HIV-1对N融合肽抑制剂抵抗机制的研究

Peptides derivated from the N- or C-terminal heptad-repeat region (HR1 or HR2) of HIV gp41 can block HIV infection in a dominant-negative manner by interfering with refolding of the viral (endogenous) HR1 and HR2 to form a six-helix bundle (6HB) that drives fusion between viral and host cell membranes. HR1 peptides can inhibit HIV, but they are less potent than HR2 peptides unless restrained by modifications that stabilize a trimeric, coiled-coil structure. HR1 peptides can form coiled-coil trimer to bind to the viral gp41 HR2 to block viral gp41 6HB formation, or interact with the viral gp41 HR1 to form a non-functional heterologous coiled coil. Addition of an isoleucine zipper (IZ)-trimerization motif to an HR1 peptide, such as N36, can facilitate the formation and stabilization of the trimeric HR1-peptides (e.g., IZN36), resulting in the enhancement of anti-HIV potency..We previously compared the antiviral activity of N36 and IZN36 against R5-tropic viruses. We found that N36 selected two genetic pathways with equal probability, each defined by an early mutation in either HR1 or HR2, while IZN36 preferentially selected the HR1 pathway. Each HR mutation enhanced the thermostability of the endogenous 6HB, potentially allowing the virus to simultaneously escape inhibitors targeting either gp41 HR1 or HR2. Since X4- and R5-tropic viruses have significant differences in the HR2 sequences, contents of α-helicity and stability of coiled-coil and 6HB, it is necessary to analyze the resistance pathways and mechanisms of X4-tropic viruses to N peptide inhibitors. .In the present study, N36- and IZN36-resistant X4-tropic viruses will be selected and the resistance mutations in envelope glycoproteins will be identified by sequence analysis. Then, the contribution of the mutations in virus resistance will be confirmed. HIV-1 isolates epidemic in China will be also used to elucidate the significance of the resistance mutations to the local virus strains. Finally, the resistance mechanisms will be investigated by kinetic assay, biophysical characterization of coiled-coil and 6HB formation and structural modeling. Based on these findings we will identify the regions of plasticity in the highly conserved gp41 that modulate virus entry and facilitate virus escape from the inhibition by HR1 peptides. We will confirm the resistance potency of these regions to HR1 peptides on HIV-1 isolates in China. These findings will provide useful information for rational design of novel HIV fusion inhibitors.

HIV-1包膜蛋白gp41 N端七重复序列（HR）的模拟肽（N36）及三聚体（IZN36）可结合HIV-1的HR2或HR1，阻断病毒六螺旋束（6HB）形成，抑制病毒融合。HIV-1根据辅助受体不同主要分为R5嗜性病毒和X4嗜性病毒。我们前期发现R5病毒可通过两种途径抵抗N36，通过其中一种途径抵抗IZN36，抵抗的主要机制均为增加6HB稳定性。R5与X4病毒的HR2序列、卷曲螺旋和6HB的稳定性均差异很大，因此X4病毒对N肽类抑制剂的抵抗途径及其机制是否与R5病毒相同尚不清楚。本项目拟筛选对N36和IZN36抵抗的X4病毒株，分析包膜蛋白序列、找出变异特征；通过病毒融合速度、卷曲螺旋和6HB的稳定性及分子构象模拟，明确X4病毒抵抗N肽的机制；找到gp41的可塑性区域，明确中国流行株这些位点对N肽抵抗的普遍意义。项目的完成将为优化设计有针对性的、更为高效广谱的肽抑制剂奠定理论基础。

HIV-1包膜蛋白gp41 N端七重复序列（HR）的模拟肽（N36）及其三聚体（IZN36）可结合HIV-1的HR2或HR1，阻断病毒六螺旋束（6HB）形成，抑制病毒融合。本项目筛选了对N36和IZN36抵抗的HIV X4嗜性病毒株(LAI株)，共获得9个抵抗突变株的包膜（Env）。Env序列比对发现三种抵抗途径，病毒以两种途径抵抗N36的抑制，分别为途径一E560K突变和途径二E648K突变的途径；而IZN36病毒抵抗突变株的包膜蛋白只利用一种途径抵抗，即途径三在CHR处的N637K位点的突变。值得注意的是，尽管N36需要高浓度达微摩（uM）水平才能抑制病毒生长，但筛选获得对N36的病毒抵抗株相对困难，当N36浓度超过起始IC90的5.5 ～ 6倍时所有培养病毒均不能生长，提示我们N36可能作为更加有效的抗病毒药物进一步研发。. 利用假病毒感染系统和细胞与细胞融合系统检测各变异株及gp41变异位点的生物学功能。IZN36筛选的Env对于同源肽有2.46 ～ 5.33倍的抵抗；N36筛选的Env只有在融合实验系统中展示出对N36较弱的抵抗（< 2倍）。所有筛选的Env均无明显抵抗T20的作用，但均显示出对sCD4更加敏感。拆分gp41的变异位点并检测其对于抵抗的贡献。IZN36筛选株按照突变出现的顺序，对IZN36的抵抗逐渐增加；而N36抵抗株，除主要变异E560K和E648K抵抗N36抑制作用外，其他伴随突变没有明显增加对N36的抵抗，这也和筛选活病毒时抵抗突变株产生相对困难相吻合；IZN36筛选的Env gp41的部分突变对T20有交叉抵抗作用。找到gp41的可塑性区域，分析对于N- 和C- 肽抑制剂的热变异位点E560K/D/G变异对于融合肽抑制剂抵抗的意义。. 明确HIV-1 X4嗜性病毒对N36和IZN36的抵抗机制，为HIV融合肽抑制剂的优化与研发提供理论基础。通过圆二色谱分析明确病毒变异途径与HR1和HR2形成的6HB的稳定性高度相关，而与HR1形成的卷曲螺旋稳定性无明显相关；分子构象模拟分析结果显示病毒抵抗突变株可能通过改变病毒gp41部分所带的电荷量从而影响病毒与融合肽抑制剂结合；病毒抵抗机制与细胞膜融合速度相关，突变的病毒包膜倾向于以更快的速度进入靶细胞。