中东呼吸综合征冠状病毒受体结合的分子机制研究

发布时间：2018-06-27 08:08

本文选题：中东地区呼吸综合征冠状病毒MERS-CoV + 受体结合区(RBD)　；参考：《安徽大学》2014年硕士论文

【摘要】：冠状病毒(coronavirus)是一类广泛存在的、对人及家畜具有重大潜在威胁的病原体。该种病毒具有广泛的组织亲嗜性,是所有RNA病毒中基因组最大的病毒(基因组约有30kb)。2012年9月在中东发现的中东呼吸综合征冠状病毒(Middle East respiratory syndrome coronavirus, MERS-Co V)可以引起人体严重的肺部疾病,其临床表现为急性呼吸窘迫综合征,并伴有多器官功能衰竭,是继SARS-CoV之后的第二大高致病性冠状病毒。该病毒自2012年9月被确认,截至今年5月8日,共有536例确诊病例,死亡率高达30.7%。因此,MERS-CoV已经成为一种严重威胁人类健康与生命安全的新型冠状病毒。国际病毒分类委员会(The International Committee on Taxonomy of Viruses, ICTV)将冠状病毒分为四个属：α,β,γ和δ冠状病毒属。p冠状病毒属中有四个亚群(A-D),通过比对MERS-CoV和其他p冠状病毒基因序列发现,MERS-CoV与SARS-CoV中较为保守的核酸复制酶序列仅有小于50%的氨基酸相同,而与从蝙蝠分离到的HKU4和HKU5亲缘关系更近,因此,ICTV将MERS-CoV归为p冠状病毒属C亚群的一个新种,也是第一种能感染人类的C亚群p冠状病毒,而SARS-CoV则属于p冠状病毒属B亚群。刺突蛋白(Spike glycoprotein, S)是冠状病毒表面参与受体结合、膜融合和病毒侵入宿主细胞的主要膜蛋白,也是宿主中和性抗体的主要靶分子。成熟的S蛋白通常会在宿主蛋白酶的作用下裂解成两个亚基：S1和S2。其中,S1负责受体的识别,而S2启动病毒与宿主的膜融合。S1亚基又包括两个相对独立的区域,N-端结构域(N-terninal domain,NTD)和C-端结构域(C-terminal domain, CTD),不同的冠状病毒通常利用这两个结构域之一作为病毒的受体结合区(recepter binding domain, RBD)。S蛋白与病毒的高致病性密切相关,也是宿主选择性的决定性因素之一。有研究表明,冠状病毒的S蛋白能够识别细胞表面的蛋白和一些糖脂的糖基,如5-N-羟乙酰神经氨酸和5-N-乙酰神经氨酸等,作为病毒的受体或共受体。目前已有两类宿主酶蛋白被鉴定为冠状病毒的受体,包括血管紧张素转换酶2(ACE2)作为SARS-CoV和人类冠状病毒HCoV-NL63的受体,氨肽酶N(APN)作为猪呼吸冠状病毒PRCV的受体。有关这些受体和病毒S蛋白的相互作用模式都得到了详细的阐述。我们首先利用哺乳动物细胞表达了MERS-CoV S蛋白的S1亚基、S1亚基的NTD和CTD的Fc融合表达蛋白。用流式方法检测了这三种蛋白与表达在BHK细胞表面的CD26分子的结合。结果显示S1蛋白和S1蛋白的CTD结合CD26分子,而S1蛋白的NTD不结合。这表明MERS-CoV S蛋白的受体结合区位于S1蛋白的C端,由此我们将该区域定义为MERS-CoV RBD。然后,我们用杆状病毒表达系统表达了MERS-CoV RBD蛋白,并解析了其晶体结构。结构显示MERS-CoV RBD结构由一个核心区和一个外展区域构成,核心区与先前解析的SARS-CoV的RBD有极高的结构相似性,而外展区结构则完全不同。我们进一步用杆状病毒表达系统表达了CD26的胞外段蛋白,并通过实时表面等离子共振技术测定了MERS-CoV RBD和CD26之间的亲和力,KD=16.7nM,随后MERS-CoV RBD与CD26复合体晶体结构也被成功解析。结构显示：MERS-CoVRBD以其外展区域与CD26分子的“p-螺旋桨样”结构域结合,呈现出一种典型的反式互作模式。CD26分子以二聚体形式存在于细胞表面,每个CD26分子结合一个MERS-CoV RBD;而两者的结合主要是通过一系列亲水氨基酸介导的氢键和盐桥实现的。进一步分析表明,MERS-CoV RBD在CD26分子上的结合位点与ADA在CD26中的识别位点高度重叠。因ADA作为CD26的天然配体,在T细胞激活过程中发挥重要作用,因此,我们的结果暗示MERS-CoV S蛋白对CD26的结合很可能对细胞免疫具有一定的影响。在本研究中,我们对MERS-CoV NTD以及SARS-CoV NTD的结构与功能进行了初步探索。任何病毒入侵宿主细胞时都需要特异的病毒配体与宿主受体的识别和互作,而这一相互作用在很大程度上决定了此种病毒的组织亲嗜性和人群易感性。因此,MERS-CoV RBD与其受体的复合物结构的解析,为以此为结构基础的小分子抑制剂的设计,以及以RBD为基础的预防性疫苗的设计,都具有重要的指导意义。这将为预防像SARS、MERS这样的突发性传染病建立良好的预报预警机制,从而在应对冠状病毒再次引起的突发公共卫生事件提供帮助。
[Abstract]:Coronavirus (Coronavirus) is a kind of widespread, potential threat to human and domestic animals. This virus has extensive tissue affinity and is the largest genome of all RNA viruses (genome 30KB) of the Middle East respiratory syndrome coronavirus (Middle East respiratory) found in the Middle East in September of.2012. Syndrome coronavirus, MERS-Co V) can cause serious lung disease with acute respiratory distress syndrome and multiple organ failure. It is the second most highly pathogenic coronavirus after SARS-CoV. The virus was confirmed in September 2012 and was cut to May 8th this year, with a total of 536 confirmed cases and high mortality rate. As a result of 30.7%., MERS-CoV has become a new coronavirus which seriously threatens human health and life safety.
The The International Committee on Taxonomy of Viruses (ICTV) divides the coronavirus into four genera: alpha, beta, gamma and delta coronaviruses of the genus.P coronavirus, four subgroups (A-D), which are found by comparison to MERS-CoV and other p coronavirus gene sequences. The enzyme sequence is only less than 50% of the same amino acid, but closely related to HKU4 and HKU5 isolated from bats. Therefore, ICTV belongs to a new species of C subgroup of the P coronavirus, the first C subgroup P coronavirus that can infect human, and SARS-CoV belongs to the B subgroup of the P coronavirus.
