风疹病毒包膜糖蛋白细胞融合活性位点的定位及衣壳蛋白对融合活性的影响

发布时间：2018-08-06 09:29

【摘要】： 风疹病毒(rubella virus,RV)是披膜病毒科风疹病毒属的唯一成员,人类是RV的唯一自然宿主。RV自然感染仅引起轻微的临床症状,许多感染是无症状的亚临床感染。一般于感染后16～20天内出现皮疹,首先于面部出现,然后扩散到躯干及四肢,其余症状还包括低热、淋巴结肿大和咽痛。RV感染的并发症以关节炎和关节痛最为常见,而且多发生于妇女。 RV引起的主要问题是它的致畸性,即母亲孕期感染RV会导致胎儿发生先天性风疹综合征(congenital rubella syndrome,CRS)。CRS的临床表现多种多样,其中耳聋最为常见,还包括心脏疾病、精神发育迟滞和眼部疾患如白内障和青光眼。妊娠期间母亲感染RV越早,胎儿损害越严重。成熟的RV病毒颗粒是直径为60nm的球形,核心是衣壳蛋白和单股正链基因组40S RNA组成的核衣壳,其外包绕脂质双层膜,膜上是长度为5～6nm的刺突——由糖蛋白E2和E1组成。衣壳蛋白(capsid protein,CP)是非糖基化的磷酸蛋白,靠二硫键形成同源二聚体。CP富含脯氨酸和精氨酸,与RV基因组RNA的结合有关。包膜糖蛋白E1和E2都是Ⅰ型膜糖蛋白,在病毒表面以异二聚体的形式存在。E1和E2都含有一段跨膜区(TM),长度分别为22和39个氨基酸。在E2中,TM之后是一段带正电荷的七氨基酸序列和E1的20个氨基酸信号肽。E1和E2都富含半胱氨酸残基,E1胞外功能区的20个半胱氨酸都形成二硫键,E2总共含有14个半胱氨酸残基。E1有3个N-联糖基化位点,E2的N-联糖基化位点数目在不同的毒株间有差别,除N-联糖基化位点外,E2还含有O-联糖基化位点。 E1和E2的功能研究较为广泛,单克隆抗体研究发现E1至少含有6个不重复的抗原位点,与血凝和中和活性有关。E1还与病毒和细胞的吸附有关,是主要的表面蛋白。E2的功能研究显得较为困难,因为它与E1结合后,几乎不暴露在细胞表面,也就无法用单克隆抗体识别其抗原位点。但是,E2也含有部分血凝表位和中和表位,还有株特异性表位。细胞融合是包括RV在内的许多有膜病毒侵入细胞、复制、释放、传播、致病的重要生物过程,也是细胞间信息传递的重要步骤。RV入侵细胞的途径还没有弄清楚,但是有证据表明是通过内吞途径进入细胞。Katow和Sugiura发现pH在6.0以下时,E1和E2会发生构象改变,这种改变有利于病毒包膜与内含体膜的融合。若能弄清RV引起细胞融合的机制,从而改变RV的基因组,改变它的表达产物和生物学活性,就可以消除或降低RV的致畸性;也可以通过改变其侵入细胞或复制的特性来消除或减少胎儿感染的危险性;还可以为研制更安全有效的RV新型基因工程疫苗(基因缺失活疫苗、蛋白工程疫苗等)和特异性抗病毒的多肽类药物奠定基础。细胞融合由位于细胞表面的蛋白引起,因此细胞融合功能的检测需要所研究的蛋白能够在细胞表面表达。RV E2的信号肽在病毒结构蛋白的加工及转运过程中发挥重要作用,为了使所表达蛋白能够在细胞内正确加工及顺利转运至细胞表面,本研究构建了重组质粒pBSK-SPE2E1,即将E2的信号肽序列、E2和E1的全基因序列克隆到载体pBluescriptⅡSK~+的EcoRⅠ和XbaⅠ酶切位点之间。然后利用定点突变和同源重组的方法,构建一系列突变体,Giemsa染色和指示基因法检测它们的细胞融合活性变化,流式细胞术(FACS)检测蛋白在细胞表面表达效率,Western blot检测总表达量的改变,血吸附实验检测受体识别活性,分析突变位点对RV包膜糖蛋白细胞融合活性的影响,以确定具有细胞融合活性的位点。本研究还构建了RV CP的重组载体pBSK-C,并检测CP对包膜糖蛋白细胞融合活性的影响。一、E1胞外功能区二硫键对RV细胞融合活性的影响 RV E1包膜糖蛋白胞外功能区含有20个半胱氨酸残基,而且都形成分子内二硫键。本研究利用定点突变和同源重组相结合的方法,将RV JR23株E1蛋白胞外功能区的20个半胱氨酸中的11个突变为其它氨基酸,构建了11个突变体Cys2、Cys3、Cys4、Cys5、Cys6、Cys8、Cys9、Cys12、Cys13、Cys17和Cys20,每个突变体去除E1的1个二硫键,检测单个二硫键的消失对E1细胞融合活性的影响。 Western blot结果显示所有突变体蛋白在细胞内的总表达量都没有降低,但是Cys5和Cys8在细胞表面表达量剧烈下降,所以二硫键C(5)-C(8)可以影响E1和E2的相互作用,使它们不能正确地形成异二聚体,从而不能顺利转运到细胞表面。突变体Cys2、Cys6、Cys9、Cys12、Cys17和Cys20的E1、E2蛋白细胞表面表达效率与野毒株相比有所下降,所以这些半胱氨酸形成的二硫键可能影响E1、E2的相互作用,也可能影响它们在细胞内的转运。突变体Cys3、Cys4和Cys13中,E1和E2蛋白的表达效率分别为野毒株的121%、107%和114%,因此半胱氨酸C(3)、C(4)和C(13)形成的二硫键对包膜糖蛋白的相互作用及在细胞内的转运没有影响。尽管11个突变体中大部分都有一定数量的突变蛋白表达在细胞表面,但是所有突变体转染的细胞中都没有检测到多核巨细胞,所以E1胞外功能区的10个二硫键都对RV的细胞融合活性有重要作用。二、E2中半胱氨酸对RV细胞融合活性的影响 RV包膜糖蛋白E2中含有14个半胱氨酸,其中12个位于胞外功能区,1个位于跨膜区,1个位于胞质区。