丙型肝炎病毒高变区1中存在介导病毒入侵和免疫逃避的三个不同结构域

发布时间：2018-06-22 01:01

本文选题：丙型肝炎病毒 + 高变区1　；参考：《第二军医大学》2013年博士论文

【摘要】：丙型肝炎病毒(hepatitis C virus, HCV)属于黄病毒科、丙型肝炎病毒属,为有包膜的单正链RNA病毒。目前全球约有1.8亿人感染HCV,大多数为慢性感染,部分感染者可发展为肝硬化和肝细胞癌。HCV的RNA聚合酶缺少校对功能,致使HCV整个基因组具有很高的变异性,但HCV基因的变异呈不平衡分布.位于包膜蛋白2(E2)氨基端的27个氨基酸残基变异性最为突出,即HVRl。HIVR1高度暴露于包膜蛋白表面,其中含有中和抗体表位,可以诱导宿主产生针对该区域的抗体,产生的抗体可以高效中和HCV的感染,但HVR1的基因序列高度变异,先前由宿主免疫细胞产生的抗体不能识别变异后的HVRl,因此病毒得以逃避宿主细胞的免疫监控并在病人体内持续感染进而使感染慢性化。众多实验室都将HVR1作为HCV疫苗研发的重要靶标,但自1989年丙型肝炎病毒首次命名至今,仍没有一个真正有效的保护性疫苗问世。 HCV感染会引起肝内生化代谢紊乱,包括脂质代谢障碍和载脂蛋白合成异常,与其他病毒性肝炎相比,HCV感染慢性化后,中重度肝细胞脂肪变性包括胞质内脂滴聚集是其典型组织病理学特征。HCV是有包膜RNA病毒,其表面有不同数量的脂蛋白包裹,所以病人血清中纯化得到的HCV病毒颗粒呈现不同的密度分布,并且不同密度的病毒颗粒感染性也不同。有研究发现HVR1的删除会导致低密度病毒颗粒数目下降,也就是减弱了HCV的脂蛋白结合能力,可见HVR1在病毒与脂蛋白结合的过程中亦发挥重要的作用。多项研究证明HVR1同时还在与宿主SR-B1受体结合以及介导病毒入侵的过程中起关键作用,HVR1具有如此重要的生物学功能,那么其结构必然具有相对稳定性和保守性,虽然HVR1是HCV基因组中变异频率最高的部分之一,但对HCV一系列分离株的序列分析表明一些氨基酸残基的位点是高度、或者相对保守的。尽管人们已经发现HVR1同时行驶着诱导宿主免疫反应,脂蛋白结合以及介导病毒与受体结合的多重功能,但目前尚不清楚其结构与生物学功能的对应关系。一、HCV高变区1中介导病毒细胞入侵关键氨基酸残基的鉴定首先我们以HCVpp作为研究模型,以HCVla亚型的H77株为模式株,用单一氨基酸残基删除扫描的方法分析HVR1中27个氨基酸残基分别对HCV细胞入侵的影响,实验结果明确的鉴定出HVRl中介导HCV细胞入侵的关键氨基酸残基(Ala-14,Gly-15, Lys-25, Gln-26以及Asn-27),其中任意一个残基的删除对HCVpp感染性的影响都是致命的,相比之下,其他22个氨基酸残基的删除对HCVpp感染性并未造成明显影响。再用HCV入侵相关受体结合实验进行机制分析,我们证明这5位氨基酸残基介导了病毒包膜蛋白与宿主细胞SR-B1受体的结合。氨基酸残基合并删除实验进一步的证明了我们的结论,HVRl氨基端13个残基的删除以及第16-24位9个氨基酸残基的删除不会显著降低HCVpp的感染力。除了决定HCV细胞入侵的5个氨基酸残基之外,HVR1氨基端的13个氨基酸残基可以通过适当的突变,增强病毒与受体的结合能力从而增强病毒的细胞入侵能力,并且这一区域适当的突变还可以增强病毒的抗中和能力从而更利于病毒的免疫逃避和持续感染。二、HCV高变区1中诱导中和抗体产生的抗原表位的鉴定我们将HVR1分为氨基段,中段和羧基段三部分肽段分别与TRX融合表达后免疫家兔来检测HVR1的27个氨基酸残基在诱导宿主免疫反应过程中是否存在差异,我们发现HVRl的氨基端13个氨基酸残基不能诱导宿主产生抗体,而第16到24位的9个氨基酸残基是HVRl中唯一的中和表位,这9个氨基酸残基对于病毒细胞入侵无关紧要,但含有肝素结合位点,有利于HCV的粘附,更为重要的是,高密度脂蛋白(HDL)介导的HCV感染增强作用依赖于该9个氨基酸残基的存在,该九个残基还可通过与HDL的作用显著下调E2抗体的中和活性,小鼠DNA免疫试验也进一步验证了我们的结论。三、HCV高变区1中介导病毒颗粒与脂蛋白结合关键区域的鉴定将浓缩的HCVcc进行碘克沙醇密度梯度离心,发现密度偏低的病毒颗粒具有强感染性,而随着HCVcc病毒颗粒密度的升高,其感染性也逐渐降低。此外,HDL对不同密度HCVcc感染的促进作用差异显著,其对低密度以及中等密度HCVcc感染的促进作用不明显,却显著增强高密度HCVcc的感染性。先前我们已证明HVRl中存在一个由9个氨基酸残基组成的功能结构域是诱导宿主产生抗体的主要抗原表位,并且HDL介导的HCV感染增强作用依赖于该9个氨基酸残基的存在,本部分实验我们发现删除了这9个氨基酸残基后,HCVcc病毒颗粒的密度分布曲线发生了明显改变,低密度的病毒颗粒数目明显下降,并且感染性也显著降低,而更多的病毒颗粒则集中在密度偏高几层,这和删除了整个HVRl的HCVcc所呈现出的病毒密度曲线趋势几乎是一致,这说明在HVRl中,决定HCV病毒颗粒与脂蛋白结合的关键部位恰恰就是这个由9个氨基酸残基组成的抗原决定区。在HDL对不同密度病毒颗粒的感染增强试验中,删除了9个氨基酸残基的任何密度层病毒颗粒的感染性均不再被HDL所增强。小结我们鉴定出HVR1中至少存在三个功能结构域,它们之间相互协调发挥着微妙的作用：HVR1的抗原决定区在病毒颗粒表面形成一个高度凸起,在脂蛋白结合过程中行驶一个非特异性“脂抓手”作用,结合在病毒颗粒表面的脂蛋白又通过与宿主SR-BI受体的结合拉近病毒颗粒与靶细胞的空间距离,使得抗原决定区两侧的SR-BI结合位点更易于与SR-BI受体结合并启动HCV细胞入侵的一系列步骤；更为有意思的是抗原决定区的空间凸起又是诱导宿主产生抗体的关键抗原表位,但其自身又呈现了高度的变异,使得宿主产生的抗体不能识别宿主体内新释放出的病毒颗粒,从而达到免疫逃避和持续感染的目的；而恰恰也是这样一个抗原表位,因为其自身的空间高度凸起,使得HCV病毒颗粒表面包裹着大量的脂蛋白,脂蛋白覆盖住了HCV病毒颗粒表面其他真正意义上的保守中和抗体表位,使得宿主细胞不能大量产生可以识别病毒颗粒表面保守位点的中和抗体,我们认为这个“脂抓手”同时又起到了“免疫诱饵”的作用。人们往往将HVR1的27个氨基端残基作为一个整体进行研究,我们的实验对HVR1的功能意义进行了深刻的剖析与阐释,并精确地鉴定出HVR1结构与功能之间的对应关系,这为今后保护性疫苗以及抗病毒药物的研发都提供了重要的启示。