Spike glycoprotein (S) is the main membrane protein of the coronavirus on the surface of the coronavirus, which participates in receptor binding, membrane fusion and virus invasion of host cells. It is also the main target molecule of the host neutralization antibody. The mature S protein usually cleavages into two subunits under the action of the host protease: S1 and S2., and S1 is responsible for the recognition of the receptor, and S2 The membrane fusion.S1 subunit of the virus and the host consists of two relatively independent regions, the N- terminal domain (N-terninal domain, NTD) and the C- terminal domain (C-terminal domain, CTD). The different coronavirus usually uses one of these two domains as the receptor junction of the virus (recepter binding domain,) and the high virus The pathogenicity is closely related and also one of the decisive factors of host selectivity. Some studies have shown that the S protein of the coronavirus can identify the protein on the surface of the cell and some glycosylated glycosyl groups, such as 5-N- hydroxyacetyl neuroammonia and 5-N- acetyl neuroammonia, as a receptor or co receptor of the virus. Currently, two types of host enzyme proteins have been identified. The receptor for the coronavirus, including angiotensin converting enzyme 2 (ACE2), is a receptor for SARS-CoV and human coronavirus HCoV-NL63, and aminopeptidase N (APN) acts as a receptor for the PRCV of porcine respiratory coronavirus. The interaction patterns of these receptors and viral S proteins have been described in detail.
We first used mammalian cells to express the S1 subunit of MERS-CoV S protein and the Fc fusion protein of S1 subunit NTD and CTD. The combination of these three proteins with CD26 molecules expressed on the surface of BHK cells was detected by flow method. The results showed that the CTD binding molecules of S1 protein and S1 protein were not combined. The receptor binding area of the S-CoV S protein is located at the C end of the S1 protein, thus we define the region as MERS-CoV RBD., and we express the MERS-CoV RBD protein with the baculovirus expression system and analyze its crystal structure. The structure shows that the MERS-CoV RBD structure is made up of a core area and an abduction region, and the core area and the S analytic S. The RBD of ARS-CoV has very high structural similarity, and the abductor region structure is completely different. We further express CD26 extracellular segment protein by baculovirus expression system, and determine the affinity between MERS-CoV RBD and CD26 by real time surface plasmon resonance technique, KD=16.7nM, and then the crystal structure of MERS-CoV RBD and CD26 complex also The structure shows that MERS-CoVRBD combines its abduction area with the "p- propeller like" domain of the CD26 molecule and presents a typical trans interaction model that exists on the cell surface in the form of two polymer, and each CD26 molecule combines a MERS-CoV RBD, and the combination of the two is mainly through a series of hydrophilic ammonia. Basic acid mediated hydrogen bonds and salt bridges are implemented. Further analysis shows that the binding site of MERS-CoV RBD on CD26 is highly overlapping with the identification site of ADA in CD26. Because ADA acts as a natural ligand for CD26, it plays an important role in the activation of T cells. Therefore, our results suggest that MERS-CoV S proteins are likely to be compatible with CD26. Cell immunity has a certain influence.
In this study, we preliminarily explored the structure and function of MERS-CoV NTD and SARS-CoV NTD. When any virus invades the host cells, the identification and interaction of specific viral ligands and host receptors are needed, and this interaction determines the tissue affinity and susceptibility to the population to a large extent. Therefore, MER The analysis of the complex structure of S-CoV RBD and its receptor is of great guiding significance for the design of small molecular inhibitors based on the structure and the design of a preventive vaccine based on RBD. This will establish a good prediction and early warning mechanism for the prevention of sudden infectious diseases such as SARS and MERS, so as to respond to the coronavirus. The public health incident caused by the accident is again helpful.
【学位授予单位】：安徽大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R373

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