HIV-1 env基因准种适应性变异在HIV相关神经认知紊乱中的作用及机制研究

The mechanism and impact of HIV-1 env quasispecies variation on HIV associated neurocognitive disorder (HAND) remains poorly understood. Our previous studies indicated that viral molecular heterogeneity between specific tissues is greater in patients with HAND compared to non-HAND patients, and discordant patterns of genetic evolution and CNS-related mutation sites occur in the HAND patients. Therefore, we believe that the CNS-specific HIV-1 strains in HAND patients were induced through pressures of immune and CNS environment, which result in new adaptive evolution and get compensatory HAND related adaptive mutations, and then induce apoptosis by inhibiting autophagy to affect the development of HAND. In this project, firstly, to screen the potential HAND-related adaptive mutation sites using deep sequencing and bioinformatics technology; Secondly, to obtain the HAND-related and CNS-specific adaptive mutation sites which changed in infectivity, fitness and sensitivity against neutralizing antibodies using point mutations, pseudovirus and structure mapping analysis methods. Thirdly, to explore characteristics and mechanism for neurotoxicity of envelope protein gp120 and gp41 that included HAND-related and CNS-specific adaptive mutation sites, disclose the impact of HIV-1 genetic variation on envelope protein neurotoxicity and mechanism for the role of autophagy play in apoptosis induced by mutation sites. This study reveals the mechanism for development of HAND from the perspective of viral fitness, which will provide a scientific data for clinical treatment and vaccine development.

中枢神经系统(CNS)中HIV-1进化变异对HIV相关神经认知紊乱(HAND)产生的影响及作用机制尚不清楚。我们前期研究发现HAND患者组织间病毒分子异质性较高，其CNS中的HIV-1 env准种存在不一致进化模式及特征性突变。HIV-1 env编码蛋白能够诱导神经细胞凋亡，其介导的自噬为触发细胞凋亡所需。因此我们提出：HIV-1准种在机体免疫和CNS共同压力下不断进化并获得新的适应性突变，通过抑制神经细胞自噬来诱导细胞凋亡，从而影响HAND发生发展。本研究1)利用深度测序及信息分析筛选与HAND相关的HIV-1 env准种潜在适应性位点；2)将候选位点构建重组假病毒进行病毒体外适应性改变的实验验证；3)利用基因重组技术对携带2)中确证位点的包膜蛋白神经毒性特点及自噬在突变位点诱发凋亡改变中作用机制进行探讨。该研究从病毒适应性角度揭示HAND发生机制，为临床治疗方案调整和疫苗研发提供建议。

HAND患者中枢神经系统(CNS)作为病毒潜藏库存在特征性突变HIV-1毒株， HIV-1进化变异对HIV相关神经认知紊乱(HAND)产生的影响及作用机制尚不清楚。HIV-1 在中枢神经系统中的适应性可通过env 基因的改变而获得，存在经进化阳性选择的中枢特异的突变位点，而这些突变位点的出现可能会导致病毒适应性变化。因此我们筛选与HAND相关的HIV-1 env准种潜在适应性位点，将候选位点构建重组假病毒进行病毒体外适应性改变的实验验证，并利用基因重组技术对携带适应性突变位点的包膜蛋白的神经毒性转录组特征及介导的自噬凋亡予以探讨。我们利用深度测序及信息分析结合前期一代测序结果筛选出T399N、S446T、F396N、K429G、K337G、T319A、Q389K、N407T、G410E及T413N为HAND相关的中枢特征性HIV-1 env 准种适应性突变位点。携带不同神经特征性突变的包膜假病毒感染能力各异，部分突变假病毒保持了与野生型同样或更高的感染性，并对PGT121、PGT135、VRC01、2G12、10E8和12A21等代表性bmNAbs产生中和抗性，而分子构象模拟结果显示氨基酸侧链折叠可能导致空间位阻增加以影响其可接触残基与广谱中和抗体发挥中和作用。Q389K、T399N、T413N和G410E和野生型构建表达载体转染U87神经细胞系进行神经毒性转录组特征分析，携带突变的包膜蛋白神经毒性凋亡和自噬水平存在差异。Q389K和T413N相对于WT差异基因的GO和/或KEGG富集结果无统计学意义。对多个中和抗体具有中和抗性的T397N包膜蛋白可通过凋亡信号通路、自噬通路和P53信号通路使RIPK2、RPS6KA2、GABARAPL1、GADD45A及MCM4等基因失调以发挥神经毒性作用。本研究从病毒适应性角度揭示HIV包膜蛋白神经毒性作用机制，为临床治疗方案调整和疫苗研发提供线索。