[Abstract]:Hepatitis C virus (hepatitis C virus, HCV) belongs to the family of the family yellirid family, hepatitis C virus and the envelope of single positive chain RNA virus. At present, about 180 million people are infected with HCV in the world, most of them are chronic infection. Some infected people can develop the RNA polymerase of liver cirrhosis and hepatocellular carcinoma.HCV without proofreading function, resulting in the whole genome of HCV. High variability, but the variation of the HCV gene is unevenly distributed. The 27 amino acid residues in the amino terminal of the envelope protein 2 (E2) are most prominent, that is, HVRl.HIVR1 is highly exposed to the surface of the envelope protein, which contains neutralizing antibody epitopes, which can induce the host to produce the antibody against the region, and the antibody produced can effectively neutralize the HCV. Infection, but the gene sequence of HVR1 is highly variable, and the antibodies previously produced by the host immune cells cannot identify the variant HVRl, so the virus can escape the monitoring of the host cells and keep the infection in the patient and cause the chronicity of the infection. Many laboratories have taken HVR1 as an important target for the development of the HCV vaccine, but since 1989 Since the first name of the hepatitis C virus, there is still no effective protective vaccine.
HCV infection causes biochemical metabolic disorders in the liver, including lipid metabolism disorders and apolipoprotein synthesis. Compared with other viral hepatitis, after chronic HCV infection, fatty degeneration of medium and severe hepatocytes, including intracellular lipid droplet aggregation, is a typical histopathological feature of.HCV, a enveloped RNA virus with different amounts of fat eggs on the surface. White parcels, so the HCV virus particles purified from the patient's serum show a different density distribution, and the virus particles of different densities are different. Studies have found that the deletion of HVR1 leads to a decrease in the number of low density virus particles, that is, the ability to reduce the lipoprotein binding capacity of HCV, and it can be seen that HVR1 is combined with the virus and lipoprotein. Many studies have shown that HVR1 also plays a key role in the combination of the host SR-B1 receptor and the invasion of the virus. HVR1 has such important biological functions that its structure is bound to be relatively stable and conserved, although HVR1 is the highest variation in the HCV genome. The sequence analysis of a series of isolates of HCV shows that some amino acid residues are highly or relatively conservative. Although it has been found that HVR1 is driving simultaneously the host immune response, lipoprotein binding, and the multiple functions of the combination of the virus and the receptor, it is not yet clear about its structure and biological function. The corresponding relationship.
