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。
Rubella virus,the etiological cause of German measles,is the only member of the Rubivirus genus within the Togaviridae family.Humans are the only natural host for the virus.The clinical symptoms of RV infections acquired postnatally are usually mild, and many infections are asymptomatic.The first clinical manifestation of rubella is usually the appearance of a macropapular rash some 16 to 20 days after exposure.The rash first appears on the face and then spreads over the trunk and later over the extremities;.Other symptoms typically include low-grade fever,lymphadenopathy,sore throat,and general malaise.Rubella can cause complications,with transient joint involvement such as arthritis and arthralgia being the most frequent.Interestingly,these symptoms are more prevalent and severe in RV-infected women than in RV-infected men.
The major public health concern posed by rubella is its teratogenicity,with maternal infection early in pregnancy leading to the congenital rubella syndrome(CRS) in infants.The clinical manifestations of CRS are numerous and varied,with deafness being the most common.Other clinical features include cardiac disease,mental retardation,and ocular conditions such as cataracts and glaucoma.The time at which infection occurs during gestation can influence the outcome.The earlier in gestation the maternal infection occurs,the more severe is the damage to the fetus.
The mature RV virion is a round or ovoid particle approximately 60 nm in diameter.The virion contains an electronlucent spherical core composed of multiple copies of the RV capsid protein and a positive,single copy of the viral 40S RNA genome.The RV core is surrounded by a host-derived lipid bilayer containing 5- to 6-nm-long spikes which project from the virion surface;the spikes are composed of the E2 and E1 glycoproteins.The capsid protein is a nonglycosylated,phosphorylated, disulfide-linked homodimer.The capsid protein contains clusters of proline and arginine residues,which have been postulated to be involved in binding to the RV genomic RNA to form the viral nucleocapsids.
The virion envelope proteins,E1 and E2,are typeⅠmembrane glycoproteins observed as spikes in the form of E1/E2 heterodimers on the virion surface.The E1 and E2 proteins each contain a putative transmembrane(TM) domain which is 22 and 39 residues in length,respectively.For E2,the putative TM domain is followed by a positively charged 7-residue sequence,RRACRRR,and a 20-residue region which acts as a signal sequence for E1.E1 and E2 are rich in cysteine residues.There are 20 cysteines in E1 ectodomain,all of which formed into disulfide bridges.E2 has 14 cysteine residues.Amino acid sequence analysis of the E1 protein has since revealed that it contains three N-linked glycosylation sites for all strains so far sequenced.In contrast,the number of N-linked glycosylation sites of the E2 protein appears to vary depending on the strain.In addition to N-linked sugars,the RV E2 protein contains O-linked carbohydrates.
The functions of the RV E1 and E2 glycoproteins have been studied extensively. Using monoclonal antibodies,it has been shown that the E1 protein contains at least six nonoverlapping epitopes,some of which are associated with hemagglutination and neutralization.E1 appears to be the main surface protein,with domains involved in the attachment of the virus to the cell.The function of E2 has been more difficult to determine.E2 is disulfide-linked to E1 in the mature virion and is poorly exposed. Therefore,the antigenic sites of E2 are less accessible to characterization by monoclonal antibodies.However,E2 does contain partial hemagglutination and neutralizing epitopes and may also carry strain-specific epitopes.
Cell fusion is an important biological process for many enveloped viruses including RV in their intrusion,replication,releasing,transmission and pathopoiesis.It is also a critical step for information transmission among cells.The route of RV entry into the host cell is not well understood.There is some evidence to suggest that RV enters cell,;via the endocytic pathway.Early biochemical studies by Katow and Sugiura showed that exposure of the RV E1 and E2 glycoproteins to pH 6.0 or less induced a conformational change within the glycoproteins that favored the fusion of the viral envelope to the endosomal membrane.
If we can interpret the mechanism of cell fusion caused by RV,the expression product and the biological activity may be changed by altering the RV genome.So the possibility of teratogenicity caused by RV can decrease,and the infection of the fetus can be reduced or eliminated by changing the invading pathway of cells or replication characteristics.It can also lay the foundation for the development of a more safe and effective RV genetic engineering vaccine(gene-deleted vaccine and protein engineering vaccine) and specific anti-virus polypeptides.
