膜融合过程是HIV-1病毒入侵宿主靶细胞的首要步骤，而HIV-1包膜蛋白上的gp41亚单位是抗病毒治疗中的一个重要靶点。源自病毒CHR序列的多肽可以与病毒NHR区结合形成外源性的六螺旋束，从而有效阻止病毒膜融合过程的发生。恩夫韦肽（T20）是目前为止唯一美国FDA批准的可以用于临床治疗的多肽类膜融合抑制剂，近年来研究者们也设计了很多高效广谱的新型膜融合抑制剂，然而耐药问题始终是膜融合抑制剂发展中一个亟待解决的重要问题。在对新型短肽类膜融合抑制剂MTSC22的耐药筛选中，我们发现了两个位于gp41蛋白CHR区的代偿性氨基酸突变N126K和E136G，这两个突变经常伴随NHR一级耐药突变位点的产生而出现，并能提高病毒耐药性。本论文主要研究了N126K和E136G两个代偿性突变对包膜蛋白结构和功能的影响，并对膜融合抑制剂的耐药机制进行了探索。
本研究首先鉴定了N126K和E136G两个代偿性突变对多种膜融合抑制剂的敏感性，发现N126K单点突变能够产生轻度耐药（2-5倍），E136G能够产生中度耐药（5-10倍），而N126K/E136G双点突变能够产生较强的耐药效果（>10倍）。同时我们还选取了HIV-1病毒中不同嗜性不同亚型的8个毒株NL4-3，92RW020，JRFL，AC10.29，REJO4541，QH0692，ZM53M.PB12，ZM109F.PB4，分别构建了带有N126K和E136G突变的假毒，确定了N126K和E136G对多肽类膜融合抑制剂的敏感性的影响。功能研究结果表明，代偿性突变不影响包膜蛋白的表达和加工，但可以影响病毒感染靶细胞的能力；从结构生物学的角度出发，我们首先以病毒C34序列为模板合成了N126K和E136G突变多肽，圆二色谱及等温量热滴定检测二级结构及其稳定性，结果表明N126K突变形成的6-HB稳定性和结合常数均高于野生型，而E136G突变形成的6-HB稳定性下降，结合常数增大。晶体结构解析直观反映gp41结构的变化：N126K突变后可以改变周围氨基酸侧链的构象，赖氨酸K取代天冬酰胺N后，可以与上游氨基酸E123形成氢键，使CHR自身稳定性增强，E123侧链位置的变化可以使6-HB疏水口袋与口袋结合区结合更紧密；同时，Y127与H53之间形成π键，稳定六螺旋束，这些构象变化共同导致了N126K与病毒NHR结合更加稳定。E136G突变导致E136和H132间的离子键破坏，螺旋稳定性下降，但同时E49和E136间的电离排斥作用消失，NHR和CHR结合力增强。其次，本研究还探索了N126和E136位点自然突变对病毒功能的影响，发现N126Y能够显著提高六螺旋稳定性，导致其对膜融合抑制剂产生较强的耐药作用。E136位点自然突变对病毒功能影响很大，多数E136自然突变均导致病毒入侵和融合能力严重下降。最后，我们鉴定了带有N126K和N126Y突变的C34多肽和T20多肽对于不同亚型的HIV-1病毒以及T20耐药株的抑制活性，发现这两个突变都能够使C34多肽的抗病毒活性略有升高，N126Y能够提高T20活性。
对于包膜糖蛋白gp41结构和功能的研究是阐明膜融合抑制剂作用机制和耐药机制的重要前提。本文以CHR区N126K和E136G两个代偿性突变作为主要研究对象，阐明该点突变对病毒功能的影响及对膜融合抑制剂敏感性的影响，从结构生物学角度分析其作用机制，并对N126和E136位点的自然突变也进行了扩展研究，另外还基于N126K和N126Y对六螺旋稳定性的影响，鉴定了带有N126K和N126Y突变的C34及T20抑制活性。这样的研究不仅丰富了人们对HIV-1包膜蛋白的结构与功能的认识，而且能够加深对膜融合抑制剂的作用机制和耐药机制的理解，有助于研究者从多角度全面审视病毒和抑制剂之间的相互作用，为多肽类膜融合抑制剂的研发、改进和应用提供新的思路。
 

Membrane fusion is the first step for HIV-1 to invade host target cells, and the gp41 subunit of HIV-1 envelope glycoprotein is an important target for antiviral therapy. Peptides derived from the CHR sequence can bind to the NHR region of gp41, forming an exogenous six-helix bundle, which can effectively prevent the viral membrane fusion process. Enfuvirtide (T20) is the only fusion inhibitor approved for clinical use by FDA in the United States so far.  In recent years, researchers have designed many novel membrane fusion inhibitors with high efficiency and broad spectrum. However, drug resistance is always an important problem to be solved in the development of fusion inhibitors. In the resistance screening of the novel short peptide membrane fusion inhibitor MTSC22, we found two compensatory mutations N126K and E136G, which located in the CHR region of gp41. These two mutations are often accompanied by NHR resistance mutations, and can improve the degree of drug resistance. In this paper, we mainly studied the effects of N126K and E136G compensatory mutations on the structure and function of envelop proteins, and explored the drug resistance mechanism of membrane fusion inhibitors.
