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首页> 外文期刊>Biochemistry >Metastable HIV-1 Surface Protein Env Sensitizes Cell Membranes to Transformation and Poration by Dual-Acting Virucidal Entry Inhibitors
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Metastable HIV-1 Surface Protein Env Sensitizes Cell Membranes to Transformation and Poration by Dual-Acting Virucidal Entry Inhibitors

机译：亚稳态 HIV-1 表面蛋白 Env 通过双作用杀病毒进入抑制剂使细胞膜对转化和穿孔敏感

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Dual-acting virucidal entry inhibitors (DAVEIs) have previously been shown to cause irreversible inactivation of HIV-1 Env-presenting pseudovirus by lytic membrane transformation. This study examined whether this transformation could be generalized to include membranes of Env-presenting cells. Flow cytometry was used to analyze HEK293T cells transiently transfected with increasing amounts of DNA encoding JRFL Env, loaded with calcein dye, and treated with serial dilutions of microvirin (Q831K/M83R)-DAVEI. Comparing calcein retention against intact Env expression (via Ab 35O22) on individual cells revealed effects proportional to Env expression. "Low-Env" cells experienced transient poration and calcein leakage, while "high-Env" cells were killed. The cell-killing effect was confirmed with an independent mitochondrial activity-based cell viability assay, showing dose-dependent cytotoxicity in response to DAVEI treatment. Transfection with increasing quantities of Env DNA showed further shifts toward "High-Env" expression and cytotoxicity, further reinforcing the Env dependence of the observed effect. Controls with unlinked DAVEI components showed no effect on calcein leakage or cell viability, confirming a requirement for covalently linked DAVEI compounds to achieve Env transformation. These data demonstrate that the metastability of Env is an intrinsic property of the transmembrane protein complex and can be perturbed to cause membrane disruption in both virus and cell contexts.
机译：双效杀病毒进入抑制剂 （DAVEI） 先前已被证明可通过裂解膜转化导致 HIV-1 Env 呈递假病毒的不可逆灭活。这项研究检查了这种转化是否可以推广到包括Env呈递细胞的膜。流式细胞术分析瞬时转染编码JRFL Env的DNA的HEK293T细胞，上载钙黄绿素染料，并用微病毒蛋白（Q831K/M83R）-DAVEI的连续稀释液处理。比较钙黄绿素保留与单个细胞上完整 Env 表达（通过 Ab 35O22）显示与 Env 表达成正比的影响。“低环境”细胞经历瞬时穿孔和钙黄绿素泄漏，而“高环境”细胞被杀死。通过独立的基于线粒体活性的细胞活力测定证实了细胞杀伤作用，显示出对 DAVEI 治疗的反应的剂量依赖性细胞毒性。随着Env DNA数量的增加，转染显示出进一步向“高Env”表达和细胞毒性的转变，进一步加强了观察到的效应的Env依赖性。具有未连接的 DAVEI 组分的对照显示对钙黄绿素泄漏或细胞活力没有影响，证实了需要共价连接的 DAVEI 化合物来实现 Env 转化。这些数据表明，Env的亚稳态是跨膜蛋白复合物的固有特性，可以在病毒和细胞环境中受到干扰以引起膜破坏。

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