本研究利用定点突变和体内同源重组的方法,用突变的寡核苷酸为引物,构建了14个E2的半胱氨酸突变体,每个突变体去除一个半胱氨酸,这些突变体分别为C69T、C82S、C91S、C124G、C132A、C139P、C152G、C157R、C172A、C196G、C207G、C219T、C255W和C259G。 Western blot表明,E2的14个半胱氨酸残基中,第132位和219位半胱氨酸的改变会导致E1蛋白总表达量降低,其细胞融合活性的降低可能与此有关。其余12位半胱氨酸的改变不影响E1蛋白总表达量,其中C69T、C82S、C124G、C132A、C139P、C152G、C157R、C172A、C196G、C207G、C219T和C255W 10个突变体几乎丧失细胞融合活性,说明在这些突变体中,E1不能有效运输到细胞表面,或者虽然能够到达细胞表面,但是构象改变,从而不能有效暴露融合活性位点。突变体C91S和C259G的细胞融合活性与野毒株相近,说明E2第91和259位半胱氨酸的改变不影响E1、E2的相互作用,从而使E1能有效转运到细胞表面发挥其细胞融合作用。实验中所用的多克隆抗体检测不到突变的E2蛋白,可见E2的半胱氨酸残基对维持蛋白的构象有重要作用,半胱氨酸的去除直接导致抗原性的消失,所以推测E2的半胱氨酸可能大部分形成二硫键,从而间接影响RV的细胞融合活性。三、E1关键氨基酸突变体对细胞融合活性的影响 E1胞外功能区的半胱氨酸突变分析显示,第3、4和13位半胱氨酸突变之后,在细胞表面表达量与野毒株相比没有降低,但是却检测不到融合活性,这3个半胱氨酸所形成的二硫键集中在E1的213～285位氨基酸之间,而这一段区域富含RV中和表位和血凝抑制表位,具有较重要的生物学活性,我们在此区域选择了一些保守的或结构上比较特殊的氨基酸,构建了12个突变体H226Q、H238Q、R252S、P253T、R254Q、R256T、L257T、D259G、D261G、P263A、R266Q和P269S。将各突变体质粒转染BHK21细胞,24h后Giemsa染色定性检测各突变体蛋白引起的细胞融合情况,发现突变体H226Q、R252S和R254Q引起的细胞融合程度与野毒株相近,而突变体R256T和P263A也能引起细胞融合,但是细胞融合强度与野毒株相比有所降低,突变体H238Q、P253T、L257T、D259G、D261G、R266Q和P269S只引起很轻微的细胞融合甚至不能引起融合。四、衣壳蛋白CP对RV包膜糖蛋白细胞融合活性的影响 RV衣壳蛋白CP在病毒复制、组装及感染过程中都有作用,本研究检测其对包膜糖蛋白的细胞融合活性的影响。RV JR23株感染BHK21细胞6天后提取病毒RNA,利用上游引物C1和下游引物C2反转录扩增C基因,引物中分别含有EcoRⅠ和SacⅠ酶切位点,扩增的片断酶切后,与经相同酶切的载体pBluescriptⅡSK~+片断连接,测序证实成功构建重组载体pBSK-C。将pBSK-C单独转染至BHK21细胞中,间接免疫荧光(IFA)检测表达蛋白活性,结果在核周区可见到较强的荧光。将pBSK-C与RV糖蛋白重组质粒pBSK-SPE2E1共同转染BHK21细胞,Giemsa染色发现细胞几乎全部发生融合,与单独转染pBSK-SPE2E1的细胞相比,细胞融合灶数量增多而且每个融合灶的细胞核数量增加,用指示基因法定量细胞融合显示共转染引起的细胞融合为糖蛋白单独转染的137%。可见CP可以促进RV包膜糖蛋白的细胞融合活性。从本实验结果可得出结论: RV包膜糖蛋白E1胞外功能区的10个二硫键都是维持E1细胞融合活性不可缺少的,其中C(5)-C(8)影响E1和E2的相互作用。 RV包膜糖蛋白E2序列中的14个半胱氨酸中,胞外区有1个半胱氨酸及胞质区的唯一1个半胱氨酸对E1的细胞融合活性没有影响,其余12个都对E1的细胞融合功能有重要作用,它们可能通过二硫键的形成间接对其产生影响。 RV E1的213～285aa区域含有一些重要的细胞融合活性位点,是维持RV融合活性的关键氨基酸,如H238、P253、L257、D259G、D261、R266和P269。成功构建了RV CP的重组载体,并在BHK21细胞中成功表达出CP,表达产物具有良好的生物学活性,能够促进酸性条件下RV包膜糖蛋白的细胞融合活性。本实验为阐明RV引起细胞融合的分子机制、包膜糖蛋白的结构与功能研究奠定了坚实的基础,也可以为RV致畸机制的研究提供帮助。
[Abstract]:The rubella virus (rubella virus, RV) is the only member of the family rash virus of the family apovirus. Human is the only natural host of RV,.RV natural infection only causes minor clinical symptoms. Many infections are asymptomatic subclinical infections. A rash occurs within 16~20 days of infection, first in the face, and then spread to the trunk and limbs, Other symptoms include low fever, enlarged lymph nodes, and sore throat. The most common complications of RV infection are arthritis and arthralgia, and most often occur in women.
