BackgroundHIV infection is enhanced by cell adhesions that form between infected and uninfected T cells called virological synapses (VS). VS are initiated by an interaction between Env and CD4 on cell surfaces and result in the recruitment of virus assembly to the site of cell–cell contact. However, the recruitment of Env to the VS and its relationship to Gag recruitment is not well defined.ResultsTo study the trafficking of HIV-1 Env through the VS, we constructed a molecular clone of HIV carrying a green fluorescent protein-Env fusion protein called, HIV Env-isfGFP-∆V1V2. The Env-isfGFP-∆V1V2 fusion protein does not produce virus particles on its own, but can be rescued by cotransfection with full-length HIV constructs and produce virus particles that package the fluorescent Env. These rescued fluorescent Env can participate in VS formation and can be used to directly image CD4-dependent Env transfer across VS from donor to target cells. The movements of fluorescently tagged Gag and Env to the VS and transfer into target cells can be also tracked through live imaging. Time lapse live imaging reveals evidence of limited Env accumulation at the site of cell–cell contact shortly after cell adhesion, followed by Gag re-distribution to contact area. Both Gag and Env can be recruited to form button-like spots characteristic of VS.ConclusionsEnv and Gag are recruited to the VS in a coordinated temporal sequence and subsequently transfer together across the synapse into the target cell. Env accumulations, when observed, are earlier than Gag re-distribution to the contact area during formation of VS.

中文翻译：

---

实时成像揭示 Env 和 Gag 向 HIV-1 T 细胞病毒学突触的顺序运输

背景 HIV 感染通过在被感染和未感染的 T 细胞之间形成的称为病毒学突触 (VS) 的细胞粘附而增强。VS 由细胞表面 Env 和 CD4 之间的相互作用启动，并导致病毒组装募集到细胞 - 细胞接触位点。然而，Env 向 VS 的募集及其与 Gag 募集的关系尚未明确定义。结果为了研究通过 VS 贩运 HIV-1 Env，我们构建了一个携带绿色荧光蛋白-Env 融合蛋白的 HIV 分子克隆，称为, HIV Env-isfGFP-ΔV1V2。Env-isfGFP-ΔV1V2 融合蛋白本身不会产生病毒颗粒，但可以通过与全长 HIV 构建体共转染来拯救，并产生包装荧光 Env 的病毒颗粒。这些获救的荧光 Env 可以参与 VS 形成，并可用于直接对 CD4 依赖性 Env 从供体到靶细胞的 VS 转移进行成像。还可以通过实时成像跟踪荧光标记的 Gag 和 Env 到 VS 并转移到靶细胞中的运动。延时实时成像揭示了细胞粘附后不久在细胞-细胞接触部位有限的 Env 积累的证据，随后 Gag 重新分布到接触区域。Gag 和 Env 都可以被募集以形成 VS 的按钮状斑点特征。结论 Env 和 Gag 以协调的时间序列被募集到 VS，随后一起穿过突触转移到目标细胞中。在 VS 形成期间，观察到 Env 积累早于 Gag 重新分布到接触区域。