Study On The Struture And Fuction Of The Linker Domains Of The Fusion Protein Of Paramyxovirus

Section ?Study on the structure and function of the HRB linker domain of HPIV3 F proteinBackground:Human parainfluenza virus type 3(human parainfluenza virus type 3,HPIV3)is an important member of the Paramyxoviridae family,which can cause lower respiratory tract infections in infants and young children under 5 years old,leading to bronchitis and pneumonia.Among patients with viral respiratory tract infection,the clinical positive detection rate is second only to respiratory syncytial virus(respiratory syncytial virus,RSV).HPIV3 is a kind of enveloped virus with a non-segmented single-negative-stranded RNA as genetic material.There are two kind of proteins closely related to virus virulence,anchoring on the HPIV3 viral envelope,hemagglutinin-neuraminidase(HN)and fusion protein(F).The fusion of virus envelope with cell membrane is an important step for the virus infection,and this process is mediated by F protein and assisted by HN protein.After the HN protein binds to the receptor on the host cell membrane,sialic acid,the F protein is activated,and undergoes a series of irreversible conformational changes,which drives the fusion of the virus envelope and the host cell membrane.The three-dimensional crystal structure of pre-fusion F protein is a "mushroom-like" homotrimer.The domains DI,DII,and D? constitute the main body of the head,and HRB constitutes the stem.Some literatures have reported that there is a low electron density region between DII and HRB in 3D structure,which is defined as HRB linker region.Although there have been numerous reports on the function of fusion peptides(FP),transmembrane regions(TM),D?,D?,and D?,few studies on the function of HRB linker have been reported.This section of the study takes the entire HRB linker of HPIV3 F as the research object to discuss its structural characteristics and the important role it plays in mediating membrane fusion.Objectives:To study the function of the HRB linker domain of the HPIV3 F protein,to clarify the role of the HRB linker of HPIV3 F protein in the process of membrane fusion,to provide a reference for the design of specific drugs targeting the HRB linker region in the future,and to construct live attenuated vaccine modified in HRB linker,so as to lay the theoretical foundation for the treatment and prevention of paramyxovirus-associated infections.Materials and methods:1.Construction of chimeric and deletion mutants of HRB linker of HPIV3 F protein:Based on the 3D structure of the known prefusion conformation of F protein of PIV5(parainfluenza virus type 5)virus,the relative position and amino acid range of the HRB linker region of HPIV3F protein on the whole polypeptide chain were determined by the method of SWISS-MODEL homology modeling.Then the recombinant plasmids of chimeric and deletion mutants in HRB linker of HPIV3 F protein were constructed by overlapping stretch PCR.All the mutants involved in the study were confirmed by sequencing.2.Expression of mutant F protein:pBSK+vector,wild type F plasmid,and each mutant plasmid were transfected into BHK-21 cells with Thermo Fisher Scientific's cationic transfection reagent TurboFect Transfection Reagent.The corresponding mutant proteins of F protein were expressed by the recombinant vaccinia virus expression system.3.Detection of the fusion activity of mutant F proteins:Dye transfer assay and syncytia formation assay were performed for semi-quantitative detection of membrane fusion activity,and content mixing assay was performed for quantitative detection of membrane fusion activity.4.Detection of expression of mutant F proteins:Indirect immunofluorescence was used to qualitatively detect the expression of mutant proteins.Flow cytometry was performed to quantitatively detect the expression efficiency of mutant proteins on the cell surface.5.Detection of the proteolytic activity of mutant F protein:Western blotting was used to detect the enzymatic hydrolytic activation of each mutant.6.Statistical method:GraphPad Prism7.0 was used to statistically analyze the experimental data.Unpaired t-test was used to statistically compare the results of mutant F protein with wild-type F protein,and P<0.05 was considered statistically significant.Results:1.Four mutant plasmids of chimerism and deletion of HRB linker region,Ch-NDV-L3,Ch-MV-L3,Ch-HPIV1-L3,De-L3 were successfully