Identification of key amino acid residues in HCV mediated hypervariable region 1 mediated virus cell invasion
First of all, we use HCVpp as the model, using the H77 strain of HCVla subtype as the model plant, and using the single amino acid residue deletion scanning method to analyze the effect of 27 amino acid residues in HVR1 on the invasion of HCV cells respectively. The experimental results clearly identify the key amino acid residues of the HVRl mediated HCV cell invasion (Ala-14, Gly-15, Lys-25, Gln-26). As well as Asn-27), the deletion of any one of the residues was fatal to the infection of HCVpp. In contrast, the deletion of the other 22 amino acid residues had no significant effect on HCVpp infection. The HCV invasion related receptor binding assay was used for mechanism analysis. We proved that the 5 amino acid residues mediate the envelope protein of the virus. The combination of the SR-B1 receptor with the host cell. The combined deletion experiment of amino acid residues has further demonstrated our conclusion that the deletion of 13 residues in the HVRl amino terminal and the deletion of the 16-24 site 9 amino acid residues will not significantly reduce the infection force of HCVpp. In addition to determining the 5 amino acid residues of the HCV cell invasion, 13 of the HVR1 amino terminus Amino acid residues can enhance the virus's cell invasiveness by increasing the binding ability of the virus to the receptor by appropriate mutations, and the appropriate mutation in this region can also enhance the anti neutralization ability of the virus and thus benefit the immune escape and persistent infection of the virus.
Two, identification of epitopes produced by neutralizing antibodies in HCV hypervariable region 1.
We divided HVR1 into amino group, the three part of the middle segment and the carboxyl segment were fused with TRX after the fusion expression of the three amino acid residues to detect the difference between the 27 amino acid residues of HVR1 in the induction of the host immune response. We found that the 13 amino acid residues of the amino terminal of HVRl did not induce the host to produce the antibody, and the sixteenth to 24 bits of the 9 ammonia. Base acid residues are the only neutralization epitopes in HVRl. These 9 amino acid residues are insignificant for virus cell invasion, but contain heparin binding sites, which are beneficial to HCV adhesion and, more importantly, the enhancement of HCV infection mediated by high density lipoprotein (HDL) depends on the existence of the 9 amino acid residues, and the nine residues can also be passed through HDL The neutralizing activity of E2 antibody was significantly reduced, and the DNA immunization test in mice further confirmed our conclusion.
Three, identification of 1 critical regions of HCV mediated hypervirion and lipoprotein binding.
The concentrated HCVcc was centrifuged with iodipol density gradient. It was found that the virus particles with low density were strongly infective, and with the increase of HCVcc virus particle density, its infectivity gradually decreased. In addition, the promoting effect of HDL on different density HCVcc infection was significantly different, and its promoting effect on low density and medium density HCVcc infection was promoted. It was not obvious, but significantly enhanced the infectivity of high density HCVcc. Previously, we have shown that a functional domain consisting of 9 amino acid residues in HVRl is the main epitope that induces the host to produce antibodies, and the enhancement of HDL mediated HCV infection depends on the presence of the 9 amino acid residues. In addition to the 9 amino acid residues, the density distribution curve of HCVcc virus particles changed significantly, the number of low density virus particles decreased significantly, and the infection was significantly reduced, while more virus particles were concentrated in a few layers of density, and this and the deletion of the virus density curve of the entire HVRl HCVcc was almost the same. It is consistent, which indicates that in HVRl, the key site to determine the binding of HCV virus particles to lipoprotein is precisely the antigen determining area consisting of 9 amino acid residues. In the HDL infection enhancement test for different density virus particles, any of the 9 amino acid residues of any dense lamina virus particles are no longer increased by HDL Strong.
Summary
We identified that there are at least three functional domains in HVR1 that play a subtle role in coordinating each other: the antigen determinant of HVR1 forms a high elevation on the surface of the virus particles, driving a nonspecific "fat gripper" in the process of lipoprotein binding, and by combining the lipoproteins on the surface of the virus particles. The combination of the host SR-BI receptor close to the space distance between the virus particles and the target cells makes the SR-BI binding site on both sides of the antigen determinant more easily combined with the SR-BI receptor and initiates a series of steps for the invasion of the HCV cells; more interesting is that the spatial protruding of the antigen determining region is the key epitope of the host to induce the host to produce antibodies. It has a high variation in itself, making the antibody produced by the host can not identify the newly released virus particles in the host, so as to achieve the purpose of immune escape and continuous infection. It is also an epitope of the antigen, because of its high elevation of its own space, so that a large amount of lipoprotein is wrapped on the surface of the HCV virus particles. Lipoproteins cover the other true conserved and antibody epitopes on the surface of the HCV virus particles, making the host cells unable to produce a large number of neutralizing antibodies that can identify the conservative sites on the surface of the virus particles. We think the "fat grab" also plays the role of "immune bait". People tend to take the 27 amino groups of HVR1. As a whole, the end residues are studied as a whole. Our experiments have deeply analyzed and explained the functional significance of HVR1, and accurately identified the corresponding relationship between the structure and function of HVR1, which provides important inspiration for the future protection of vaccine and the development of antiviral drugs.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R512.63

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