Racah Institute of Physics, The Hebrew University, Jerusalem, 91904, Israel.
Department of Biology, Molecular Biophysics, Humboldt Universität zu Berlin, 10115, Berlin, Germany.
Sci Rep. 2018 Jun 27;8(1):9747. doi: 10.1038/s41598-018-28114-5.
The HIV-1 glycoprotein gp41 critically mediates CD4 T-cell infection by HIV-1 during viral entry, assembly, and release. Although multiple immune-regulatory activities of gp41 have been reported, the underlying mechanisms of these activities remain poorly understood. Here we employed multi-colour single molecule localization microscopy (SMLM) to resolve interactions of gp41 proteins with cellular proteins at the plasma membrane (PM) of fixed and live CD4 T-cells with resolution of ~20-30 nm. We observed that gp41 clusters dynamically associated with the T cell antigen receptor (TCR) at the immune synapse upon TCR stimulation. This interaction, confirmed by FRET, depended on the virus clone, was reduced by the gp41 ectodomain in tight contacts, and was completely abrogated by mutation of the gp41 transmembrane domain. Strikingly, gp41 preferentially colocalized with phosphorylated TCRs at the PM of activated T-cells and promoted TCR phosphorylation. Gp41 expression also resulted in enhanced CD69 upregulation, and in massive cell death after 24-48 hrs. Our results shed new light on HIV-1 assembly mechanisms at the PM of host T-cells and its impact on TCR stimulation.
HIV-1 糖蛋白 gp41 在 HIV-1 进入、组装和释放过程中对 CD4 T 细胞感染起着至关重要的作用。尽管已经报道了 gp41 的多种免疫调节活性，但这些活性的潜在机制仍知之甚少。在这里，我们采用多色单分子定位显微镜（SMLM）解析了固定和活 CD4 T 细胞的质膜（PM）上 gp41 蛋白与细胞蛋白之间的相互作用，分辨率为~20-30nm。我们观察到，gp41 簇在 TCR 刺激时在免疫突触处与 T 细胞抗原受体（TCR）动态相关。这种相互作用通过 FRET 得到证实，取决于病毒克隆，被 gp41 外域在紧密接触时减少，并被 gp41 跨膜域的突变完全消除。引人注目的是，gp41 在激活的 T 细胞的 PM 上优先与磷酸化的 TCR 共定位，并促进 TCR 磷酸化。gp41 的表达还导致 CD69 的上调增加，并在 24-48 小时后导致大量细胞死亡。我们的研究结果为宿主 T 细胞 PM 中 HIV-1 的组装机制及其对 TCR 刺激的影响提供了新的认识。