constructed.A-tag was added to the C-terminus of wt F and four mutant proteins.2.Results of the detection of membrane fusion activity of deletion and chimeric mutants:in dye transfer assays,the average number of dye transfer events caused by wild-type wt F was 98.0,while Ch-HPIV1-L3 decreased to 29.7.The other three mutants failed to cause significant dye transfer events.In the content mixing assay,the ability of the four mutants to cause cell content mixing was reduced from 16.5%to 23.5%of wt F.In the syncytia formation assay,except Ch-HPIV1-L3's ability to form syncytia was slightly weaker than wt F,the syncytia-forming abilities of the other three mutants dropped to less than 5%of wt F.3.Indirect immunofluorescence assay showed that all the mutants tagged with His-tag were detected by anti-His-tag monoclonal antibody,while only wt F was detected by anti-HPIV3 F monoclonal antibody.In the flow cytometry assay,the expression of mutant proteins on the cell surface was detected with anti-His-tag monoclonal antibody.It was found that the mutant proteins with His-tag were successfully expressed on the cell surface,there was no significant statistical difference compared with wt F-His.4.Western blotting assay showed that HPIV3 wt F-His,Ch-MV-L3-His,and Ch-HPIV1-L3-His could be hydrolyzed into active F1 fragments,while Ch-NDV-L3-His and De-L3-His could not be hydrolyzed,and remained an inactive FO state.Conclusions:The mutation in the HRB linker domain of HPIV3 F protein has a significant effect on the membrane fusion activity mediated by F protein,indicating that the HRB linker domain plays an important role in the normal function of F protein in mediating membrane fusion.The effect of the HRB linker domain on membrane fusion activity is achieved by changing the conformational stability of F protein before fusion.At the same time,the HRB linker domain can also affect the exposure of protease recognition sites on F protein precursor F0,and the hydrolysis of F 0 to active F1 is a prerequisite for F to perform membrane fusion function.Section ?Study on the structure and function of the D?-D? linker domain of HPIV3 F proteinBackground:Human parainfluenza virus(HPIV)is one of the major members of the paramyxovirus family,and is an important pathogen causing community-acquired pneumonia,especially causing infant pneumonia and lower respiratory tract diseases.According to its genetics and serological characteristics,HPIV can be divided into types 1 to 4,in which human parainfluenza virus type 3(HPIV3)infection is the main cause of lower respiratory tract infection in infants and young children under 5 years of age,second only to respiratory syncytial virus infection.At present,there is still no effective means of prevention and treatment.HPIV3 is an enveloped virus with a non-segmented,negative-sense,and single-stranded RNA genome.The fusion of the virus envelope and the target cell membrane is a key step in virus infection,and then the virus injects its genome into the cytoplasm and initiate the viral replication cycle.The fusion protein(F)and hemagglutinin neuraminidase(HN)are the decisive factors for the virus to enter the cells.HN binds to receptors,sialic acid on the cell membrane,and causes conformational changes of homologous F protein to catalyze membrane fusion.The F protein consists of several important protein domains.There is a "hinge"-like linker region between DI and D? structural domains,namely the D?-D? linker or LI linker,which may be involved in the conformational conversion of F protein-mediated membrane fusion.The key amino acids of the D?-D? linker of HPIV3 F protein has been located previously,the function of overall D?-D? linker has not been clarified.Objective:To determine the role of D?-D? linker of HPIV3 F protein in mediating membrane fusion,and to explore the mechanism of the effect of D?-D? linker on the fusion activity of F protein membrane.Materials and methods:Based on the 3D structure of the pre-fusion conformation of PIV5 F protein,the amino acid range of D?-D? linker of HPIV3 F protein was determined by SWISS-MODEL homology modeling method,the corresponding base sequence of D?-D?linker of NDV,MV,HPIVIF protein was determined by multiple sequence alignment,and the recombinant plasmids of deletion or chimerism mutation of D?