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.

Recombinant AAV vectors containing the foot and mouth disease virus 2A sequence confer efficient bicistronic gene expression in cultured cells and rat substantia nigra neurons

“…[6][7][8][9][10][11][12][13][14] Recently, progress has been made in identifying positions within the AAV capsid proteins that tolerate small insertions of targeting ligands without significantly affecting resultant viral titers. [15][16][17] Moreover, rAAV genomes can be packaged into capsids derived from other AAV serotypes, rendering them more efficient in infecting certain cell types. 18,19 It is expected that these developments and ongoing efforts to obtain detailed structural information on the AAV capsid will expand the number of tissues and cells that can be targeted by AAV in the future.…”

Monoclonal Antibodies against the Adeno-Associated Virus Type 2 (AAV-2) Capsid: Epitope Mapping and Identification of Capsid Domains Involved in AAV-2–Cell Interaction and Neutralization of AAV-2 Infection

“…However, conflicting results have raised doubts about the general role of these coreceptors in the AAV-2 infection process (28,29). Analysis of insertion mutants of AAV-2 capsids suggests that the heparin binding site resides within the VP3 portion of the capsid proteins (30). After binding, AAV-2 enters the cell by a dynamin-dependent endosomal pathway (4,10).…”

Adeno-associated virus vectors: potential applications for cancer gene therapy

“…89 A second approach for receptor targeting is to genetically alter the capsid-coding region. Prior to the elucidation of the AAV crystal structure, three strategies have been employed to determine domains of AAV surface that can be modified: 54 (a) sequence alignment of AAV2 and other parvoviruses with known crystal structure, 90,91 (b) randomly insertional mutagenesis of the entire AAV-2 capsid genome, [92][93][94] and (c) incubation of AAV2 neutralizing serum with AAV2 capsid peptide pools to define the peptides which represent immunogenic regions on the virion surface. 95 By aligning the capsid sequences of AAV2 and CPV, Girod et al predicted six sites (amino-acid positions 261, 381, 447, 534, 573, 587) that could tolerate the insertion of a targeting ligand.…”

“…97,98 The identification of the regions located on the surface of the capsid and the corresponding amino acids responsible for heparin sulfate binding is instrumental for subsequent genetic modifications to the AAV vector. To this end, both Rabinowitz et al 93 and Wu et al 92 have used a random site-directed mutagenesis approach, which has provided a wealth of information for later structural study of AAV2.…”