Study On The Structure And Function Of The Junctional Regions Among Key Domains Of The Envelope Glycoproteins Of Newcastle Disease Virus

Part?:Functional and structural analysis of the head-stalk linker of NDV HNBackgroundNewcastle disease virus?NDV?is an enveloped negative-strand RNA virus of the family Paramyxoviridae,which includes measles virus?MeV?,mumps virus?MuV?,parainfluenza viruses?PIVs?,canine distemper virus,Hendra virus?HeV?,Nipah virus?NiV?and so on.To infect cells,all paramyxoviruses need to fuse their viral envelopes with cell plasma membranes of their target host.In this process,most paramyxoviruses use a two-component fusion apparatus consisting of a fusion protein trimer and an attachment protein tetramer.Attachment proteins are classified into 3 categories according to their different functions:HN?have both receptor recognition and neuraminidase activities?,H or G?only have receptor recognition activitiy?.Furthermore,attachment protein can activate its homologous F protein,which carried out the fusion process by conformational change.According to the structure information and mutagenesis analysis for the headless PIV5 HN1-117,a 'stalk exposure' model for membrane fusion triggering was proposed firstly for PIV5 and now is extended to other paramyxoviruses.This model suggests that before receptor recognition,the globular heads of HN/H/G proteins physically restrict the access of the F protein to the F-activating region residing on the HN/H/G stalk domains.Binding receptor results in a molecular rearrangement of the dimer-of-dimer globular heads of attachment proteins,exposing the F-activating regions of HN/H/G stalk domains,allowing the F protein to interact with this region of HN/H/G stalk and subsequently undergo refolding process mediating the membrane fusing.Several lines of mutagenesis analysis suggested that headless attachment proteins could trigger F's activation,including PIV5 HN1-117,MeV Hl-122,NiV G1-167,MuV HN1-132 and NDV HN1-123.However,it is unclear for the reasons why only certain lengths of stalk region can activate F and why the headless stalk needs to be stable for H.A possible explanation is that a specific connecting segment existed between head and stalk,which is important for attachment protein to adopt a triggering-permissive conformation to trigger F for membrane fusion.ObjectiveTo investigate the role of the junctional region between the head and stalk domain of attachment protein in the fusion activity,the NDV HN protein was chosen as the representative to study,and the whole region of the junction between the head and stalk?aa115-122?was changed.The effects on the fusion-promoting activity along with other biological functions of full-length HN and HN 1-123 proteins are analyzed to elucidate the role of this linker region in the function of HN proteins,especially the fusion promotion activity.Methods1.Construction for mutants of HN:The sequences of attachment proteins from hPIV1-4,PIV5,APMV6,HeV,NiV,SeV,MuV and MeV were aligned with that of NDV HN.The HN head stalk linker region?115-NGAANNSG-122?was replaced with the corresponding sequence of other paramyxoviruses.In addition,a HN mutant without this segment was also constructed.All mutants were constructed using site-directed mutagenesis and bacteria homologous recombination method.2.Expression of HN mutant proteins:HN mutant proteins were expressed by a transient expression system of vaccinia virus-T7 RNA polymerase in BHK-21 cells.3.Expression efficiency detection of HN mutant proteins:The expressions of each chimeric protein on the cell membrane surface were qualitatively and quantitatively detected using indirect immunofluorescence assay?IIFA?and fluorescence-activated cell sorter?FACS?,respectively.4.Functional detections of HN mutant proteins:The Giemsa staining method,the reporter gene method,and the R18 dyer transfer were utilized to determine the fusion-promoting ability of HN mutant proteins.The hemadsorption test?HaD?was used to detect the changes in the receptor recognition activity of mutant HN proteins.Changes in neuraminidase activity?NA?of each mutant protein were tested using the NA kit.5.Construction for mutants of HN1-123 protein and functional detections:4 HN mutants with obvious changes in fusion promotion activity were re-constructed by deleting the head region?aal24-571?,and the fusion-promotion ability of these head deleting HN 1-123 mutants are determined by the Giemsa staining and reporter gene method.6.Western Blot assay was used to detect the oligomeric state of mutant HN proteins and the glycosylation modification state of the N119 site of each HN 1-123 protein.7.Statistical analysis:Statistical analysis was calculated by SPSS 17.0 using Student's t test with a significance level of P<0.05.Results1.Residues from 115 to 122 of NDV HN were replaced by those of other 11 paramyxoviruses to form the following chimeras named by the source of virus type:Chi_hPIV1,Chi_hPIV2,Chi_hPIV3,Chi_hPIV4,Chi_PIV5,Chi_APMV6,Chi_HeV,Chi NiV,Chi_SeV,Chi_MuV and Chi_MeV.2.Results from IIFA and FACS showed that chimeras in the linker had no effect on the cell