Mapping Of Cell Fusion Sites In Rubella Virus Glycoproteins And Effects Of Capsid Protein On Fusion Activity

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 typeâ… membrane 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 pBluescriptâ…¡SK⁺ between the EcoRâ… and Xbaâ… sites.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 RVThere 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 RVRV 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 actitvityThe 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 glycoproteinsRV 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.EcoRâ… and Xbaâ… sites were included in the primers,respectively.PCR products were ligated into vector pBluescriptâ…¡SK⁺.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.