We previously discovered a class of recombinant lectin conjugates, denoted lectin DLIs (‘dual-acting lytic inhibitors’) that bind to the HIV-1 envelope (Env) protein trimer and cause both lytic inactivation of HIV-1 virions and cytotoxicity of Env-expressing cells. To facilitate mechanistic investigation of DLI function, we derived the simplified prototype microvirin (MVN)-DLI, containing an MVN domain that binds high-mannose glycans in Env, connected to a DKWASLWNW sequence (denoted ‘Trp3’) derived from the membrane-associated region of gp41. The relatively much stronger affinity of the lectin component than Trp3 argues that the lectin functions to capture Env to enable Trp3 engagement and consequent Env membrane disruption and virolysis. The relatively simplified engagement pattern of MVN with Env opened up the opportunity, pursued here, to use recombinant glycan knockout gp120 variants to identify the precise Env binding site for MVN that drives DLI engagement and lysis. Using mutagenesis combined with a series of biophysical and virological experiments, we identified a restricted set of residues, N262, N332 and N448, all localized in a cluster on the outer domain of gp120, as the essential epitope for MVN binding. By generating these mutations in the corresponding HIV-1 virus, we established that the engagement of this glycan cluster with the lectin domain of MVN*-DLI is the trigger for DLI-derived virus and cell inactivation. Beyond defining the initial encounter step for lytic inactivation, this study provides a guide to further elucidate DLI mechanism, including the stoichiometry of Env trimer required for function, and downstream DLI optimization.

中文翻译：

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通过基于凝集素的重组工程蛋白结合物鉴定gp120中不可逆的HIV-1裂解失活所必需的聚糖簇

我们先前发现了一类重组凝集素结合物，称为凝集素DLI（“双作用裂解抑制剂”），可与HIV-1包膜（Env）蛋白三聚体结合，并引起HIV-1病毒体的裂解失活和Env-的细胞毒性。表达细胞。为了促进DLI功能的机理研究，我们衍生了简化的原型微病毒蛋白（MVN）-DLI，其中包含与Env中的高甘露糖聚糖结合的MVN域，并连接到源自膜相关膜的DKWASLWNW序列（表示为“ Trp3”） gp41的区域。凝集素成分比Trp3相对更强的亲和力认为，凝集素的功能是捕获Env，从而使Trp3参与并随后引起Env膜破裂和病毒水解。MVN与Env的相对简化的参与模式为我们提供了机会，使用重组聚糖敲除gp120变体来识别驱动DLI参与和裂解的MVN的精确Env结合位点。使用诱变结合一系列的生物物理和病毒学实验，我们确定了一组受限的残基N262，N332和N448，它们全部位于gp120外域的簇中，作为MVN结合的必要表位。通过在相应的HIV-1病毒中产生这些突变，我们确定该聚糖簇与MVN * -DLI的凝集素结构域的结合是DLI衍生病毒和细胞失活的触发因素。除了定义裂解失活的初始步骤外，本研究还提供了进一步阐明DLI机制的指南，包括功能所需的Env三聚体的化学计量以及下游DLI优化。