Aires da Silva Frederico, Santa-Marta Mariana, Freitas-Vieira Acilino, Mascarenhas Paulo, Barahona Isabel, Moniz-Pereira José, Gabuzda Dana, Goncalves Joao
URIA - Centro de Patogénese Molecular, Faculdade de Farmácia, Universidade de Lisboa, Lisboa 1649-019, Portugal.
J Mol Biol. 2004 Jul 9;340(3):525-42. doi: 10.1016/j.jmb.2004.04.062.
We recently developed a specific single-chain antibody from immunized rabbits to HIV-1 Vif protein that was expressed intracellularly and inhibited reverse transcription and viral replication. The Vif of HIV-1 overcomes the innate antiviral activity of a cytidine deaminase Apobec3G (CEM15) that induces G to A hypermutation in the viral genome, resulting in enhancement of viral replication infectivity. Here, we have developed a minimal scaffold VH fragment with intrabody properties derived from anti-Vif single-chain antibody that was engineered to mimic camelid antibody domains. Non-specific binding of VH by its interface for the light chain variable domain (VL) was prevented through amino acid mutations in framework 2 and 4 (Val37F, G44E, L45R, W47G and W103R). Our results demonstrate that all constructed anti-Vif VH single-domains preserve the antigen-binding activity and specificity in the absence of the parent VL domain. However, only the most highly camelized domains had high levels of intracellular expression. The expression in eukaryotic cells showed that VH single-domains could correctly fold as soluble proteins in the reducing environment. The results demonstrated an excellent correlation between improvements in protein solubility with gradually increasing camelization. Camelized single-domains efficiently bound Vif protein and neutralized its infectivity enhancing function, by reducing late reverse transcripts and proviral integration. The activity of the anti-Vif single-domains was shown to be cell-specific, with inhibitory effects only in cells non-permissive that require Vif for HIV-1 replication. Moreover, cell specificity of anti-Vif intrabodies was correlated with an increase of Apobec3G, which potentiates viral inhibition. The present study strongly suggests that camelization of rabbit VH domains is a potentially useful approach for engineering intrabodies for gene therapy.
我们最近从免疫兔中开发出一种针对HIV-1 Vif蛋白的特异性单链抗体，该蛋白在细胞内表达，可抑制逆转录和病毒复制。HIV-1的Vif可克服胞苷脱氨酶Apobec3G（CEM15）的固有抗病毒活性，后者可诱导病毒基因组中G到A的超突变，从而增强病毒复制的感染性。在此，我们开发了一种具有胞内抗体特性的最小支架VH片段，其源自抗Vif单链抗体，并经过工程改造以模拟骆驼科动物抗体结构域。通过框架2和4中的氨基酸突变（Val37F、G44E、L45R、W47G和W103R），可防止VH通过其轻链可变域（VL）界面发生非特异性结合。我们的结果表明，所有构建的抗Vif VH单结构域在没有亲本VL结构域的情况下仍保留抗原结合活性和特异性。然而，只有骆驼化程度最高的结构域具有高水平的细胞内表达。真核细胞中的表达表明，VH单结构域在还原环境中可正确折叠为可溶性蛋白。结果表明，随着骆驼化程度的逐渐增加，蛋白质溶解度的改善之间存在极好的相关性。骆驼化的单结构域通过减少晚期逆转录产物和前病毒整合，有效地结合Vif蛋白并中和其感染性增强功能。抗Vif单结构域的活性显示具有细胞特异性，仅在需要Vif进行HIV-1复制的非允许细胞中具有抑制作用。此外，抗Vif胞内抗体的细胞特异性与Apobec3G的增加相关，Apobec3G可增强病毒抑制作用。本研究强烈表明，兔VH结构域的骆驼化是一种潜在有用的方法，可用于工程化胞内抗体以进行基因治疗。
