Study On The Indentification Of The Key Amino Acids And The Mechanism Of Paramyxovirus Hemagglutinin-neuraminidase Glycoprotein-mediated Membrane Fusion Promotion

Section I "α"-position-mutated and N481-mutated HN proteins of Newcastle disease virus impair fusion and HN-F interaction by different mechanismsBackground:Newcastle disease virus (NDV) belongs to the family Paramyxoviridue, a group of enveloped viruses with nonsegmented, negative-sense, single-stranded RNA genome. NDV causes series of severe respiratory and neurological diseases named Newcastle disease (ND) in the bird species world widely. NDV also causes a spectrum of human diseases, ranging from conjunctivitis to lymphadenitis by contact with sick birds. NDV is usually used as a model for paramyxovirus research. There are no therapeutic weapons for preventing and treating ND. The hurdle for developing modes of preventing and treating ND has been in large part a result of gaps in our understanding of the interaction of NDV with its hosts during pathogenesis. Thus, deeper understanding of the interplay between virus-mediated pathology and host cells will be vital for developing safe and effective vaccines strategies.Membrane fusion is not only a key progress of NDV proliferation and infection, but also pathological characteristics of infected cells. Optimum membrane fusion requires both NDV HN and F proteins. Therefore, some researchers proposed that a type-specific interaction between the paramyxovirus HN and F proteins was necessary for fusion of virus with cell membrane. Previous studies of chimeric FIN proteins revealed that the putative stalk domain of paramyxoviruses was important for interaction with F protein and promotion of cell fusion. Flowever, the mechanism of the HN-F interaction is controversial. Heptad repeat regions (HRs) are often involved in protein-protein interactions and could form alpha helices. There are two characterized HRs in the stalk domain of NDV FIN protein, HR1(74-88aa) and HR2(96-110aa). L74, V81and V88are a-position residues in HR1, and L96,1103and L110are a-position residues in HR2. Mutational analyses of the HRs of paramyxoviruses F proteins have shown that a-position residues were important in fusion activity as well as the folding of their F proteins. However, the effect of these substitutions on the HN-F interaction was not determined.N-linked glycosylation has some influences on correct folding of HN into its biologically active conformation and protein activity. NDV HN protein has six potential N-linked glycosylation sites and only four (Gl to G4at residues119,341.433,481, respectively) of which were utilized. Loss of N-linked glycosylation from HN protein altered the functions of HN protein and the virulence oi-NDV. But the effect of elimination of N-linked carbohydrate addition sites on the HN-F interaction was not determined.Objectives:To study the fuctions of a-position residues in FIR and N-linked carbohydrate addition sites; to illustrate the mechanisms of glycoprotein-mediated membrane fusion promotion and HN-F interaction; to lay the foundation for the design of effective vaccines for preventing ND. Materials and methods:1. Construction and transfection of NDV HN mutants:Mutants were constructed by combination of fusion PCR and recombination. The presence of the desired mutation was confirmed by DNA sequencing. LipofectamineTM2000(invitrogen) transfections were performed in the vaccinia virus-T7RNA polymerase expression system, using1μg of each plasmid for transfection.2. Radiolabeling, immunoprecipitation and co-immunoprecipitation assay:In immunoprecipitation assay, the total HN and F proteins were labeled with100μCi of Expre35S35S labeling mix. One aliquot of each sample was digested with PNGase F, and the other was not. In co-immunoprecipitation assay, cell surface proteins were labeled Expre35S35S labeling mix and biotinylated with sulfo-NHS-SS-biotin. Proteins were eluted by boiling in protein loading buffer and then analyzed by10%sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE). The percentage of total cell surface HN that co-immunoprecipitated with F was quantified with a Perkin Elmer Cyclone Plus.3. Cell surface expression and function characteristics assay of mutants:The cell surface expression (CSE) was quantitated by flow cytometry with a mixture of two MAbs specific for the NDV HN protein. Qualitative and quantitative assay of fusion promotion function was performed by Giemsa staining and the reporter gene method, respectively. The hemadsorption (HAD) activity was determined by the ability of HN protein to adsorb erythrocytes. The neuraminidase (NA) activity was determined colorimetrically using sialyllactose.4. Statistical analysis:Statistical analysis was performed by SPSS16.0software. The results are expressed as mean±the standard deviation for three independent experiments. T-test was used to compare the data of mutated HN protein with the data of wild-type (wt) HN protein and P<0.05demonstrated the difference was statistically significant.Results:1. Six a-position residues in the HRs were mutated to alanine (L74A, V81A, V88A, L96A,1103A and L110A) successfully. HN proteins carrying the L74A, V81A, V88A, L96A,1103A, and L110A mutations showed significant reductions in fusion activity, which was22.6%,29.9%,24.7%,70.4%9.1%, and17.8%relative activity compared to that of the wt HN, respectively. Each of the mutated proteins had a cell surface expression level similar to that of wt HN but reduced the HN-F interaction. L96A substitution exhibited the similar level of HAD activity to wt HN. Other mutated proteins had the decreased HAD activities and1103A reduced most. HN proteins carrying L74A, V81A or L96A mutations had the similar NA activity