Functional neutralization of HIV-1 Vif protein by intracellular immunization inhibits reverse transcription and viral replication
Functional neutralization of HIV-1 Vif protein by intracellular immunization inhibits reverse transcription and viral replication
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DOI：
10.1074/jbc.m201906200
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发表时间：
2002-08-30
影响因子：
4.8
通讯作者：
Barbas, C
中科院分区：
文献类型：
--
作者：
Goncalves, J;Silva, F;Barbas, C
人类免疫缺陷病毒 1 型 (HIV-1) 编码的 Vif 蛋白对于病毒复制和感染性非常重要。 Vif 是一种细胞质蛋白，在病毒组装过程中通过未知机制发挥作用，增强病毒的感染性。 Vif 在生产细胞中的作用对于病毒进入后完成前病毒 DNA 合成至关重要。因此，Vif被认为是在病毒组装和逆转录水平上抑制病毒感染性的重要替代治疗靶点。为了深入了解这一过程，我们开发了一种 Vif 特异性单链抗体，并在细胞质中进行细胞内表达。这种胞内抗体有效结合 Vif 蛋白并中和其增强感染性的功能。研究表明，体内表达细胞对于不同毒株的 HIV-1 和 HIV-1 感染细胞的攻击具有高度抵抗力。在供体细胞中通过体内表达抑制 Vif 产生的病毒颗粒在受体细胞中不能完成逆转录。抗 Vif scFv 对 Vif 蛋白具有特异性，因为它的功能仅在不允许的细胞（H9、CEM 和 U38）中观察到。此外，用体内表达 Vif 的 HIV 衍生逆转录病毒载体转导外周血单核细胞，可赋予对实验室适应型和原发性 HIV 毒株的抵抗力。这项研究为 Vif 在 HrV-1 生命周期中的作用提供了生化证据，并验证了 Vif 作为控制 HIV-1 感染的靶点。
Human immunodeficiency virus type 1 (HIV-1)-encoded Vif protein is important for viral replication and infectivity. Vif is a cytoplasmic protein that acts during virus assembly by an unknown mechanism, enhancing viral infectivity. The action of Vif in producer cells is essential for the completion of proviral DNA synthesis following virus entry. Therefore, Vif is considered to be an important alternative therapeutic target for inhibition of viral infectivity at the level of viral assembly and reverse transcription. To gain insight into this process, we developed a Vif-specific single-chain antibody and expressed it intracellularly in the cytoplasm. This intrabody efficiently bound Vif protein and neutralized its infectivity-enhancing function. Intrabody-expressing cells were shown to be highly refractory to challenge with different strains of HIV-1 and HIV-1-infected cells. Inhibition of Vif by intrabody expression in the donor cell produced viral particles that do not complete reverse transcription in the recipient cell. The anti-Vif scFv was shown to be specific for Vif protein because its function was observed only in nonpermissive cells (H9, CEM, and U38). Moreover, transduction of peripheral blood mononuclear cells with an HIV-derived retroviral vector expressing Vif intrabody was shown to confer resistance to laboratory-adapted and primary HIV strains. This study provides biochemical evidence for the role of Vif in the HrV-1 lifecycle and validates Vif as a target for the control of HIV-1 infection.