Mutations on the external surfaces of adeno-associated virus type 2 capsids that affect transduction and neutralization - PubMed (original) (raw)

Comparative Study

Mutations on the external surfaces of adeno-associated virus type 2 capsids that affect transduction and neutralization

Michael A Lochrie et al. J Virol. 2006 Jan.

Abstract

Mutations were made at 64 positions on the external surface of the adeno-associated virus type 2 (AAV-2) capsid in regions expected to bind antibodies. The 127 mutations included 57 single alanine substitutions, 41 single nonalanine substitutions, 27 multiple mutations, and 2 insertions. Mutants were assayed for capsid synthesis, heparin binding, in vitro transduction, and binding and neutralization by murine monoclonal and human polyclonal antibodies. All mutants made capsid proteins within a level about 20-fold of that made by the wild type. All but seven mutants bound heparin as well as the wild type. Forty-two mutants transduced human cells at least as well as the wild type, and 10 mutants increased transducing activity up to ninefold more than the wild type. Eighteen adjacent alanine substitutions diminished transduction from 10- to 100,000-fold but had no effect on heparin binding and define an area (dead zone) required for transduction that is distinct from the previously characterized heparin receptor binding site. Mutations that reduced binding and neutralization by a murine monoclonal antibody (A20) were localized, while mutations that reduced neutralization by individual human sera or by pooled human, intravenous immunoglobulin G (IVIG) were dispersed over a larger area. Mutations that reduced binding by A20 also reduced neutralization. However, a mutation that reduced the binding of IVIG by 90% did not reduce neutralization, and mutations that reduced neutralization by IVIG did not reduce its binding. Combinations of mutations did not significantly increase transduction or resistance to neutralization by IVIG. These mutations define areas on the surface of the AAV-2 capsid that are important determinants of transduction and antibody neutralization.

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Figures

FIG. 1.

FIG. 1.

Structure of AAV-2 capsid and location of mutations. (A) Space-filling diagram of AAV-2 capsid. Amino acids are colored as follows: red, residues D and E; pink, W; brown, P; orange, S and T; yellow, C and M; dark blue, R and K; medium blue, N and Q; light blue, F and Y; light green, H; dark green, I, L, and V; gray, A; and white, G. The black triangle defines the boundary of one asymmetric structural unit. There are 60 asymmetric structural units per capsid. Approximately 145 amino acids out of 735 amino acids in VP1 are on the surface. (B) Positions

FIG. 2.

FIG. 2.

Heparin binding. (A) Heparin binding by capsids with mutations in or near the heparin binding site. Wild-type AAV-2 and wild-type AAV-8 were used as positive and negative controls, respectively. (B) Heparin binding by mutants with mutations in the dead zone. These mutants have <10% of the wild type's transduction in vitro. The R588A mutant and wild-type AAV-2 were used as negative and positive controls, respectively. B, bound to heparin; UB, unbound. Only VP3 is shown.

FIG. 3.

FIG. 3.

Model of AAV-2 capsid binding to a fibroblast growth factor receptor 1/basic fibroblast growth factor/heparin complex. (A) Two trimers of the AAV-2 capsid monomer. Axes of symmetry (2, 3, and 5) are indicated. The AAV-2 capsid is shown in space-filling format and colored as follows: red, acidic amino acid; blue, basic; yellow, polar; and gray, hydrophobic. The dimple is centered over the twofold axis of symmetry and is located at the top of the figure. (B) Docking of a fibroblast growth factor receptor 1/basic fibroblast growth factor/heparin complex into the dimple. The receptor complex is shown in the wire frame format. (C) Location of heparin in the model. Two trimers of the AAV-2 capsid monomer are shown in white and rotated 90° toward the reader relative to their positions in Fig. 3A. The heparin is shown in Corey, Pauling, and Koltun colors. The five basic amino acids that comprise the AAV-2 heparin binding site are colored blue.

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