surface expression of protein.Each of the mutant HN proteins displayed a similar expression level to that of the wt HN protein,ranging from 81.8%to 114.1%of the wt HN level?P>0.05?.3.Function detection results:Most chimeras had similar levels to wt HN in both the NA activity and the receptor recognition activity.Only one chimera,Chi_MeV,dropped its receptor recognition activity to 72.7%?P<0.05?of wt HN level without affecting neuraminidase activity.Contrast to these two functions,the fusion-promoting activity changed differently.The quantitative results of the reporter gene method showed that the fusion promoting abilities of Chi_hPIV1,Chi_hPIV2,Chi PIV5,Chi_SeV and Chi_MeV decreased to 29.4%,42.4%,30.1%,42.7%,and 27.1%of wild-type HN,and the syncytia observed under the microscope were smaller and less than wt HN.The R18 hemi-fusion observation also showed that the dye transfer fluorescence intensities caused by these chimeras were weaker.In contrast,Chi_APMV6 and Chi HeV showed an increased ability to promote cell fusion,and more and larger syncytia could be observed under the microscope.Meanwhile,the fluorescence of the R18 dye transfer became stronger.The remaining chimeras maintained a similar fusion-promoting ability to the wild type?P>0.05?.4.A deletion mutant Del₁15-122 was successfully constructed.This mutant protein successfully expressed on the surface of the transfected cell membrane,which was approximately 84.9%of expression level of the wild-type HN protein.There was no significant difference in the expression efficiency between Del₁15-122 and wt HN?P>0.05?.Del₁15-122 lost the ability to promote fusion completely.However,the receptor recognition and NA activities retained 109%and 96.1%of that of wt HN,respectively?P>0.05?.5.NDV HN1-123?HNs?and 4 chimeras NDV HN1-123-Chi_PIV5?HNs-PIV5?,HNM23-Chi_APMV6?HNs-APMV6?,HN1-123-Chi_HeV?HNs-HeV?and HN1-123-Chi_MeV?HNs-MeV?were constructed.The fusion ability of HN1-123 chimeras was divided into two groups compared with the HNs:the fusion-promoting ability of HNs_PIV5 and HNs_MeV decreased;while HNs_APMV6 and HNs HeV showed comparable levels with HN 1-123?P>0.05?.6.The results of oligomeric state of HN mutant proteins showed that sequence changes in this linker region had a certain influence on the oligomeric state and stability of HN protein.A certain difference was found in the ratio of monomeric bands of HN mutant proteins under the non-reducing state.After DTT was introduced,5 mutants?Chi_hPIV1,Chi_hPIV4,Chi_PIV5,Chi_MuV and Del₁15-122?still retain the dimeric form of HN,suggesting higher resistance to cleavage of disulfide bond by DTT compared with wt HN.7.The glycosylation condition of N119 site for the HN 1-123 protein and its chimera showed that substitution of the amino acid sequence resulted in a change in the glycosylation of the N119 site.Loss of glycosylation at this site has an effect on the cell surface expression of the HN1-123 protein.ConclusionsThe head-stalk junction region?aal 15-122?of NDV HN has a significant effect on the fusion-promoting function without affecting the NA activity and receptor recognition activity.The head-stalk junction region may influence the fusion promotion activity by altering the oligomeric state of HN protein and its stability,also by affecting the structure of the stalk 4HB.Part ?:Functional and structural analysis of the D?-D? linker region of NDV F proteinBackgroundNewcastle disease?ND?is a highly contagious disease in chickens,causing huge economic losses to the poultry industry.Current vaccination strategies are not fully efficacious and the development of new concepts for vaccine generation are needed since multiple outbreaks still occur worldwide and NDV strains have been isolated from vaccinated chicken flocks.To develop new vaccines or therapeutic drugs,it is necessary to clarify further the fusion process caused by NDV.NDV infection is achieved by membrane fusion,which is initiated by receptor recognition and viral binding to the host cell surface.This process is mediated by the interaction between HN and F glycoproteins.The F protein of NDV is a class I integral membrane protein,showing as a trimer at the virion surface or expressing cell.Initially,an inactive precursor FO is synthesized and subsequently cleaved by the cellular proteases into disulfide-linked F1-F2 complex.The F1 subunit,from the N-terminus to the C-terminus,sequentially contains the fusion peptide?FP?,two hydrophobic heptad repeat?HR?domains?HRA and HRB?,and the trans-membrane?TM?domain.Upon triggering,the hydrophobic FP released from F trimer firstly,then causing HRA extension and forming a triple-stranded coiled coil that mediates insertion of the FP into the target membrane,thus forming the so-called transient intermediates.Subsequently,HRB binds with HRA coiled coil in an anti-parallel orientation,forming a six-helix bundle?6HB?that is critical for pulling the target and effector membranes together.With the acquisition of the crystal structure of the F protein,different domains are divided for F protein including D?,D?,D?,HRA and HRB.Besides,certain unstructured junctions,such as the DI-DII linker and the HRB linker,exist among these domains,which may act as a flexible hinge in maintaining the conformation of F protein during the fusion process.In our previous study on the role of the DI-DII linker of hPIV3 F,the mutants introduced into this DI-DII linker region mediated a significant reduction in fusogenic activity.To date,it is not known whether the DI-DII linker of the NDV F protein has similar effects on fusion activity to that of hPIV3 F protein.ObjectiveTo explore the role of this linker in fusion activity induced by NDV F protein,to compare the function of this DI-DII linker between NDV F and hPIV3 F proteins.To provide clues for explaining the mechanism of fusion process.Methods1.Amino acid sequence alignment was used to determine the position of NDV F DI-DII region corresponding to the hPIV3 F protein.2.Single-point mutants of this region were constructed by site-directed mutagenesis and homologous recombination in bacteria.A transient expression system of vaccinia virus-T7 RNA polymerase was used to express the wt or mutated F proteins.3.Cell surface expression detection:The cell surface expression of the F mutant proteins was detected with IIFA and FACS.4.Fusion ability detection co-expressed with HN:Giemsa staining and reporter gene method were used to qualitatively and quantitatively determine the effect of each mutant protein on membrane fusion function.In addition,a R18 dye transfer method was used to observe the F protein-mediated hemi-fusion.5.Fusion ability detection without HN:Giemsa staining was used to observe the formation of syncytia of each mutant F protein expressed alone.6.The cleavage activation conditions were detected with Western Blot assay.7.Statistical analysis:Statistical analysis was calculated by SPSS 17.0 using Student's t test with a significance level of P<0.05.Results1.The DI-DII linker is located between domains DI?yellow?and DII?orange?at residues from 376 to 381 in the NDV F protein.Twelve mutants?E376A,E376K,G377A,G377S,A378D,L379A,L379I,T380A,T380V,T380P,T381A and T381P?were constructed including alanine mutants and the substitution of corresponding residues of the hPIV3 F protein by amino acid sequence alignment.2.All the mutant F proteins were detected at the cell surface,with similar levels to wt F,ranging from 82.8%to 106.8%?P>0.05?.However,mutants G377S,A378D,L379A and T380P-expressing cells formed obvious mass of green fluorescence observed in IIFA.3.Most of the mutated F proteins lead to obvious alteration in fusion activity except for E376K,T380V and T381A.Syncytium formation showed that the syncytia formed by mutants G377A,L379I,T380A and T381P in the presence of HN protein were not only smaller but also fewer than those produced by wt F and HN proteins.However,mutants E376A,G377S,L379A and T380P induced much larger syncytia than wt F.The quantitative results of the reporter gene method are consistent with the results of syncytium observation.The G377A,L379I,T380A and T381P mutations reduced their abilities to form syncytia also resulted in weakened content mixing,which retained only 44%,41.9%,18.8%,and 29.9%of the wt F level,respectively,when coexpressed with HN.Mutants E376A,G377S and A378D increased the levels of content mixing to 142.7%,167.3%and 130.8%of that of wt F proteins.As expected,L379A and T380P showed much higher levels in content mixing,almost 2.5 folds of that of the wt F and HN.Mutants E376K,T380V and T381A mediated approximately 99.1%,110.3%and 90.8%of the levels of content mixing of the wild-type F protein,respectively?P>0.05?.The microscopic observations results of hemi-fusion were almost identical to the Giemsa staining results.Mutants?i.e.G377A,L379I,T380A and T381P?that reduced the ability to form syncytia also showed less and weak fluorescence.In contrast,mutants E376A,G377S,A378D,L379A and T380P that enhanced the fusion ability had stronger fluorescence than wt F.4.Mutants G377S,A378D,L379A and T380P proteins were able to mediate the fusion in the absence of HN protein,although the fusion activities were much lower than that of wt F and HN.However,the HN-independent fusion results obtained in BHK-21 cells were not observed in Vero cells.5.Results from Western Blot assay showed all F mutants were cleaved efficiently.Besides,an extra slower migrating band than F0 was detected for mutants G377S,A378D,L379A and T380P,which may be due to structural changes.ConclusionsThe mutations introduced into the DI-DII linker of the NDV F glycoprotein exhibited different levels in the membrane fusion,and mutants G377S,A378D,L379A and T380P in this region,to some extent,changed the requirement of HN for promoting membrane fusion,suggesting that this region may be involved in the structural switch of F protein.Part ?:Roles of conserved amino acids around the D?/D?junctional region of the NDV F proteinBackgroundNDV F mediates membrane fusion,which is essential to the processes of viral entry,replication,and dissemination.A large intervening region with important functions exists between HRA and HRB,and current crystallographic structures divide this region into three distinct domains,?,?,?,from the N-terminus to the C-terminus,which constitute the head of F.From the top view of the F protein of NDV and human parainfluenza virus 3?hPIV3?,it was found that the key domains of the heads were roughly of the same shape,but there were some differences in the orientation.This difference in orientation may attribute to the different arrangements of domains D?,D?,D?,which may relate to the different junction regions among these domains.Though no obvious or direct linkage as DI-DII linker exits between domains D?and DI,several important amino acids were found around this junction.Also,the amino acids around this segment are considered to be a key region that constitutes the radial channels of the head of the F protein,which play an important role in the docking of FP.Conserved sites were found around this junction region by sequence alignment of the F protein from NDV,hPIV3,and PIV5,and we speculated that these amino acids were conserved due to important roles in the folding or fusion function of the F protein.ObjectiveTo address the functional roles of conserved amino acids at the D? and D?junctions and found out key conserved sites important for protein function at the junction of the Dill and DI domains.Methods1.Amino acid sequence alignment was used to find out the conserved amino acids around the Dill and DI junctions.2.Spatial position display of conserved amino acids:The pre-fusion structure of NDV F protein was constructed by homology modeling,and the position of each conserved amino acid was displayed on the pre-fusion conformation of NDV F protein.3.Site-directed mutagenesis was utilized to substitute the 8 conserved amino acids with alanine.A vaccinia virus-T7 RNA polymerase transient expression system was employed to express the wt or mutated F proteins.4.Three different types of membrane fusion assays were performed to analyze the fusogenic activity.5.IIFA and FACS analysis were utilized to examine the cell surface expression.6.The cleavage activation conditions were detected with Western Blot assay.7.Statistical analysis:Statistical analysis was calculated by SPSS 17.0 using Student's t test with a significance level of P<0.05.Results1.Eight conserved amino acid residues were found and site-directed mutagenesis was utilized to mutate to alanine at these sites?1269,1274,D277,Q286,V287,P290,L295 and N297?.These conserved sites were exhibited on the NDV F homology modeling structure.Most of these sites were located within the F protein,while sites L295 and N297 were situated on the surface of this protein.2.Syncytium formation showed that most of these substitutions displayed reduced fusion activity except for mutants Q286A and N297A.Mutant I269A formed fewer syncytia compared with wt F.However,mutants I274A,P290A and L295A had very limited ability to form syncytia and no syncytia were observed for mutants D277A and V287A.Only mutant Q286A produced significantly larger syncytia than wt F,while N297A did not affect the formation of syncytia.3.The quantitative results of the reporter gene method are consistent with the results of syncytium observation.The F protein carrying the individual I269A mutation resulted in weakened content mixing,retaining only 57.1%of the wt F level,when co-expressed with the NDV HN protein.Mutants I274A and L295A reduced the levels of content mixing to extremely lower levels,21.9%and 24.2%of that of wt F proteins.Additionally,background levels of fusion activity,5.6%,7.8%and 3.0%of wt F,respectively,were detected for mutants D277A,V287A and P290A.Mutant Q286A produced fusion activity about 1.6 fold of the level of the wt F,whereas mutant N297A showed a similar amount of fusion,approximately 97.7%of the wt F?P>0.05?.4.The microscopic observations results of hemi-fusion almost mirrored the Giemsa staining results.Mutants I274A,D277A,V287A,L295A and P290A produced only a very small number of fusion events,almost similar to the negative control level.In addition,mutant I269A showed less and weak fluorescence.However,mutant Q286A showed a stronger fluorescence than wt F,while mutant N297A produced a similar level in lipid mixing.Therefore,these alanine substitutions also resulted in defects in mediating lipid mixing except for Q286A and N297A.5.According to the results of IIFA and FACS,these F mutants showed different expression levels.Q286A,L295A and N297A were very well expressed,94%,100%,and 86.8%of that of wt F,respectively?P>0.05?.Mutants I269A and P290A showed a reduced expression level,only about one-thirds of that of wt F.However,mutants I274A,D277A and V287A could not be detected with anti-NDV serum.6.The results of Western Blot showed that most mutants were cleaved efficiently and both the F0 and F1 were present,except for I274A,D277A and V287A.The total expression levels of mutants I269A and P290A increased compared with their cell surface expression levels,suggesting that these two mutants led to transport defects.ConclusionsThe conserved amino acids near the D? and DI domain of the NDV F protein play an important role in the folding and transport of the F protein and participate in fusion process.