HIV-1 gp120来源的天然降解淀粉样多肽的发现及其促进HIV-1感染的研究

发布时间：2018-09-19 12:12

【摘要】：研究背景:在HIV病毒感染靶细胞的过程中,人体内天然存在的多种淀粉样多肽,如SEVI(精液源性的病毒增强因子),Aβ(老年痴呆蛋白)等可显著提高病毒感染靶细胞的效率。本实验室在前期研究中发现人工合成的位于gp120MNβ区的多肽能形成淀粉样纤维并能促进HIV病毒的感染,提高ARV药物的IC50。在人体复杂的生理环境中,是否存在来源于病毒本身的感染增强因素?淀粉样多肽在HIV感染的过程中是否天然存在?诱导其形成的因素是什么?gp120在体内多种酶的作用下能否降解成为小的多肽片段并产生一定的生理效应?研究目的:拟采用多肽组学结合高分辨质谱法(LC-MS/MS)分别从gp120体外酶解产物,病毒感染靶细胞上清液,HIV阳性患者的血浆及淋巴漏液中寻找HIV包膜蛋白gp120来源的淀粉样多肽。分别使用免疫荧光,免疫胶体金技术确定病毒感染上清液及患者淋巴漏液中淀粉样纤维的存在与组成,免疫组化结合刚果红染色对患者的淋巴结进行分析,确定患者淋巴结中淀粉样纤维的存在与组成。通过病毒感染增强实验评价所鉴定的多肽对病毒感染靶细胞及临床抗病毒药物IC50的影响。本研究为深入理解HIV病毒的非基因耐药新机制,优化HIV临床治疗方案提供理论参考。方法与结果:为检测体液中酶对gp120稳定性的影响,分别选用α-L-AFU、Thrombin B、Plasmin、CathepsinB四种酶对gp120JRFL蛋白进行体外酶促反应,10%SDS-PAGE电泳结合考马斯亮蓝染色发现gp120蛋白在不同时间点均存在不同程度降解,透射电镜(TEM)下酶解产物可自发形成淀粉样纤维。为验证HIV病毒感染靶细胞的过程中能否自发产生gp120来源的多肽,我们在体外模拟了病毒体内的感染过程。分别采用不同嗜性病毒X4嗜性(HIVIIIB)感染MT-2细胞及R5嗜性(HIVSF162)病毒感染CEMx174 5.25M7细胞,感染7天后离心除去细胞及细胞碎片收取上清。免疫荧光及免疫胶体金法检测到感染上清中存在gp120来源的淀粉样纤维。为验证临床样本中是否存在gp120来源多肽,是否存在促进病毒感染的因素。免疫荧光与免疫胶体金检测到淋巴漏液中存在gp120来源的淀粉样纤维。免疫组化与刚果红染色实验表明HIV阳性患者淋巴结中gp120蛋白表达量较高且与淀粉样物质存在共定位。进一步实验发现灭活的淋巴漏液可以浓度依赖性的促进R5嗜性(HIVSF162)克隆病毒感染靶细胞。为鉴定多肽具体序列,分别使用超滤法处理gp120体外酶解产物,OMC材料吸附,免疫亲和,乙腈沉淀法结合超滤法对感染上清,淋巴漏液进行样本预处理。高分辨质谱LC-MS/MS鉴定并发现了一系列gpl20来源多肽。序列同源比对分析多肽在gp120上的定位及可能的二级结构,选择性合成了具有β结构域的部分多肽。采用刚果红,硫磺素T特异性染色,透射电镜TEM,圆二色谱CD等验证多肽是否可形成淀粉样纤维并具有典型的β折叠。病毒感染活性实验表明 GAP380-396、GAP229-244、GAP354-369 多肽对克隆病毒,GAP380-396、GAP229-244多肽对临床株病毒具有感染增强作用。GAP380-396、GAP229-244多肽使四种临床抗病毒药物的IC50升高。为解析多肽增强病毒感染的机制,用Zetasizer Nano ZS电势测量仪检测了GAP380-396、GAP229-244多肽的电势,竞争性结合实验检测了电荷因素对病毒感染的作用。发现CS对带有正电荷的GAP229-244多肽的病毒增强感染能力具有部分拮抗作用,对负电荷GAP380-396及几乎不带电荷的EP2多肽无影响。采用Virus pull-down进一步检测多肽纤维沉淀与上清对病毒的感染性,发现仅有沉淀部分可增强病毒感染,说明多肽纤维可捕获并结合病毒。Cell-binding及荧光病毒结合实验发现多肽可通过结合病毒与细胞膜来增强病毒感染。荧光多肽显示不同多肽之间可形成杂合多肽且仍保持增强病毒感染能力,多肽之间可能具有叠加效应。XTT实验显示多肽与病毒对Tzm-bl细胞无毒性。结论:分别从HIV gp120的体外酶解产物,感染上清,临床样本淋巴漏液中鉴定出来源于gp120β-片层能形成淀粉样纤维的多肽GAPs及能促进病毒感染的淀粉样纤维GEVI。体液中存在的酶的催化作用可能是gp120降解形成多肽的原因之一。GAPs多肽可促进HIV病毒感染及并拮抗抗病毒药物活性,且不同多肽之间具有增强感染的叠加效应。这种感染增强效应可能是静电及其疏水效应共同作用的结果。本研究为深入理解HIV病毒感染增强效应,非基因突变引发的耐药问题提供理论依据,为HIV的临床治疗提供了改进方向。
[Abstract]:BACKGROUND: Many amyloid peptides, such as SEVI (semen-derived viral enhancer factor) and A-beta (alzheimer's disease protein), can significantly improve the efficiency of HIV infection in target cells. In our previous study, we found that synthetic peptides located in gp120MN-beta region can form. Amyloid fibers can promote HIV infection and increase IC50 of ARV drugs. Is there an infection-enhancing factor originating from the virus itself in the complex physiological environment of the human body? Is amyloid polypeptides present naturally in the process of HIV infection? What are the factors inducing the formation of amyloid polypeptides? Can gp120 be reduced by a variety of enzymes in the body? Objective: To search for amyloid peptides from the enzymatic hydrolysate of gp120 in vitro, the supernatant of target cells infected by virus, the plasma and lymphatic leak of HIV-positive patients, respectively, by using high resolution mass spectrometry (LC-MS/MS). The presence and composition of amyloid fibers in virus infection supernatant and lymphatic leak were determined by fluorescence and immunogold assay. The presence and composition of amyloid fibers in lymph nodes were determined by immunohistochemistry combined with Congo red staining. Methods and Results: In order to detect the influence of enzymes in body fluid on the stability of gp120, four enzymes, alpha-L-AFU, Thrombin B, Plasmin and CathepsinB, were used to determine the stability of gp120 JR. FL protein was enzymatically hydrolyzed in vitro. 