宿主限制因子APOBEC3F抑制HIV-1病毒相关机制的研究

发布时间：2018-02-11 08:35

本文关键词： 人免疫缺陷病毒 APOBEC3F Vif 逆转录酶　出处：《北京协和医学院》2013年博士论文　论文类型：学位论文

【摘要】：HIV-1病毒的感染是引起AIDS的主要原因,对HIV-1的治疗和预防也因此成为治疗AIDS的根本途径。HIV-1感染的有效治疗已经取得了显著成就,但仍面临一些挑战,如病毒耐药性以及病毒感染难以完全治愈等,因而继续研究更为有效的治疗策略十分必要。宿主中存在可以对HIV-1病毒产生限制作用的分子,被称为宿主限制因子,同时病毒也具有相对的拮抗措施而使限制因子无效。有关宿主限制因子与病毒间相互拮抗机制的研究能够为HIV-1的防治提供新的研究途径。以APOBEC3F和APOBEC3G为代表的APOBEC3蛋白家族是一类重要的HIV-1的宿主限制因子,它们可通过脱氨酶的致突变作用和非脱氨酶作用的机制限制病毒复制。但HIV-1可通过自身的Vif蛋白与APOBEC3F的相互作用引导其被降解限制其作用的产生。APOBEC3F被包装进入病毒颗粒是其发挥抗病毒作用的前提条件,本文首先对APOBEC3F的包装机制进行研究。对APOBEC3F缺失突变包装效果的分析显示,APOBEC3F的104-157位序列是其进入病毒颗粒的关键区域。同时,对HIV-1病毒Gag结构缺失突变的研究显示,与APOBEC3F包装进入病毒直接相关的区域是Gag的NC结构域。进一步的结果说明,NC的锌指结构以及第一锌指结构两侧的碱性氨基酸对APOBEC3F和APOBEC3G进入病毒均重要,同时竞争性包装实验结果也说明二者的包装机制存在较高相似性。上述工作阐明了APOBEC3F的104-156氨基酸与病毒Gag NC结构域的相互作用对包装过程起决定作用。除依赖脱氨酶机制外,APOBEC3蛋白也具有其他抑制HIV-1病毒的机制。APOBEC3F可以阻断HIV-1逆转录过程,但其具体机制还不明确。本研究中首先确定了在病毒颗粒内APOBEC3F和RT存在相互作用。APOBEC3F和RT各自的缺失突变结果明确了APOBEC3F的104-1 56位氨基酸以及RT的Thumb结构域是APOBEC3F和RT与相互作用关键区域。APOBEC3F 1-156仍具有较强抗病毒能力,且该活性与其脱氨酶活性无关。上述结果提示APOBEC3F的不依赖嘧啶脱氨酶活性的HIV-1抗病毒能力可能源于与RT的相互作用而影响逆转录过程。HIV-1的Vif蛋白可通过与APOBEC3F的相互作用引导其被降解等机制而消除其抗病毒作用。本文中采用模拟计算方式预测关键作用点。根据已报道的APOBEC3C的结构,通过同源性分析,建立了APOBEC3F碳端结构的模型。再通过与Vif模型模拟相互作用,可以对Vif与APOBEC3F作用面上的关键位点进行分析预测。预测结果通过生物学研究方法加以证实,确定了部分未见报道的APOBEC3F结构中与Vif作用的重要位点。
[Abstract]:The infection of HIV-1 virus is the main cause of AIDS. Therefore, the treatment and prevention of HIV-1 have become the basic way to treat AIDS. HIV-1 infection has made remarkable achievements, but it still faces some challenges. It is necessary to continue to study more effective treatment strategies, such as virus resistance and the difficulty of virus infection to be completely cured. There are molecules in the host that can limit the effect of HIV-1 virus, so they are called host limiting factors. At the same time, the virus also has relatively antagonistic measures to make the limiting factor ineffective. The study on the antagonistic mechanism between the host limiting factor and the virus can provide a new way for the prevention and treatment of HIV-1. The APOBEC3 eggs represented by APOBEC3F and APOBEC3G can be used as the representative. White family is an important host limiting factor of HIV-1. They can limit virus replication through the mutagenicity of deaminase and the mechanism of non-deaminase action. However, HIV-1 can lead it to be degraded by its own Vif protein interacting with APOBEC3F. APOBEC3F can be packaged into the disease. Du granule is the precondition for its antiviral effect. In this paper, we first studied the packaging mechanism of APOBEC3F. The analysis of the packaging effect of APOBEC3F deletion mutation showed that the 104-157 position sequence of APOBEC3F was the key region to enter the virus particles. Meanwhile, the study on the Gag deletion mutation of HIV-1 virus showed that, The direct correlation between APOBEC3F packaging and virus entry is the NC domain of Gag. The further results show that the zinc finger structure of Gag and the basic amino acids on both sides of the first zinc finger structure are important for APOBEC3F and APOBEC3G to enter the virus. At the same time, the competitive packaging experiment results also showed that the packaging mechanism was similar. The above work indicated that the interaction between APOBEC3F 104-156 amino acids and the Gag NC domain of the virus played a decisive role in the packaging process. APOBEC3 protein also has other mechanisms of inhibiting HIV-1 virus. APOBEC3F can block the reverse transcriptional process of HIV-1. However, the specific mechanism is not clear. In this study, we first identified the interaction between APOBEC3F and RT in virus particles. The deletion mutation of APOBEC3F and RT determined that the 104-156 amino acid of APOBEC3F and the Thumb domain of RT were APOBEC3F and RT. APOBEC3F1-156 still has strong antiviral ability. The above results suggest that the antiviral ability of HIV-1, which is independent of pyrimidine deaminase activity, may be due to the interaction with RT and the effect of Vif protein on the reverse transcription process. HIV-1 may be related to the phase of APOBEC3F. The interaction leads to the mechanism of degradation and eliminates the antiviral effect. In this paper, the key points of application are predicted by simulation. According to the reported structure of APOBEC3C, By homology analysis, the carbon terminal structure model of APOBEC3F was established. By simulating the interaction with Vif model, the key sites on the interaction surface between Vif and APOBEC3F could be analyzed and predicted. The predicted results were confirmed by biological methods. Some unreported important sites of APOBEC3F structure interacting with Vif were identified.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R512.91

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