Cell fusion is caused by proteins on cell surface,so the proteins should be able to be transported to the cell surface when we study their activity of cell fusion.The signal peptide of RV E2 plays an important role in the processing and transport of the structural proteins.To facilitate the expression of E1 on the cell surface,the recombinant plasmid(pBSK-SPE2E1) has been constructed in our laboratory.The genes encoding the signal peptide of E2,E2 and E1 of RV JR23 strain is subcloned into the vector pBluescriptⅡSK~+ between the EcoRⅠand XbaⅠsites.Then series mutants were constructed using site-directed mutagenesis and homologous recombination.Their fusogenic and hemadsorption activities in addition to a potential of cell surface expression of E1 and E2 were assayed by Giemsa staining,reporter gene method, hemadsorption,and FACS,respectively.Western blot was applied to assay the total expression of the mutants.The effects of the mutated sites on the fusogenic activity of RV envelope glycoprotein were analyzed.A recombinant vector of RV capsid protein named pBSK-C was constructed in this study,and the ability of the capsid protein to promoting the fusion activity of envelope glycoprotein was detected when it was co-transfected with pBSK-SPE2E1.
1.Effects of disulfide bridges in E1 ectodomain on fusogenic activity of RV
There are 20 cysteine residues in the ectodomain of RV E1.All of the 20 cysteine residues are involved in the formation of disulfide bridges.Site-directed mutagenesis and homologous recombination were used to substitute 11 of the 20 cysteines in the ectodomain of E1 with other amino acids individually in the recombinant plasmid pBSK-SPE2E1.The mutants were named as Cys2,Cys3,Cys4,Cys5,Cys6,Cys8, Cys9,Cys12,Cys13,Cys17,and Cys20 according to the sequence of the mutated cysteines in E1,respectively.One disulfide bridge in E1 was eliminated in each mutant, and then the influence of a single disulfide bridge to the fusion activity was analyzed.
Western blot analysis did not detect obvious reduction in the total protein production of any mutant.Mutants Cys5 and Cys8 were poorly expressed on the cell surface.We speculated that the disulfide bridge C(5)-C(8) played an important role in the interaction of E1 and E2.The mutation in Cys5 and Cys8 might lead to a conformational change in E1,which could result in the failure of recognition by E2 and the defect of transport in the cells.The cell surface expression of Cys2,Cys6,Cys9, Cys12,Cys17,and Cys20 mutants was decreased compared to the wild type plasmid. The disulfide bridges formed by these cysteines may affect the interaction between E2 and E1 or the transport of the mutated proteins.Mutants Cys3,Cys4,and Cys13 showed 121%,107%and114%cell surface expression of the wt plasmid,respectively. The disulfides formed by C(3),C(4) and C(13) have no effects on the interaction of the glycoproteins and their transport in the cells.
Although a certain amount of mutant proteins could be detected in most mutants, no syncytia were detected in cells transfected with any of the mutated plasmids,Cys 2 to 20.We can conclude that all the 10 disulfide bridges in ectodomain of E1 are important for the fusogenic activity of RV.
2.Effects of cysteines in E2 on fusogenic activity of RV
RV envelope glycoprotein E2 contains 14 cysteines,12 of which are located in the ectodomain,one on the transmembrane domain,and one on the cytoplasmic domain.14 cysteine mutants of E2 were constructed using site-directed mutagenesis and in vivo homologous recombination.The mutants are C69T,C82S,C91S,C124G,C132A, C139P,C152G,C157R,C172A,C196G,C207G,C219T,C255W and C259G.One cysteine was removed in each mutant.
Western blot showed that the change of the C132 and C139 resulted in lower production in E1.The decreased fusion activity may be related to the low production of the mutated protein.The change of the rest 12 cysteines had no effect on the total expression of E1.Cell fusion activity was almost lost in mutants C69T,C82S,C124G, C132A,C139P,C152G,C157R,C172A,C196G,C207G,C219T and C255W.The loss of the fusion activity may be due to the defective transport to the cell surface of these mutants.There is another possibility that the fusion activity sites could not be exposed because of the conformational change of the protein.
