A New Approach to Produce HIV-1 Envelope Trimers BOTH CLEAVAGE AND PROPER GLYCOSYLATION ARE ESSENTIAL TO GENERATE AUTHENTIC TRIMERS
A New Approach to Produce HIV-1 Envelope Trimers BOTH CLEAVAGE AND PROPER GLYCOSYLATION ARE ESSENTIAL TO GENERATE AUTHENTIC TRIMERS
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DOI：
10.1074/jbc.m115.656611
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发表时间：
2015-08-07
影响因子：
4.8
通讯作者：
Rao, Venigalla B.
中科院分区：
文献类型：
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作者：
AlSalmi, Wadad;Mahalingam, Marthandan;Rao, Venigalla B.
HIV-1的三聚体包膜刺突介导病毒进入人类细胞。三聚体的暴露部分gp140由两个非共价结合的亚基gp120和gp41外结构域组成。一种模仿天然三聚体的重组疫苗可能会引发进入阻断抗体并预防病毒感染。然而，真正的HIV-1三聚体的制备一直具有挑战性。最近，一个包含广泛中和抗体2G12的亲和柱被用于捕获重组gp140，并从进化枝A BG505中制备自然产生稳定三聚体的三聚体。然而，这种基于抗体的方法可能对具有不同表位特征的不同HIV-1毒株不那么有效。在这里，我们报告了一种新的和简单的方法来产生HIV-1包膜三聚体。gp140的C端连接到Strep-tag II上，用一个长连接体将标签与大质量的三聚体碱基和聚糖屏蔽体分开。这样可以直接从培养基中捕获几乎均匀的gp140。产生了来自不同进化支病毒的裂解、未裂解、完全或部分糖基化三聚体。广泛的生化表征表明，gp140的裂解对三聚体化不是必需的，但它引发了构象变化，将三聚体引导到正确的糖基化途径，产生紧凑的三叶片螺旋桨状三聚体。未裂解的三聚体进入异常途径，导致高糖基化、非特异性交联和构象异质性。即使是断裂的三聚体在gp41糖基化上也表现出微异质性。这些研究建立了一个广泛适用的HIV-1三聚体生产系统，并对它们的组装和成熟产生了新的见解，这些系统共同影响HIV-1疫苗的设计。
The trimeric envelope spike of HIV-1 mediates virus entry into human cells. The exposed part of the trimer, gp140, consists of two noncovalently associated subunits, gp120 and gp41 ectodomain. A recombinant vaccine that mimics the native trimer might elicit entry-blocking antibodies and prevent virus infection. However, preparation of authentic HIV-1 trimers has been challenging. Recently, an affinity column containing the broadly neutralizing antibody 2G12 has been used to capture recombinant gp140 and prepare trimers from clade A BG505 that naturally produces stable trimers. However, this antibody-based approach may not be as effective for the diverse HIV-1 strains with different epitope signatures. Here, we report a new and simple approach to produce HIV-1 envelope trimers. The C terminus of gp140 was attached to Strep-tag II with a long linker separating the tag from the massive trimer base and glycan shield. This allowed capture of nearly homogeneous gp140 directly from the culture medium. Cleaved, uncleaved, and fully or partially glycosylated trimers from different clade viruses were produced. Extensive biochemical characterizations showed that cleavage of gp140 was not essential for trimerization, but it triggered a conformational change that channels trimers into correct glycosylation pathways, generating compact three-blade propeller-shaped trimers. Uncleaved trimers entered aberrant pathways, resulting in hyperglycosylation, nonspecific cross-linking, and conformational heterogeneity. Even the cleaved trimers showed microheterogeneity in gp41 glycosylation. These studies established a broadly applicable HIV-1 trimer production system as well as generating new insights into their assembly and maturation that collectively bear on the HIV-1 vaccine design.