Despite efforts to develop effective treatments for eradicating HIV-1, a cure has not yet been achieved. Whereas antiretroviral drugs target an actively replicating virus, latent, nonreplicative forms persist during treatment. Pharmacological strategies that reactivate latent HIV-1 and expose cellular reservoirs to antiretroviral therapy and the host immune system have, so far, been unsuccessful, often triggering severe side effects, mainly due to systemic immune activation. Here, we present an alternative approach for stimulating latent HIV-1 expression via direct protein delivery of cell-penetrating zinc-finger activators (ZFAs). Cys₂-His₂ zinc-fingers, fused to a transcription activation domain, were engineered to recognize the HIV-1 promoter and induce targeted viral transcription. Following conjugation with multiple positively charged nuclear localization signal (NLS) repeats, protein delivery of a single ZFA (3NLS-PBS1-VP64) efficiently internalized HIV-1 latently infected T-lymphocytes and specifically stimulated viral expression. We show that short-term treatment with this ZFA protein induces higher levels of viral reactivation in cell line models of HIV-1 latency than those observed with gene delivery. Our work establishes protein delivery of ZFA as a novel and safe approach toward eradication of HIV-1 reservoirs.

尽管人们努力开发有效的治疗方法来根除 HIV-1，但尚未实现治愈。虽然抗逆转录病毒药物针对的是活跃复制的病毒，但在治疗过程中潜伏的、非复制的病毒形式仍然存在。迄今为止，重新激活潜伏的 HIV-1 并使细胞储库暴露于抗逆转录病毒治疗和宿主免疫系统的药理学策略尚未成功，通常会引发严重的副作用，这主要是由于全身免疫激活所致。在这里，我们提出了一种通过细胞穿透锌指激活剂 (ZFA) 的直接蛋白质递送来刺激潜在 HIV-1 表达的替代方法。 Cys ₂ -His ₂锌指与转录激活结构域融合，经过改造可识别 HIV-1 启动子并诱导靶向病毒转录。与多个带正电荷的核定位信号 (NLS) 重复序列缀合后，单个 ZFA (3NLS-PBS1-VP64) 的蛋白质递送可有效内化 HIV-1 潜伏感染的 T 淋巴细胞，并特异性刺激病毒表达。我们发现，与基因递送观察到的情况相比，在 HIV-1 潜伏细胞系模型中，使用这种 ZFA 蛋白进行短期治疗可诱导更高水平的病毒再激活。我们的工作将 ZFA 的蛋白质递送确立为一种消灭 HIV-1 病毒库的新颖且安全的方法。