The main problem caused by RV is its teratogenicity, the mother's pregnancy infection of RV can lead to a variety of clinical manifestations of the congenital rubella syndrome (CRS).CRS, the most common of which are deafness, including heart disease, mental retardation, and eye disorders such as cataracts and glaucoma. The earlier the infection of RV, the more serious the fetus is.
The mature RV virus particle is a spheroid with a diameter of 60NM. The core is the nucleocapsid of the capsid protein and the single strand positive chain genome 40S RNA, which is outsourced around the lipid bilayer membrane, the membrane is 5 to 6nm, which consists of glycoprotein E2 and E1. The capsid protein (capsid protein, CP) is a non glycosylated phosphate protein and forms the same by the two sulfur bond. Source two dimer.CP is rich in proline and arginine, and is related to the binding of RV genomic RNA.
Both E1 and E2 are type I membrane glycoproteins. The presence of.E1 and E2 in the form of different two polymers on the virus surface contains a segment of transmembrane region (TM), with a length of 22 and 39 amino acids. In E2, TM is a positive charge seven amino acid sequence and E1's 20 amino acid signal peptide.E1 and E2 are rich in cysteine residues and E1 extracellular All 20 cysteines in the functional region form a two sulfur bond, and E2 contains 14 cysteine residues.E1 with 3 N- glycosylation sites, and the number of N- glycosylation sites of E2 is different among different strains. In addition to N- glycosylation sites, E2 also contains O- linked glycosylation sites.
The function of E1 and E2 is widely studied. The study of monoclonal antibodies found that E1 contains at least 6 non repeated antigen sites, and that.E1 is related to the adsorption of hemagglutination and neutralization activity, which is also related to the adsorption of viruses and cells. It is difficult to study the function of the main surface protein.E2, because it is almost not exposed to the cell surface after it is combined with E1. Monoclonal antibodies could not be used to identify the antigenic sites. However, E2 also contained some hemagglutination epitopes and neutralization epitopes, as well as strain-specific epitopes.
