Mutational analysis of the adeno-associated virus type 2 (AAV2) capsid gene and construction of AAV2 vectors with altered tropism

Adeno-associated virus type 2 (AAV2) has proven to be a valuable vector for gene therapy. Characterization of the functional domains of the AAV capsid proteins can facilitate our understanding of viral tissue tropism, immunor eactivity, viral entry, and DNA packaging, all of which are important issue s for generating improved vectors. To obtain a comprehensive genetic map of the AAV capsid gene, we have constructed 93 mutants at 59 different positi ons in the AAV capsid gene by site directed mutagenesis. Several types of m utants were studied, including epitope tag or ligand insertion mutants, ala nine scanning mutants, and epitope substitution mutants. Analysis of these mutants revealed eight separate phenotypes. Infectious titers of the mutant s revealed four classes. Class 1 mutants were viable, class 2 mutants were partially defective, class 3 mutants were temperature sensitive, and class 4 mutants were noninfectious. Further analysis revealed some of the defects in the class 2, 3, and 4 mutants. Among the class I mutants, a subset comp letely abolished capsid formation. These mutants were located predominantly , but not exclusively, in what are likely to be P-barrel structures in the capsid protein VP3. Two of these mutants were insertions at the N and C ter mini of VP3, suggesting that both ends of VP3 play a rule that is important for capsid assembly or stability. Several class 2 and 3 mutants produced c apsids that were unstable during purification of viral particles. One mutan t, R432A, made only empty capsids, presumably due to a defect in packaging viral DNA. Additionally, five mutants were defective in heparan binding, a step that is believed to be essential for viral entry. These were distribut ed into two amino acid clusters in what is likely to be a cell surface loop in the capsid protein VP3. The first cluster spanned amino acids 509 to 52 2; the second was between amino acids 561 and 591. In addition to the hepar an binding clusters, hemagglutinin epitope tag insertions identified severa l other regions that mere on the surface of the capsid. These included inse rtions at amino acids 1, 34, 138, 266, 447, 591, and 664. Positions 1 and 1 38 were the N termini of VP1 and VP2, respectively; position 34 was exclusi vely in VP1; the remaining surface positions were located in putative loop regions of VP3. The remaining mutants, most of them partially defective, we re presumably defective in steps of viral entry that were not tested in the preliminary screening, including intracellular trafficking, viral uncoatin g, or coreceptor binding. Finally, in vitro experiments showed that inserti on of the serpin receptor ligand in the N-terminal regions of VP1 or VP2 ca n change the tropism of AAV. Our results provide information on AAV capsid functional domains and are useful for future design of AAV vectors for targ eting of specific tissues.