Enzyme Digests Eliminate Nonfunctional Env from HIV-1 Particle Surfaces, Leaving Native Env Trimers Intact and Viral Infectivity Unaffected
Enzyme Digests Eliminate Nonfunctional Env from HIV-1 Particle Surfaces, Leaving Native Env Trimers Intact and Viral Infectivity Unaffected
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DOI：
10.1128/jvi.00154-11
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发表时间：
2011-06-01
影响因子：
5.4
通讯作者：
Binley, James M.
中科院分区：
文献类型：
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作者：
Crooks, Ema T.;Tong, Tommy;Binley, James M.
HIV-1病毒和病毒样颗粒（VLP）携带非天然的“垃圾”形式的包膜（Env）糖蛋白，其可能破坏针对功能性gp 120/gp 41三聚体的抗体应答的发展，从而减弱颗粒引发中和抗体的能力。在这里，我们试图更好地了解垃圾环境的性质，以期制定战略，以消除它。最初的研究表明，天然三聚体在恶劣条件下惊人地稳定，这表明垃圾Env不太可能通过三聚体解离或gp 120脱落而产生。此外，在原代HIV-1分离株Envs合成后立即发生的有限gp 120脱落不是由串联gp 120/gp 41切割位点的异常切割引起的，发现串联gp 120/gp 41切割位点以共依赖方式切割。一个主要的VLP污染物被发现由早期的，单体形式的gp 160，是糖基化的内质网（gp 160 ER），然后绕过蛋白质成熟和交通直接进入颗粒。发现gp 160 ER结合单克隆抗体（MAb）2G 12的两个拷贝，与其专有的高甘露糖聚糖谱一致。这些发现促使我们评估酶抑制剂作为去除异常Env的一种方法。值得注意的是，连续的糖苷酶-蛋白酶酶解导致从许多病毒株中完全或接近完全去除垃圾Env，留下三聚体和病毒感染性基本上完好无损。“三聚体VLP”可以是有用的中和抗体免疫原。
HIV-1 viruses and virus-like particles (VLPs) bear nonnative "junk" forms of envelope (Env) glycoprotein that may undermine the development of antibody responses against functional gp120/gp41 trimers, thereby blunting the ability of particles to elicit neutralizing antibodies. Here, we sought to better understand the nature of junk Env with a view to devising strategies for its removal. Initial studies revealed that native trimers were surprisingly stable in the face of harsh conditions, suggesting that junk Env is unlikely to arise by trimer dissociation or gp120 shedding. Furthermore, the limited gp120 shedding that occurs immediately after synthesis of primary HIV-1 isolate Envs is not caused by aberrant cleavage at the tandem gp120/gp41 cleavage sites, which were found to cleave in a codependent manner. A major VLP contaminant was found to consist of an early, monomeric form of gp160 that is glycosylated in the endoplasmic reticulum (gp160ER) and then bypasses protein maturation and traffics directly into particles. gp160ER was found to bind two copies of monoclonal antibody (MAb) 2G12, consistent with its exclusively high-mannose glycan profile. These findings prompted us to evaluate enzyme digests as a way to remove aberrant Env. Remarkably, sequential glycosidase-protease digests led to a complete or near-complete removal of junk Env from many viral strains, leaving trimers and viral infectivity largely intact. "Trimer VLPs" may be useful neutralizing antibody immunogens.