In previous studies, we demonstrated that single-chain variable fragments (scFvs) from anti-human immunodeficiency virus (HIV) Env monoclonal antibodies act as entry inhibitors when tethered to the surface of target cells by a glycosyl-phosphatidylinositol (GPI) anchor. Interestingly, even if a virus escapes inhibition at entry, its replication is ultimately controlled. We hypothesized that in addition to functioning as entry inhibitors, anti-HIV GPI-scFvs may also interact with Env in an infected cell, thereby interfering with the infectivity of newly produced virions. Here, we show that expression of the anti-HIV Env GPI-scFvs in virus-producing cells reduced the release of HIV from cells 5- to 22-fold, and infectivity of the virions that were released was inhibited by 74% to 99%. Additionally, anti-HIV Env GPI-scFv X5 inhibited virion production and infectivity after latency reactivation and blocked transmitter/founder virus production and infectivity in primary CD4⁺ T cells. In contrast, simian immunodeficiency virus (SIV) production and infectivity were not affected by the anti-HIV Env GPI-scFvs. Loss of infectivity of HIV was associated with a reduction in the amount of virion-associated Env gp120. Interestingly, an analysis of Env expression in cell lysates demonstrated that the anti-Env GPI-scFvs interfered with processing of Env gp160 precursors in cells. These data indicate that GPI-scFvs can inhibit Env processing and function, thereby restricting production and infectivity of newly synthesized HIV. Anti-Env GPI-scFvs therefore appear to be unique anti-HIV molecules as they derive their potent inhibitory activity by interfering with both early (receptor binding/entry) and late (Env processing and incorporation into virions) stages of the HIV life cycle.

IMPORTANCE The restoration of immune function and persistence of CD4⁺ T cells in HIV-1-infected individuals without antiretroviral therapy requires a way to increase resistance of CD4⁺ T cells to infection by both R5- and X4-tropic HIV-1. Previously, we reported that anchoring anti-HIV-1 single-chain variable fragments (scFvs) via glycosyl-phosphatidylinositol (GPI) to the surface of permissive cells conferred a high level of resistance to HIV-1 variants at the level of entry. Here, we report that anti-HIV GPI-scFvs also derive their potent antiviral activity in part by blocking HIV production and Env processing, which consequently inhibits viral infectivity even in primary infection models. Thus, we conclude that GPI-anchored anti-HIV scFvs derive their potent blocking activity of HIV replication by interfering with successive stages of the viral life cycle. They may be effectively used in genetic intervention of HIV-1 infection.

在以前的研究中，我们证明了抗人免疫缺陷病毒（HIV）Env单克隆抗体的单链可变片段（scFvs）在通过糖基磷脂酰肌醇（GPI）锚栓连接至靶细胞表面时起着进入抑制剂的作用。有趣的是，即使病毒在进入时逃脱了抑制，其复制最终仍受到控制。我们假设除作为进入抑制剂起作用外，抗HIV GPI-scFvs还可与被感染细胞中的Env相互作用，从而干扰新产生的病毒体的感染性。在这里，我们证明了抗HIV Env GPI-scFvs在病毒生产细胞中的表达将HIV从细胞中的释放降低了5到22倍，并且释放的病毒体的感染性被抑制了74％至99％ 。此外，⁺ T细胞。相反，猿猴免疫缺陷病毒（SIV）的产生和感染性不受抗HIV Env GPI-scFv的影响。HIV感染力的丧失与病毒体相关的Env gp120数量的减少有关。有趣的是，对细胞裂解物中Env表达的分析表明，抗Env GPI-scFv干扰了细胞中Env gp160前体的加工。这些数据表明，GPI-scFvs可以抑制Env的加工和功能，从而限制了新合成的HIV的产生和感染性。因此，抗Env GPI-scFv似乎是独特的抗HIV分子，因为它们通过干扰HIV生命周期的早期（受体结合/进入）和晚期（Env加工并掺入病毒体）来获得强大的抑制活性。

重要信息未经抗逆转录病毒疗法治疗的HIV-1感染者的免疫功能恢复和CD4 ⁺ T细胞的持久性需要一种增加CD4 ⁺抵抗力的方法T细胞受R5-和X4-tropic HIV-1感染。先前，我们报道了通过糖基磷脂酰肌醇（GPI）将抗HIV-1单链可变片段（scFvs）锚定到允许细胞的表面，在进入水平赋予了对HIV-1变异体的高水平抗性。在这里，我们报告说抗HIV GPI-scFvs也部分地通过阻止HIV产生和Env加工而获得了有效的抗病毒活性，因此即使在原发感染模型中也抑制了病毒的感染性。因此，我们得出结论，GPI锚定的抗HIV scFvs通过干扰病毒生命周期的连续阶段而获得了有效的HIV复制阻断活性。它们可以有效地用于HIV-1感染的遗传干预。