In this study, the sensitivity of two compensatory mutations, N126K and E136G, to multiple membrane fusion inhibitors was identified. It was found that N126K mutation could cause mild resistance (2-5 times), E136G could cause moderate resistance (5-10 times), and N126K/E136G two-point mutation could cause strong resistance (more than 10 times). At the same time, eight strains of HIV-1 virus with different relapses and subtypes NL4-3, 92RW020, JRFL, AC10.29, REJO4541, QH0692, ZM53M.PB12, and ZM109F.PB4 were selected to construct plasmids with N126K and E136G compensatory mutations, respectively.  And the effects of N126K and E136G on the sensitivity of fusion inhibitors were determined. Studies have shown that the compensatory mutation does not affect the expression and processing of gp41, but can affect the infection ability to target cells. In the study of structural biology, N126K and E136G mutant peptides were synthesized using C34 as the template, and the secondary structure and stability were detected by circular dichromatography and isothermal calorimetry. The results showed that the stability and binding constant of 6-HB contain N126K was higher than wild type, while the 6-HB contain E136G mutant was less stable but binding constant increased. The crystal structure analysis reflects the structural changes of gp41 directly: N126K can change the conformation of surrounding amino acids; after lysine K replaces asparagine N, it can form a hydrogen bond with the upstream residue E123, which enhances the stability of CHR itself. The change of side chain position of E123 can make the hydrophobic pocket bind to the pocket binding region more closely. At the same time, the π bond formed between Y127 and H53 can stabilize the 6-HB. E136G mutation leads to the destruction of the ionic bond between E136 and H132 and the decline of helical stability, but at the same time, the ionization repulsion between E49 and E136 disappears, and the binding force of NHR and CHR is enhanced. Second, this study also explored the effect of natural mutations at N126 and E136 sites on virus function, and found that N126Y can significantly improve the stability of the 6-HB, leading to strong drug resistance against membrane fusion inhibitors. Finally, we identified the inhibitory activities of C34 peptides and T20 peptides with N126K and N126Y mutations against different subtypes of HIV-1 virus and T20 resistant strains, and found that both mutations could slightly increase the antiviral activity of C34 peptides. N126Y mutation could increase the T20 activity, while N126K may decrease the T20 activity.
The study on the structure and function of gp41 is an important precondition for elucidating the mechanism of action and drug resistance of membrane fusion inhibitors. We clarified the influence of N126K and E136G on the virus function and membrane fusion inhibitors’ sensitivity, and we also analyzed their mechanism with the structural biology. In addition, based on the effect of N126K and N126Y on the stability of the six helix, the inhibitory activities of C34 and T20 with N126K and N126Y mutations were also identified. Such research will enrich the understanding of the structure and function relationship of envlope of HIV-1, deepen to the mechanism of fusion inhibitors, helped to understand the mechanism of drug resistance, re-examine the interaction of virus and fusion inhibitors from multiple perspectives, and provide a new idea for the development and application of peptide membrane fusion inhibitors.