10% SDS-PAGE electrophoresis combined with Coomassie brilliant blue staining showed that gp120 protein was degraded in varying degrees at different time points, and amyloid fibers could be formed spontaneously by enzymatic hydrolysates under transmission electron microscopy (TEM). The infection process in vivo was simulated in vitro. MT-2 cells and CEMx174 5.25M7 cells were infected with different trophotropic viruses X4 (HIVIIIB) and R5 (HIV SF162) respectively. After 7 days of infection, CEMx174 5.25M7 cells were centrifuged to remove cells and cell debris for supernatant collection. Immunohistochemistry and Congo red staining showed that the expression of gp120 protein in lymph nodes of HIV-positive patients was higher than that in lymph nodes of HIV-positive patients. It was found that inactivated lymphatic leakage could promote R5 tropism (HIV SF162) in a concentration-dependent manner. To identify the specific sequence of the polypeptide, the enzymatic hydrolysates of gp120 were treated by ultrafiltration, OMC adsorption, immunoaffinity, acetonitrile precipitation and ultrafiltration respectively. A series of polypeptides derived from gpl20 were identified by high resolution mass spectrometry LC-MS/MS. Homology alignment analysis showed that the polypeptides were localized on gp120 and possibly secondary structures. Some polypeptides with beta domain were selectively synthesized. Specific staining of Congo red, sulfur T, TEM and circular dichroism were used. The results of virus infection activity test showed that GAP380-396, GAP229-244, GAP354-369 polypeptides could enhance the infection of cloned viruses, GAP380-396, GAP229-244 polypeptides to clinical strains of viruses. GAP380-396, GAP229-244 polypeptides increased the IC50 level of four clinical antiviral drugs. In order to elucidate the mechanism of polypeptide-enhanced viral infection, the potential of GAP380-396 and GAP229-244 peptides was detected by Zetasizer Nano-ZS potentiometer, and the effect of charge factor on viral infection was tested by competitive binding assay. Virus pull-down was used to further detect the viral infectivity of polypeptide fiber precipitation and supernatant. It was found that only part of the precipitation could enhance viral infection, indicating that polypeptide fibers could capture and bind to viruses. Cell-binding and fluorescent viral binding experiments showed that polypeptides could bind to viruses. The fluorescent polypeptides showed that different peptides could form heterozygous polypeptides and still enhance the viral infection ability. The polypeptides may have superposition effect. XTT test showed that the polypeptides and viruses were non-toxic to Tzm-bl cells. Conclusion: The enzymatic hydrolysates of HIV gp120 in vitro, the supernatants of HIV gp120 infection, and clinical samples were obtained. The polypeptide GAPs derived from gp120 beta-lamellar amyloid fibers and the amyloid fibers GEVI that can promote virus infection were identified in the lymphatic leakage. The catalysis of enzymes in body fluid may be one of the reasons for the degradation of gp120 to form polypeptides. GAPs can promote HIV infection and antagonize the activity of antiviral drugs, and different polypeptides can promote the degradation of gp120 to form polypeptides. This study provides a theoretical basis for further understanding of HIV infection enhancement effect and drug resistance caused by non-gene mutation, and provides a direction for clinical treatment of HIV.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R512.91

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