Peptides derived from the C-terminal heptad repeat (CHR) region of HIV-1 gp41 is potent viral membrane fusion inhibitors, such as the first clinically approved peptide drug T20 and a group of newly-designed peptides. The resistance profiles of various HIV-1 fusion inhibitors were previously characterized, and the secondary mutation N126K in the gp41 CHR was routinely identified during the in vitro and in vivo selections. In this study, the functional and structural relevance of the N126K mutation has been characterized from multiple angles. First, we show that a single N126K mutation across several HIV-1 isolates conferred mild to moderate cross-resistances. Second, the N126K mutation exerted different effects on Env-mediated HIV-1 entry and cell-cell fusion. Third, the N126K mutation did not interfere with the expression and processing of viral Env glycoproteins, but it disrupted the Asn126-based glycosylation site in gp41. Fourth, the N126K mutation was verified to enhance the thermal stability of 6-HB conformation. Fifth, we determined the crystal structure of a 6-HB bearing the N126K mutation, which revealed the interhelical and intrahelical interactions underlying the increased thermostability. Therefore, our data provide new information to understand the mechanism of HIV-1 gp41-mediated cell fusion and its resistance mode to viral fusion inhibitors.

中文翻译：

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由各种HIV-1融合抑制剂选择的次级突变N126K的结构和功能表征。

源自HIV-1 gp41的C端七肽重复序列（CHR）区域的肽是有效的病毒膜融合抑制剂，例如首个临床批准的肽药物T20和一组新设计的肽。先前已鉴定了各种HIV-1融合抑制剂的抗药性，并在体外和体内选择过程中常规鉴定了gp41 CHR中的二级突变N126K。在这项研究中，已从多个角度表征了N126K突变的功能和结构相关性。首先，我们证明了跨多个HIV-1分离株的单个N126K突变赋予了轻度到中度的交叉耐药性。其次，N126K突变对Env介导的HIV-1进入和细胞融合具有不同的作用。第三，N126K突变不会干扰病毒Env糖蛋白的表达和加工，但会破坏gp41中基于Asn126的糖基化位点。第四，验证了N126K突变可增强6-HB构象的热稳定性。第五，我们确定了带有N126K突变的6-HB的晶体结构，该结构揭示了热稳定性增加背后的螺旋间和螺旋内相互作用。因此，我们的数据提供了新的信息，以了解HIV-1 gp41介导的细胞融合的机制及其对病毒融合抑制剂的耐药模式。这揭示了增加的热稳定性背后的螺旋间和螺旋内相互作用。因此，我们的数据提供了新的信息，以了解HIV-1 gp41介导的细胞融合的机制及其对病毒融合抑制剂的耐药模式。这揭示了增加的热稳定性背后的螺旋间和螺旋内相互作用。因此，我们的数据提供了新的信息，以了解HIV-1 gp41介导的细胞融合的机制及其对病毒融合抑制剂的耐药模式。