of distinguishing AAV2 from AAV1 and AAV5, as seen by differential susceptibility and unique fragment patterns. These data demonstrate a novel approach for studying the structure of AAV capsids in solution and should be valuable in the testing and engineering of AAV vectors for gene transfer.
Van Vliet
Kim
K
Department of Molecular Genetics and Microbiology, University of Florida, College of Medicine, 1600 SW Archer Road, Gainesville, FL 32610-0266, USA.
Blouin
Veronique
V
Agbandje-McKenna
Mavis
M
Snyder
Richard O
RO
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002213:Capsid / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D036022:Capsid Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D002918:Chymotrypsin / Q000378:metabolism; D000229:Dependovirus / Q000378:metabolism* / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D015151:Immunoblotting; D046529:Microscopy, Electron, Transmission; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D014357:Trypsin / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D014771:Virion / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure
D036022:Capsid Proteins; D014764:Viral Proteins; D006493:Heparin; D010450:Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin