Study On Molecular Mechanism Of Cell Fusion Caused By Rubella Virus Envelope Glycoprotein

Rubella virus(RV),the only member of the genus Rubivirus in the family Togaviridae,is one of the most common and important pathogens of TORCH syndromes.Rubella is usually a mild disease in children.The hazard of RV infection is mainly in congenital infection.Intrauterine infection can be caused by infection of RV in pregnant women,especially in the first three months of pregnancy.The congenital infection can result in congenital rubella syndrome(CRS),including multi-organ deformity of the infant or functional abnormalities such as cataracts, deafness,and congenital heart disease,etc.And stillbirth or abortion may occur in serious infection.There is no specific therapy for rubella up to now.Clinical therapeutic effect of some antiviral drugs is not obvious,and it can't reduce the course or improve prognosis,especially can't prevent CRS.The most effective means in prevention of rubella and CRS currently is vaccination.At present the vaccine in clinical application are attenuated vaccines,in which the RV may infect the fetus through the placenta,so there is still the risk of teratogenicity.It was confirmed by Hofmann that rubella vaccine virus can be transported through placenta and it is involved in fetal infection, so the women in pregnancy can not be vaccinated with this vaccine to prevent rubella infection.And vaccination in adult women,especially postpartum can induce temporary arthritis(10%) and joint pain(2.5%).Subunit vaccine contains only proteins of the virus but no nucleic acids,therefore its immunogenicity is still retained and it is very safe for pregnant women.But the immunological effect of subunit vaccines isn't obvious and continual.Repeated injection is required to maintain the vaccination effect.So it is hard to promote application of subunit vaccine.If the products of RV expression and biological activity of RV is altered by changing its genome,the teratogenicity of RV will be reduced or eliminated.The risk of fetal infection may also be eliminated or reduced by changing its characteristics of invading cells or replication.For many kinds of envelope virus including RV,cell fusion is an important biological process in invading cells,reproducing,releasing, dissemination and pathogenicity;it is also an important step in transmission of intercellular information.Two results may be induced by membrane fusion in envelope viruses and target cells.(1) The core protein and genetic material of virus was transported into the host cells and replicated,then spread to offspring along with division of host cells,resulting in the death of host cells and infection in healthy cells. (2) Break the stability of the target cell membrane,so that the environment inside and outside cells is out of balance,which eventually lead to cytopathic effect or death of cells.Therefore,if molecular mechanism of cell fusion caused by RV is clear, especially active sites and non-active sites on envelope glycoprotein for membrane fusion are positioned precisely;it will lay theoretical foundation and provide technical parameters for the development of a new type of safer and more effective RV genetic engineering vaccine(gene deletion vaccine and protein engineering vaccine,etc.), development of specific anti-viral peptide drugs,prevention and treatment of RV infection.It is also helpful in studying the mechanism of RV teratogenesis and the molecular mechanism of cell fusion caused by other envelope viruses.In the three kinds of structural proteins encoded by the 3' end structural gene of RV,the capsid protein C was conjugated with genomic RNA to form nucleocapsid; envelope glycoprotein E2 and E1,typeⅠprotein and containing main biological functions,were composed of heterodimers and formed spike structure on the surface of virus.There are three or four N-linked glycosylation sites in E2 glycoprotein.E2 glycoprotein of RV JR23 strain contains three glycosylation as structure of Asn-X-Ser, which is located in N53,N71 and N129.E1 glycoprotein contains three N-linked glycosylation sites as structure of Asn-X-Thr,which is located in N76,N177 and N209.N-linked glycosylation is an important post-translational modification process of protein in eukaryotic expression system.Such glycosylation modification plays an important role in formatting and maintaining biological activity conformation of glycoproteins,protecting peptides from intracellular proteolytic enzymes,impacting antigenicity and immunogenicity of proteins.To detect the effects of RV glycoproteins glycosylation on specific cell fusion,a series of mutants were constructed to remove glycosylation of specific glycosylated sites separately in this study.The specific cell fusion activities of these mutants were observed,and it was found that efficiencies of some mutants were reduced significantly.The results indicated that glycosylation in these sites were involved in the specific cell fusion by formatting and maintaining the correct protein conformation and ensuring proteins transported to cell surface.But cell fusion can't be completely prevented in any mutant,which indicated that glycosylation of envelope proteins play a role in cell fusion together with other factors.Some specific amino acids were selected and mutated on E2 and E1 proteins to find the regions of cell fusion activity.The leucine is one of hydrophobic amino acids and it is important in formation and maintenance of protein's structure,so some leucine on extracellular region of E2 and E1 proteins were selected and mutated.And the specific cell fusion was observed both in mutants and wild type proteins.1.Effects of E2 and E1 glycosylation on specific membrane fusion in RV.Six mutants were constructed by homologous recombination in vivo in this study. And glycosylation of one corresponding mutated site was blocked in each mutant, glycosylation of E2 N53,N71 and N129 were blocked in mutant N53G,S73I and S131V,respectively.Glycosylation of E1 N76,N129 and N211 were blocked in mutant T78A,T179A and T211A,respectively. The FACS assay showed that all the mutated proteins expressed on cell surface were reduced differently except for S131V,as compared with the wild type glycoprotein.Cell fusion of different extension was observed by Giemsa staining after these mutants were transfected into BHK21 cells.Considering the fact