SC29EK is an electronically constrained α-helical peptide HIV-1 fusion inhibitor that is highly effective against both wild-type and enfuvirtide (T20)-resistant viruses. In this study, we focused on investigating the mechanism of HIV-1 resistance to SC29EK by two approaches. First, SC29EK-escaping HIV-1 variants were selected and characterized. Three mutant viruses, which possessed two (N43K/E49A) or three (Q39R/N43K/N126K and N43K/E49A/N126K) amino acid substitutions in the N- and C-terminal repeat regions of gp41 were identified as conferring high resistance to SC29EK and cross-resistance to the first-generation (T20 and C34) and newly designed (sifuvirtide, MT-SC29EK, and 2P23) fusion inhibitors. The resistance mutations could reduce the binding stability of SC29EK, impair viral Env-mediated cell fusion and entry, and change the conformation of the gp41 core structure. Further, we determined the crystal structure of SC29EK in complex with a target mimic peptide, which revealed the critical intra- and interhelical interactions underlying the mode of action of SC29EK and the genetic pathway to HIV-1 resistance. Taken together, the present data provide new insights into the structure and function of gp41 and the structure-activity relationship (SAR) of viral fusion inhibitors.

IMPORTANCE T20 is the only membrane fusion inhibitor available for treatment of viral infection, but it has relatively low anti-HIV activity and genetic barriers for resistance, thus calling for new drugs blocking the viral fusion process. As an electronically constrained α-helical peptide, SC29EK is highly potent against both wild-type and T20-resistant HIV-1 strains. Here, we report the characterization of HIV-1 variants resistant to SC29EK and the crystal structure of SC29EK. The key mutations mediating high resistance to SC29EK and cross-resistance to the first and new generations of fusion inhibitors as well as the underlying mechanisms were identified. The crystal structure of SC29EK bound to a target mimic peptide further revealed its action mode and genetic pathway to inducing resistance. Hence, our data have shed new lights on the mechanisms of HIV-1 fusion and its inhibition.

SC29EK是一种电子约束的α-螺旋肽HIV-1融合抑制剂，对野生型和恩福韦肽（T20）耐药病毒均具有很高的抵抗力。在这项研究中，我们集中于通过两种方法研究HIV-1对SC29EK的抗性机制。首先，选择并鉴定了逃逸SC29EK的HIV-1变体。在gp41的N和C末端重复区域具有两个（N43K / E49A）或三个（Q39R / N43K / N126K和N43K / E49A / N126K）氨基酸取代的三种突变病毒被鉴定为对SC29EK具有高抗性对第一代（T20和C34）和新设计的（sifuvirtide，MT-SC29EK和2P23）融合抑制剂具有交叉耐药性。抗药性突变可能会降低SC29EK的结合稳定性，损害病毒Env介导的细胞融合和进入，并改变gp41核心结构的构象。此外，我们确定了与目标模拟肽复合的SC29EK的晶体结构，揭示了SC29EK作用方式和HIV-1耐药性遗传途径的关键内部和螺旋间相互作用。两者合计，目前的数据提供gp41的结构和功能以及病毒融合抑制剂的结构活性关系（SAR）的新见解。

重要性T20是唯一可用于治疗病毒感染的膜融合抑制剂，但它具有相对较低的抗HIV活性和耐药性的遗传障碍，因此需要新的药物来阻断病毒融合过程。作为一种电子约束的α-螺旋肽，SC29EK对野生型和抗T20的HIV-1菌株均具有很高的效力。在这里，我们报告抗SC29EK和SC29EK的晶体结构的HIV-1变异的表征。鉴定了介导对SC29EK的高抗性和对第一代和新一代融合抑制剂的交叉抗性的关键突变，以及潜在的机制。与靶模拟肽结合的SC29EK的晶体结构进一步揭示了其作用方式和诱导抗性的遗传途径。因此，