Mutants C91S and C259G had similar cell fusion activity with the wild type glycoprotein.So C91 and C259 did not affect the interaction of E2 and E1,and E1 could be transported to the cell surface.
Mutated E2 could not be detected using polyclonal antibodies to RV.It was indicated that the cysteine residues of E2 had an important role for the maintenance of the protein conformation.The removal of cysteines directly led to the disappearance of antigenicity,so we speculated that the majority of the cysteines in E2 were involved in the formation of disulfide bridges.
3.Effects of key animo acids in E1 on its cell fusion actitvity
The analysis of cysteine mutagenesis in the ectodomain of E1 showed that there was a region that may make sense in the fusogenic activity of E1.The mutation of C(3), C(4),and C(13) had no effect on the cell surface expression of the mutated proteins,but no fusion activity in these mutants was detected.The disulfide bridges formed by these cysteines were located in the sequence comprising the amino acid residues 213 to 285. This region contains a cluster of epitopes binding neutralizing and hemagglutination inhibiting antibodies and is believed of importance in the maintenance of the virus biological activity.Some conservative and structurally special amino acids were selected to be mutated.The mutants were H226Q,H238Q,R252S,P253T,R254Q, R256T,L257T,D259G,D261G,P263A,R266Q and P269S.
All mutants were transfected into BHK21 cells.Syncytia formation was observed after Giemsa staining at 24h post transfection.Mutants H226Q,R252S,and R254Q caused obvious cell fusion similar to wild type E1.The fusogenic activity of mutants R256T and P263A decreased compared with pBSK-SPE2E1.The rest mutants named H238Q,P253T,L257T,D259G,D261G,R266Q and P269S could cause slight fusion in cells or did not result in any fusion at all.
4.Effects of RV CP on the fusogenic activity of envelope glycoproteins
RV CP plays a role in the process of the virus replication,assembly and infection. This study was to test whether it had effect on the fusogenic activity of envelope glycoprotein.BHK21 cells were infected with RV JR23 strain.Viral RNA was extracted from the supernatant after 6 days.Reverse transcription was performed,and the gene segment for C was PCR-amplified using primers named C1 and C2.EcoRⅠand XbaⅠsites were included in the primers,respectively.PCR products were ligated into vector pBluescriptⅡSK~+.The recombinant vector pBSK-C was constructed successfully conformed by DNA sequencing,pBSK-C was transfected into BHK21 cells and the biological activity of the products were assayed with IFA.The fluorescent signal was concentrated in the perinuclear region of the transfected cells,pBSK-C and pBSK-SPE2E1 were co-transfected into BHK21 cells.Almost all the cells were observed to fuse to each other at 24h post transfection.Reporter gene method showed that the cell fusion caused by co-expression of the two recombinant vectors is 137% compared to expression of pBSK-SPE2E1 alone.The experiments indicated that capsid protein of RV could promote the fusogenic activity of the envelope glycoprotein.
The results suggested as followings:
The disulfide bridges in the ectodomain of RV E1 are indispensable for the fusogenic activity of RV.The disulfide bridge C(5)-C(8) affect the interaction of E2 and E1.
RV glycoprotein E2 contains 14 cysteine residues,12 of which play an important role in the fusogenic activity of E1.But the activity of E1 was not affected by the 3~(rd) cysteine in the ectodomain of E2 and the unique cysteine in the cytoplasmic domain. Disulfide bridges may be formed among the cysteines to maintain their importance in the fusion process.
There are some significant sites in the sequence comprising the amino acid residues 213 to 285 in E1,including H238,P253,L257,D259G,D261,R266,and P269. The mutation of these sites will result in the loss of the fusogenic activity of E1.
The recombinant vector of RV CP was successfully constructed and expressed in BHK21 cells.The products possessed good immunoreactivity and could promote the cell fusion activity of RV envelope glycoprotein under acid conditions.
This research is important to interpret the molecular mechanism of cell fusion caused by RV and for the study of the relationship between the structure and the function of envelope glycoprotein.It also provides information for the research of the teratogenesis mechanism of RV.
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