Cell fusion is an important biological process for the invasion of cells including RV, including the invasion of cells, replication, release, transmission, and pathogenesis, and also an important step in the transmission of information between cells. The pathway of.RV invading cells is not clear, but there is evidence that E1 and E2 are found under the endocytic pathway into.Katow and Sugiura when pH is below 6. Conformational changes may occur, which is conducive to the fusion of viral envelope and inclusion membrane.
It is possible to eliminate or reduce the teratogenicity of RV by altering the mechanism of RV causing cell fusion and changing the genome of RV, changing its expression products and biological activity. It can also eliminate or reduce the risk of fetal infection by changing the characteristics of its intruding cells or replicating, and also to develop a more safe and effective new gene for RV. Engineering vaccine (gene deleted live vaccine, protein engineering vaccine, etc.) and specific antiviral polypeptide drugs laid the foundation.
Cell fusion is caused by protein on the surface of the cell, so the detection of cell fusion function requires that the protein expressed on the surface of the cell can express the signal peptide of.RV E2 on the processing and transport of the viral structural protein, in order to make the expressed protein properly processed and transshipped to the cell surface. In this study, the recombinant plasmid pBSK-SPE2E1 was constructed, and the sequence of signal peptide of E2, the whole gene sequence of E2 and E1 was cloned between the EcoR I and Xba I sites of the carrier pBluescript II SK~+, and a series of mutants were constructed by site directed and homologous recombination, and the cell melting was detected by Giemsa staining and indicator gene method. Activity changes, flow cytometry (FACS) detection of protein on the cell surface expression efficiency, Western blot detection of the total expression change, blood adsorption test to detect receptor recognition activity, analysis of mutation site on the RV envelope glycoprotein cell fusion activity, to determine the cell fusion activity site. This study also constructed the RV CP weight. The vector pBSK-C was used to detect the effect of CP on the fusion activity of the envelope glycoprotein.
1. The effect of two sulfur bonds in the extracellular domain of E1 on the fusion activity of RV cells
The extracellular function area of RV E1 envelope glycoprotein contains 20 cysteine residues and all forms intramolecular two sulfur bonds. In this study, 11 mutations of 20 cysteine in the extracellular domain of RV JR23 strain E1 protein were mutated into other amino acids by the combination of site directed and homologous recombination, and 11 mutant Cys2, Cys3, Cys4, Cys5, Cys6, were constructed. Cys8, Cys9, Cys12, Cys13, Cys17, and Cys20 were removed from each mutant to detect the effect of single disulfide bond deletion on the fusion activity of E1 cells.
Western blot results showed that the total expression of all the mutant proteins did not decrease, but the expression of Cys5 and Cys8 decreased sharply on the surface of the cells, so the two sulfur bond C (5) -C (8) could affect the interaction between E1 and E2, making them unable to form the hetero two polymer correctly, and thus could not be successfully transported to the cell surface. Mutant Cys2, C Ys6, Cys9, Cys12, Cys17 and Cys20 E1, the expression efficiency of E2 protein cells decreased compared with that of wild strains, so the two sulfur bonds formed by these cysteine may affect the interaction of E1, E2, and may also affect their intracellular transport. The expression efficiency of the mutant Cys3, Cys4 and Cys13, is 121% of the wild strain, respectively. Thus disulfide bonds formed by cysteine C(3), C(4) and C(13) have no effect on the interaction of envelope glycoproteins and their intracellular transport.
Although a large number of mutant proteins were expressed on the cell surface in most of the 11 mutants, no multinuclear giant cells were detected in all the transfected cells, so the 10 two sulfur bonds in the E1 extracellular domain were important for the cell fusion activity of RV.
Two, the effect of cysteine in E2 on the fusion activity of RV cells.
The RV envelope glycoprotein E2 contains 14 cysteines, of which 12 are located in the extracellular domain, 1 in the transmembrane region and 1 in the cytoplasm. In this study, 14 E2 cysteine mutants were constructed by site directed mutagenesis and homologous recombination in vivo, and one cysteine was removed by each mutant. The mutants were C69T, C82S, C91S, C124G, C132A, C139P, C152G, C157R, C172A, C196G, C207G, C196G, and, respectively.
Western blot showed that the changes in the 132nd and 219 cysteine residues in the 14 cysteine residues of E2 could lead to a decrease in the total expression of E1 protein, and the decrease of the cell fusion activity may be related to this. The changes in the remaining 12 cysteine did not affect the total expression of E1 protein, including C69T, C82S, C124G, C132A, C139P, C152G, C157R, C157R The 10 mutants of G, C219T and C255W almost lose cell fusion activity, indicating that in these mutants, E1 can not be transported to the cell surface effectively, or even though it can reach the cell surface, but the conformation changes can not be effectively exposed to the fusion active site.
