可溶性X4和杂交X4-R5 HIV-1包膜三聚体的抗原性及三维结构特征
Antigenic and 3D structural characterization of soluble X4 and hybrid X4-R5 HIV-1 Env trimers.
作者信息
Arnold Philipp, Himmels Patricia, Weiß Svenja, Decker Tim-Michael, Markl Jürgen, Gatterdam Volker, Tampé Robert, Bartholomäus Patrick, Dietrich Ursula, Dürr Ralf
机构信息
Molecular Virology, Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Paul-Ehrlich-Str, 42-44, 60596 Frankfurt, Germany.
出版信息
Retrovirology. 2014 May 30;11:42. doi: 10.1186/1742-4690-11-42.
BACKGROUND
HIV-1 is decorated with trimeric glycoprotein spikes that enable infection by engaging CD4 and a chemokine coreceptor, either CCR5 or CXCR4. The variable loop 3 (V3) of the HIV-1 envelope protein (Env) is the main determinant for coreceptor usage. The predominant CCR5 using (R5) HIV-1 Env has been intensively studied in function and structure, whereas the trimeric architecture of the less frequent, but more cytopathic CXCR4 using (X4) HIV-1 Env is largely unknown, as are the consequences of sequence changes in and near V3 on antigenicity and trimeric Env structure.
RESULTS
Soluble trimeric gp140 Env constructs were used as immunogenic mimics of the native spikes to analyze their antigenic properties in the context of their overall 3D structure. We generated soluble, uncleaved, gp140 trimers from a prototypic T-cell line-adapted (TCLA) X4 HIV-1 strain (NL4-3) and a hybrid (NL4-3/ADA), in which the V3 spanning region was substituted with that from the primary R5 isolate ADA. Compared to an ADA (R5) gp140, the NL4-3 (X4) construct revealed an overall higher antibody accessibility, which was most pronounced for the CD4 binding site (CD4bs), but also observed for mAbs against CD4 induced (CD4i) epitopes and gp41 mAbs. V3 mAbs showed significant binding differences to the three constructs, which were refined by SPR analysis. Of interest, the NL4-3/ADA construct with the hybrid NL4-3/ADA CD4bs showed impaired CD4 and CD4bs mAb reactivity despite the presence of the essential elements of the CD4bs epitope. We obtained 3D reconstructions of the NL4-3 and the NL4-3/ADA gp140 trimers via electron microscopy and single particle analysis, which indicates that both constructs inherit a propeller-like architecture. The first 3D reconstruction of an Env construct from an X4 TCLA HIV-1 strain reveals an open conformation, in contrast to recently published more closed structures from R5 Env. Exchanging the X4 V3 spanning region for that of R5 ADA did not alter the open Env architecture as deduced from its very similar 3D reconstruction.
CONCLUSIONS
3D EM analysis showed an apparent open trimer configuration of X4 NL4-3 gp140 that is not modified by exchanging the V3 spanning region for R5 ADA.
背景
HIV-1表面装饰有三聚体糖蛋白刺突，其通过与CD4和趋化因子共受体（CCR5或CXCR4）结合来实现感染。HIV-1包膜蛋白（Env）的可变环3（V3）是共受体使用的主要决定因素。对主要使用CCR5（R5）的HIV-1 Env在功能和结构方面进行了深入研究，而较少见但更具细胞病变性的使用CXCR4（X4）的HIV-1 Env的三聚体结构在很大程度上尚不清楚，V3及其附近的序列变化对抗原性和三聚体Env结构的影响也不清楚。
结果
可溶性三聚体gp140 Env构建体被用作天然刺突的免疫原性模拟物，以在其整体三维结构背景下分析其抗原特性。我们从一个原型T细胞系适应（TCLA）的X4 HIV-1毒株（NL4-3）和一个杂交体（NL4-3/ADA）中产生了可溶性、未切割的gp140三聚体，其中V3跨区被来自原发性R5分离株ADA的V3跨区所取代。与ADA（R5）gp140相比，NL4-3（X4）构建体显示出总体上更高的抗体可及性，这在CD4结合位点（CD4bs）最为明显，但针对CD4诱导（CD4i）表位的单克隆抗体和gp41单克隆抗体也有此现象。V3单克隆抗体对这三种构建体表现出显著的结合差异，通过表面等离子体共振（SPR）分析得以明确。有趣的是，具有杂交NL4-3/ADA CD4bs的NL4-3/ADA构建体尽管存在CD4bs表位的关键元件，但CD4和CD4bs单克隆抗体的反应性受损。我们通过电子显微镜和单颗粒分析获得了NL4-3和NL4-3/ADA gp140三聚体的三维重建，这表明这两种构建体都具有螺旋桨样结构。来自X4 TCLA HIV-1毒株的Env构建体的首次三维重建显示为开放构象，这与最近发表的R5 Env的更封闭结构形成对比。将X4 V3跨区替换为R5 ADA的跨区，并未改变从其非常相似的三维重建推断出的开放Env结构。
结论
三维电子显微镜分析显示X4 NL4-3 gp140呈现明显的开放三聚体构型，将V3跨区替换为R5 ADA并未对其进行修饰。
Zotero 插件