-D? linker were constructed with overlap extension PCR.The recombinant vaccinia virus expression system was utilized to express wild-type F protein and mutant proteins in the BHK-21 cell line.The semi-fusion activity was determined by dye transfer assay,and the cell fusion-promoting activity was determined qualitatively and quantitatively by syncytial formation assay and content mixing assay,respectively.The expression of mutant protein was analyzed qualitatively with indirect immunofluorescence,the expression efficiency of mutant proteins on cell surface was quantitatively analyzed by flow cytometry,and the proteolysis of mutant protein was detected by Western blotting.GraphPad Prism 7.0 software was used for statistical analysis.Unpaired t-test was used to compare the results of the mutant and the wild type F.P<0.05 was considered statistically significant.Results:1.Four mutant plasmids of chimerism and deletion of HRB linker region,Ch-NDV-L1,Ch-MV-L1,Ch-HPIV1-L1,De-L1 were successfully constructed.A His-tag was added to the C-terminus of wt F and four mutant proteins.2.Fusion activity determination of chimeric or deletion mutants of HPIV3 F:after co-transfection with homologous HN,all mutants showed that the level of syncytial formation decreased in different degrees compared with wild type F.The mutants,Ch-NDV-L1,Ch-MV-L1,De-L1,almost eliminated their abilities to form syncytia,and the content mixing level was less than 5.0%of the wt F level.The content mixing abilities of the four mutants,Ch-NDV-L1,Ch-MV-L1,Ch-HPIV1-L1,De-L1,retained only 13.1%,12.4%,61.8%and 12.7%of that of wt F.Ch-NDV-L1,Ch-MV-L1,De-L1 greatly reduced the dye transfer level,corresponding to less than 4.4%of that of wt F.The dye transfer degree of Ch-HPIV1-L1 was also reduced to 30.6%of that of wt F.3.Expression of mutant proteins with chimeric and deletion mutation in the D?-D? linker of HPIV-3 F:in indirect immunofluorescence assay with anti-HPIV3 wt F monoclonal antibody,it was found that,no fluorescence signal was detected in mutant Ch-NDV-L1,Ch-MV-L1 and De-L1,except for Ch-HPIV1-L1.Indirect immunofluorescence detection with anti-His-tag monoclonal antibody showed that all His-tagged mutants could be detected,indicating the successful expression of all labeled mutant proteins.FACS with anti-His-tag monoclonal antibody showed that all mutants produced a certain proportion of positive cells,which was between 100.0%?105.1%of that of wt F group,and there was no significant statistical difference.Conclusion:1.The chimeric and deletion mutations of the D?-D? linker region of HPIV-3 F protein can lead to weakened or even loss of the ability to promote the cell membrane fusion.The effects of mutations on the ability to promote cells can occur early in the fusion.2.The mutation of HPIV-3 F protein does not lead to the expression efficiency of F protein on the cell surface,does not affect the transport process of F protein in the cell,but affects the folding and/or conformational changes of F protein.Although the D?-D? linker is freely curled before fusion,it still participates in the conformational change after F protein activation,and even plays an important role.Section ?Study on the identification and function of key amino acids of the HRB linker domain of NDV FBackground:Newcastle disease(ND),caused by Newcastle disease virus(NDV),is a severe infectious disease of poultry with strong infectivity and high fatality rate,which seriously harms the poultry farming.NDV can infect almost all kinds of birds,among which chickens are the most susceptible and often fatal,while infection in waterfowl cannot cause obvious symptoms,leading to the establishment of a storage pool of the virus.Mammals usually have a strong resistance to NDV,but human beings occasionally suffer from conjunctivitis due to the NDV infection.The virulence of NDV varies greatly.According to the severity of infection in chicken,NDV strains can be divided into four pathogenic types:strong virulent strain,medium virulent strain,low virulent strain,and avirulent strain.NDV belongs to the genus of Paramyxovirus,family of Paramyxoviridae.Like the other viruses in this family,NDV is a kind of enveloped virus with a single,unsegmented,negative-stranded RNA as its genetic material.Its genome encodes six structural proteins in sequence from the 3' end to the 5' end,which are nucleocapsid protein(NP),phosphoprotein(P),matrix protein(M),fusion protein(F),hemagglutinin-neuraminidase(HN)and macropolymerase(L).In addition,with RNA editing during transcription,the P gene expresses two non-structural