title: Insertional mutagenesis of AAV2 capsid and the production of recombinant virus.
abstract: The structural genes of adeno-associated virus serotype 2 (AAV2) have been altered by linker insertional mutagenesis in order to define critical components of virion assembly and infectivity. An in-frame restriction site linker was inserted across the capsid coding domain of a recombinant plasmid. After complementation in vivo, recombinant AAV2 viruses were generated and assayed for capsid production, packaging, transduction, heparin agarose binding, and morphology. Three classes of capsid mutants where identified. Class I mutants expressed structural proteins but were defective in virion assembly. Class II mutants generated intact virions that protected the viral genome from DNase, but failed to infect target cells. The majority of these mutants bound the heparin affinity matrix, suggesting that attachment to the AAV primary receptor was not rate limiting. One class II mutant, H2634, assembled virions and bound heparin using only Vp3, indicating that this subunit is responsible for mediating AAV receptor attachment. Finally, class III mutants assembled virions, encapsidated DNA, and infected target cells. Infectivity of these mutants ranged from 5 to 100% of that of the wild-type, demonstrating for the first time the ability to alter capsid proteins without interfering with infectivity. These AAV virions with altered capsid subunits will provide critical templates for manipulating AAV vectors for cell-specific gene delivery in vivo. In summary, the AAV capsid variants described here will facilitate further study of virus assembly, entry, and infection, as well as advance the development of this versatile vector system.
authors.affiliation: The Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
authors.forename: J E
authors.initials: JE
authors.lastname: Rabinowitz
authors.forename: W
authors.initials: W
authors.lastname: Xiao
authors.forename: R J
authors.initials: RJ
authors.lastname: Samulski
mesh_terms: D002213:Capsid / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D036022:Capsid Proteins; D002461:Cell Line, Transformed; D000229:Dependovirus / Q000235:genetics / Q000254:growth & development* / Q000502:physiology / Q000648:ultrastructure; D006367:HeLa Cells; D006493:Heparin / Q000378:metabolism; D006801:Humans; D016254:Mutagenesis, Insertional; D011995:Recombination, Genetic; D014771:Virion; D014779:Virus Replication
chemical_list: D036022:Capsid Proteins; C442553:VP2 protein, Dependovirus; C442558:VP3 protein, Dependovirus; D006493:Heparin