that cell fusion activity caused by viral glycoprotein is affected by its expression efficiency on cell surface.To eliminate such effect,the percentage of cell fusion activity was normalized with the cell surface expression efficiency.It was revealed that E2 N53G,S73I and S131V had 62.73%,66.66%and 55.12%,and E1 T78A,T179A and T211A had 66.93%,87.33%and 90.18%of fusion activities,respectively,as compared with the wild type glycoprotein.The results indicated that the three N-linked glycosylation sites in E2 and the N76 glycosylaiton site in E1 were involved in their membrane fusion.On the other hand,the fusion activities were minimally affected by glycosylation modification in T179A and T211A.Hemadsorption was observed in all the mutants after they were transfected into BHK21 cells.The results indicated that the adsorption efficiencies of E2 S73I and E1 T78A were slightly decreased,as compared with wild type protein,and hemadsorptoin efficiencies of the other four mutants exhibited comparable levels to the wild type protein.A series of glycosylation sites combined mutants were constructed in this study, which are N53G-S73I,N53G-S131V,S73I-S131V,N53G-S73I-S131V,T78A-T179A, T78A-T211A,T179A-T211A,T78A-T179A-T211A,N53G-T78A,N53G-T179A and N53G-T211A,respectively.The analysis of FACS showed that the cell surface expression efficiencies of all the combined mutants were lower than 20%,except for N53G-T78A(25.30) and N53G-T211A(21.91%),as compared with the wild type protein.No cell fusion was observed by Giemsa staining in cells transfected by all the combined mutants.And no hemadsorption was found in most combined mutated proteins,except that a little of pigeon erythrocyte was observed in N53G-S131V,N53G-T78A and N53G-T211A. 2.Effects of leucine mutation in E2 glycoprotein on specific cell fusion.Mutants E2 L14A,L35A,L58A,L105A,L128A,L144A,L178A,L183A and L225A were constructed by homologous recombination,respectively,in which L14, L58,L128,L178 and L225 are located in coil structure of E2 protein,and L35,L105, L144 and L183 are located in strand structure of E2 protein.FACS assay showed that expression efficiencies of all the mutants on cell surface, except for L144A and L225A,were reduced in various degrees,as compared with the wild type protein.Cell fusion was observed by Giemsa staining after these mutants were transfected into BHK21 cells.To eliminate the effect of different cell surface expression in the mutants on cell fusion,the percentage of cell fusion activity was normalized with the cell surface expression efficiency.Compared with the wild type protein,cell fusion efficiencies of mutants L14A,L35A,L58A,L105A,L128A, L144A,L178A,L183A and L225A were 88.31%,79.55%,41.79%,49.16%,54.34%, 49.56%,72.31%,82.09%and 69.09%,respectively.The result indicated that efficiencies of specific cell fusion caused by these mutated proteins were,to some extent,reduced.Hemadsorption was observed in all the mutants after they were transfected into BHK21 cells.Compared with wild type protein,the adsorption efficiencies of L58A, L105A,L128A and L144A were slightly decreased and hemadsorptoin efficiencies of the other mutants were comparable with the wild type protein.3.Effects of leucine mutation in E1 glycoprotein on specific cell fusion.Mutants E1 L23A,L62A,L98A,L169A,L192A,L255A,L265A,L322A, L334A,L370A and L391A were constructed by homologous recombination, respectively,in which L62,L265,L322,L334 and L391 are located in coil structure of E1 protein,and L23,L98,L169,L192,L255 and L370 are located in strand structure of E1 protein.FACS analysis showed that all the mutants,except for L192A,L334A,L370A and L391A,have lower cell surface expression efficiencies than the wild type protein. Interestingly,cell surface expression efficiency of L370A is a little higher than that of the wild type protein.Cell fusion was detected by Giemsa staining in all the mutants,but the efficiencies of cell fusion in different mutants are various.When the effect of cell surface expression in the mutants on cell fusion was eliminated by normalizing the percentage of cell fusion activities with the cell surface expression efficiency,cell fusion efficiencies of mutants L23A,L62A,L98A,L169A,L192A,L255A,L265A, L322A,L334A,L370A and L391A were 60.89%,73.28%,86.41%,84.21%,91.62%, 87.00%,48.96%,74.88%,93.38%,80.25%and 73.64%,respectively,as compared with the wild type protein.The result indicated that efficiencies of specific cell fusion caused by these mutated proteins were decreased differently.Hemadsorption was observed in all the mutants.The quantitative assay showed that the hemadsorption efficiencies of L98A,L169A and L391A were comparable with the wild type protein and that of the other mutated proteins were slightly decreased,as compared with the wild type protein.The results suggested as followings:N-linked glycosylation on the three glycosylation sites of E2 and N76 of E1 are important in process of RV causing specific cell fusion.The adsorption capacity of S73I and T78A to sensitive cells is slightly decreased,which is one of the causes reducing cell fusion.N-linked glycosylation on the same one glycosylation site plays different roles in transportation and expression of protein and promotion of cell fusion, in which N71 of E2 and N177 of E1 play an important role in expression of glycoproteins on cell surface.The cell fusion phenomenon disappeared in combined mutants' expression because of their low cell surface expression efficiencies.In the secondary structure of E2 glycoprotein,the coil structure is important in transportation of glycoproteins to cell surface and effective expression on cell surface. But in extracellular domain of E2 glycoprotein,which has the main biological activity, the amino acid sequences near the N-terminal region(L58～L144) of intermediate part plays an important role in specific cell fusion. The variety in cell surface expression efficiencies and cell fusion efficiencies caused by different E1 mutants has a certain consistency,which indicated that mutation of these leucines affect cell surface expression and cell fusion simultaneously.The importance of different leucine depends on the position of the leucine in E1 protein,in which the amino acid sequences at the N-terminal region and near the carboxy-terminal region(L265 and nearby) of intermediate part play an important role in specific cell fusion.