Mutations on the external surfaces of adeno-associated virus type 2 capsids that affect transduction and neutralization
Mutations on the external surfaces of adeno-associated virus type 2 capsids that affect transduction and neutralization
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DOI：
10.1128/jvi.80.2.821-834.2006
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发表时间：
2006-01-01
影响因子：
5.4
通讯作者：
Colosi, P
中科院分区：
文献类型：
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作者：
Lochrie, MA;Tatsuno, GP;Colosi, P
在2型腺相关病毒（AAV-2）衣壳外表面预期结合抗体的区域的64个位置进行突变。127个突变包括57个单丙氨酸取代，41个单非丙氨酸取代，27个多突变和2个插入。测定突变体的衣壳合成、肝素结合、体外转导以及鼠单克隆和人多克隆抗体的结合和中和。所有突变体的衣壳蛋白的水平约20倍的野生型。除了7个突变体外，所有突变体都与肝素以及野生型结合。42个突变体转导人类细胞至少以及野生型，和10个突变体增加转导活性高达9倍以上的野生型。18个相邻的丙氨酸取代使转导减少了10至100，000倍，但对肝素结合没有影响，并限定了转导所需的区域（死区），该区域与先前表征的肝素受体结合位点不同。突变，减少结合和中和的鼠单克隆抗体（A20）的本地化，而突变，减少中和的个人血清或合并的人，静脉注射免疫球蛋白G（IVIG）分散在一个更大的区域。减少A20结合的突变也减少中和。然而，使IVIG结合减少90%的突变并不减少中和，并且减少IVIG中和的突变并不减少其结合。突变的组合没有显著增加转导或抵抗IVIG中和。这些突变限定了AAV-2衣壳表面上的区域，其是转导和抗体中和的重要决定因素。
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.