proteins,V and W proteins.The active template for viral genome transcription and replication is the ribonucleoprotein complex(RNP),which is encapsulated by NP protein with genomic RNA,bounding with P and L proteins.At present,the known NDV genome has three lengths,namely 15186,15192 or 15198nt,which are in accordance with the rule of multiples of 6 nucleotides.The fusion of the viral envelope of NDV with the host cell membrane is the key step for the virus to infect the host cell.After the fusion of the two membranes,the viral genetic material enters the cell,and the virus completes its life cycle.The fusion process is assisted by the HN protein and mediated by the fusion protein(F).As an important member of the genus Paramyxovirus,the F protein of NDV shares highly similar structural characteristics with the other viruses of the Paramyxovirus genus.This section of the study takes NDV F as the research object,and explore the important role of the NDV F HRB linker in mediating membrane fusion and to locate the key amino acids in the HRB linker at the same time.Objective:To study the function of the HRB linker region of the NDV F protein,to clarify the mechanism of the NDV F protein HRB linker region affecting F-mediated membrane fusion,and to locate key amino acids in the HRB linker region.Materials and methods:Based on the known 3D structure of the pre-F protein fusion conformation of PIV5 virus,the amino acid range of the HRB linker of NDV F protein was determined with the SWISS-MODEL homology modeling.L436,E439,1450,and S453 were identified as conserved amino acid by homologous sequence alignment.Then the plasmids of single amino acid mutation in HRB linker of NDV F protein were constructed by site-directed mutagenesis and homologous recombination in bacteria.In BHK-21 cells,transient expression system with recombinant-vaccinia virus was utilized to express mutant F proteins.Semi-quantitative and quantitative detections of fusion activity of mutant-mediated membrane were carried out with dye transfer,syncytial formation and content mixing assays.The expressions of mutant proteins were analyzed with indirect immunofluorescence.Western blot was employed to detect the hydrolytic activation of each mutant.Finally,the results of mutant and wild type F were analyzed statistically with unpaired t-test.P<0.05 was considered statistically significant.Results:1.The six kinds of recombinant plasmids with key amino acid mutations in the HRB linker of the NDV F were successfully constructed,named as L436A,L436T,E439A,I450A,S453A,S453N.2.Fusion activity determination of single amino acid mutant of NDV F.L436A and I450A almost lost the ability of syncytial formation,only 1.96%and 1.33%of wild F.The syncytium performed by L436T and S453A were smaller than the wild type F,and their abilities decreased to 31.5%and 45.6%of that of wild type F.On the contrary,the mutant S453N increased to 115.7%of the wild type.The mutant E439A did not cause significant change in its syncytial formation ability.The semi-fusion ability of L436A and I450A almost was lost,and the obvious semi-fusion phenomenon was observed in L436T,E439A,S453A and S453N.The content mixing ability of L436A,L436T,and I450A decreased to 52.14%,73.11%,and 51.93%of the wild type F.When S450 was mutated to alanine S450A,its content mixing ability was 83.65%of wild type,while S450 was mutated to the asparagine(N)corresponding to HPIV3 F protein at this site,its content mixing ability rose to 143.58%of wild type.3.Expression of mutant proteins with single amino acid mutation in the HRB linker of NDV F.In indirect immunofluorescence,bright green fluorescence was detected in the cells transfected with recombinant plasmids of L436,E439A and S453N.While no fluorescent signals were detected in the cells transfected with L436A and I450A.Non-reducing protein immunoblotting showed that all mutants could be hydrolyzed and activated into F1,but the expression level was significantly lower than that of wild-type F protein,and there were significant differences in protein expression among mutants.Conclusion:Site-directed mutation of conserved amino acids in HRB linker of NDV F protein has a significant effect on the membrane fusion activity mediated by F protein,indicating that HRB linker plays an important role in the fusion process.The HRB linker of NDV F protein is very important for its fusion,in which L436 and 1450 amino acids are the key amino acids.