title: Properties of the adeno-associated virus assembly-activating protein.
abstract: Adeno-associated virus (AAV) capsid assembly requires expression of the assembly-activating protein (AAP) together with capsid proteins VP1, VP2, and VP3. AAP is encoded by an alternative open reading frame of the cap gene. Sequence analysis and site-directed mutagenesis revealed that AAP contains two hydrophobic domains in the N-terminal part of the molecule that are essential for its assembly-promoting activity. Mutation of these sequences reduced the interaction of AAP with the capsid proteins. Deletions and a point mutation in the capsid protein C terminus also abolished capsid assembly and strongly reduced the interaction with AAP. Interpretation of these observations on a structural basis suggests an interaction of AAP with the VP C terminus, which forms the capsid protein interface at the 2-fold symmetry axis. This interpretation is supported by a decrease in the interaction of monoclonal antibody B1 with VP3 under nondenaturing conditions in the presence of AAP, indicative of steric hindrance of B1 binding to its C-terminal epitope by AAP. In addition, AAP forms high-molecular-weight oligomers and changes the conformation of nonassembled VP molecules as detected by conformation-sensitive monoclonal antibodies A20 and C37. Combined, these observations suggest a possible scaffolding activity of AAP in the AAV capsid assembly reaction.
authors.affiliation: German Cancer Research Center, Research Program Infection and Cancer, Heidelberg, Germany. m.naumer@dkfz.de
authors.forename: Matthias
authors.initials: M
authors.lastname: Naumer
authors.forename: Florian
authors.initials: F
authors.lastname: Sonntag
authors.forename: Kristin
authors.initials: K
authors.lastname: Schmidt
authors.forename: Karen
authors.initials: K
authors.lastname: Nieto
authors.forename: Christian
authors.initials: C
authors.lastname: Panke
authors.forename: Norman E
authors.initials: NE
authors.lastname: Davey
authors.forename: Ruth
authors.initials: R
authors.lastname: Popa-Wagner
authors.forename: Jürgen A
authors.initials: JA
authors.lastname: Kleinschmidt
mesh_terms: D000911:Antibodies, Monoclonal; D002213:Capsid / Q000378:metabolism* / Q000648:ultrastructure; D036022:Capsid Proteins / Q000235:genetics* / Q000378:metabolism; D000229:Dependovirus / Q000378:metabolism* / Q000648:ultrastructure; D015151:Immunoblotting; D047468:Immunoprecipitation; D008854:Microscopy, Electron; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D017422:Sequence Analysis, DNA; D019065:Virus Assembly / Q000235:genetics*
chemical_list: D000911:Antibodies, Monoclonal; D036022:Capsid Proteins