The cell fusion activity of the mutant C91S and C259G is similar to that of the wild virus, indicating that the change of E2 ninety-first and 259th - cysteine does not affect the interaction of E1 and E2, so that E1 can be effectively transported to the cell surface to play its cell fusion.
The polyclonal antibody used in the experiment did not detect the mutant E2 protein. It was found that the cysteine residues of E2 had an important role in maintaining the conformation of the protein. The removal of cysteine directly led to the disappearance of antigenicity. Therefore, it is presumed that the cysteine of E2 may form a majority of the two sulfur bonds, indirectly affecting the cell fusion activity of RV.
Three, the effect of E1 key amino acid mutants on cell fusion activity.
Cysteine mutation analysis in the E1 extracellular domain showed that after 3,4 and 13 cysteine mutations, the cell surface expression was not decreased compared with the wild virus, but the fusion activity was not detected. The two sulfur bonds formed by the 3 cysteine were concentrated between the 213~285 amino acids of E1, and the region was rich in the RV neutralization epitopes. And hemagglutination inhibition epitopes, with more important biological activity, we selected some conservative or structural specific amino acids in this area, and constructed 12 mutants H226Q, H238Q, R252S, P253T, R254Q, R256T, L257T, D259G, D261G, P263A, R266Q, and P269S..
The mutant plasmids were transfected into BHK21 cells. After 24h, Giemsa staining was used to detect the cell fusion caused by the mutant proteins, and the mutant H226Q, R252S and R254Q were found to be similar to those of the wild strain, while the mutant R256T and P263A also could cause cell fusion, but the cell fusion intensity was lower than that of the wild strain. Mutants H238Q, P253T, L257T, D259G, D261G, R266Q and P269S only cause mild cell fusion and even fail to induce fusion.
Four, the effect of capsid protein CP on the fusion activity of RV envelope glycoprotein cells.
RV capsid protein CP plays a role in the process of viral replication, assembly and infection. The effect of this study on the cell fusion activity of the envelope glycoproteins was detected. The.RV JR23 strain infected BHK21 cells after 6 days to extract the virus RNA. The upstream primer C1 and the downstream primer C2 were used to amplify the C gene. The primers contained EcoR I and Sac I enzyme cut sites, and the amplification of the DNA was expanded. After the fragment of the fragment was cut, it was connected with the carrier pBluescript II SK~+, which was cut through the same enzyme. It was confirmed that the recombinant vector pBSK-C. was successfully constructed to transfect pBSK-C alone into BHK21 cells, and the activity of expression protein was detected by indirect immunofluorescence (IFA). The results showed that strong fluorescence could be seen in the peri nuclear region. PBSK-C and RV glycoprotein were recombinant plasmid pBSK-SPE2E1. BHK21 cells were transfected by CO transfection, and almost all the cells were fused by Giemsa staining. Compared with the cells transfected with pBSK-SPE2E1 alone, the number of cell fusion foci increased and the number of cell nuclei increased in each fusion focal point. The fusion of CO transfected cells by indicator gene quantitative cell fusion showed that the 137%. of the cells transfected by glycoprotein could be seen as C. P can promote the cell fusion activity of RV envelope glycoprotein.
The results can be concluded from the results of this experiment.
Ten disulfide bonds in the extracellular domain of RV envelope glycoprotein E1 are indispensable for maintaining the fusion activity of E1 cells, and C(5)-C(8) affects the interaction between E1 and E2.
In the 14 cysteine of RV envelope glycoprotein E2 sequence, the extracellular domain of 1 cysteine and the only 1 cysteine in the cytoplasmic region has no effect on the cell fusion activity of E1, and the other 12 are important for the cell fusion function of E1, and they may indirectly affect it through the formation of the two sulfur bond.
The 213 ~ 285aa region of RV E1 contains some important cell fusion active sites, which are key amino acids for the maintenance of RV fusion activity, such as H238, P253, L257, D259G, D261, R266 and P269..
The recombinant vector of RV CP was successfully constructed and CP was successfully expressed in BHK21 cells. The expression product has good biological activity, and it can promote the cell fusion activity of RV coated glycoprotein under acid condition.
This study provides a solid basis for elucidating the molecular mechanism of RV induced cell fusion, the structure and function of the membrane glycoproteins, and can also help the research of the RV teratogenicity mechanism.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R373

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