to wt NDV HN. V88A-mutated HN still gained78.5%of wild-type (wt) HN activity. However, the remaining mutants,1103A and L110A, exhibited levels of NA that were only5.7%and5.2%of the wt amount, respectively.2. Four asparagine-to-alanine mutants in the glycosylation sites (N119A, N341A, N433A and N481A) were created by site-directed mutagenesis. The results showed that proteins with mutations in sites N119, N341, N433and N481migrated faster than wt protein and the PNGase F-digested mutant HN proteins comigrated with wt HN at a higher rate. The amount of N481A-mutated HN protein was extremely small. N481A-mutated protein and the CSE of it was only57.5%of the wt HN level. The other three mutations in the glycosylation sites expressed at the near-wt level. N-linked glycans at N119. N341, and N433from HN protein still remained the similar fusion activity to the wt level. However, the HN protein carrying the N481A substitution gained only66.2%ability to promote fusion compared to wt HN. Also.53.6%±4.2%of the total amount of the N481A-mutated HN protein at the cell surface co-immunoprecipitated with F. The remaining three mutated proteins co-immunoprecipitated with F at the near-wt level. The HN protein carrying the N481A substitution had very low HAD (33.4%) and NA (6.5%) relative to the level of wt. The N119A-mutated and N341A-mutated HN proteins had similar HAD and NA activities compared to the wt. The N433A had HAD activity similar to the wt (109.3%), but slightly lower NA activity (70.0%).Conclusions:1. Reduced fusogenic activity of six HRs-mutated HN proteins correlates with diminished capacity to interact with NDV F protein, but the amount of co-IP of HN is not directly proportional to the extent of fusion. 2. HAD activity, NA activity and HN-F interaction of HRs-mutated HN proteins formed a balanced network, which regulate the fusion promotion activity together.3."a"-position-mutated and N481-mutated HN proteins of NDV impair fusion and HN-F interaction by different mechanisms. N481A indirectly affects HN’s fusion promotion function and HN-F interaction by misfolding the important structure, while mutations in the HRs do that by altering the transmission of a signal from the globular domain to the F-specific region in the stalk. Section Ⅱ Mutational analysis of conserved amino acids of human parainfluenza virus type3hemagglutinin-neuraminidase proteinBackground:The human parainfluenza viruses (hPIV), belonging to the Paramyxoviridae family, are a major cause of croup and respiratory tract infections in infants and children. Among hPIV types1to4, the hPIV-3subtype is the most frequently detected virus causing most infections. Annual epidemics of hPIV-3infections continue to occur and the mechanism by which hPIV-3infects host cells remains unclear.hPIV-3infection cycle in host cells is divided into absorption, penetration. uncoating, replication, assembly and release. The fusion of the viral membrane with the host-cell membrane includes absorption, penetration and uncoating. The fusion process is not only the first step of hPIV-3infection, but also the key progress of viral life cycle and the major target in antiviral therapy. The envelope of hPIV-3contains two glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion (F) proteins. The F peotein, synthesized as a precursor Fo, must be cleaved into F1and F2, to activate fusion activity via interaction of hPIV-3HN with sialic acid-containing receptor on the cell surface.N-linked glycosylation is an importany type of posttranslational modification of hPIV-3HN protein. Endogenous lectins and carbohydrate-binding proteins secreted by target cells can bind to specific N-glycans on viral glycoproteins and help virus envelope-mediated cell-cell fusion, further suggesting that N-glycans on the viral envelope glycoproteins play a critical role in the pathobiology of enveloped virus entry into host cells. At present, there have been no studies on the role of N-linked glycosylation in the activities of the hPIV-3HN protein.There are two receptor binding/NA active sites on hPIV-3HN. R192, D216, E409, R424, R502, Y530and E549are conserved amino acids in site Ⅰ, and N551and H552are conserved amino acids in site Ⅱ. For NDV HN, site Ⅰ, exhibiting both NA and receptor binding activities, and site Ⅱ, possessing only receptor binding activity. We speculate that the functions of two receptor binding/NA active sites on hPIV-3HN maybe similar to that on NDV HN.One domain in particular, the sequence NRKSCS at residues252to257in hPIV-3HN, was predicted to be closest to the receptor binding/NA active site in the three-dimensional structure of the protein. This hexapeptide is also the longest linear stretch of amino acids that is totally conserved among the HN proteins of many paramyxoviruses. Therefore, the role of individual amino acid in this domain on receptor binding and NA activity may be very important.Not only NRKSCS is conserved among the HN/G/H proteins of many paramyxoviruses. but also the sequence EGR at residues409to411(in the forth β-shect), GVY at residues476to478(in the fifth β-sheet), and YTT at530to532(in the sixth β-sheet) in hPIV-3’s HN is conserved among the HN/G/H proteins of many paramyxoviruses. EGR and YTT are closest to the receptor-binding pocket, and E409and Y530are also conserved amino acids in site I. The conservation of three tripeptides suggests that they may be important for the structure and function of hPIV-3HN.Objectives:1. To determine the function of conserved amino acids in the N-linked glycosylation sites and receptor binding/NA active sites of hPIV-3HN protein.2. To determine the function of conserved amino acids in the hexapeptide and three tripeptides of hPIV-3HN protein.3. To illustrate the mechanisms of hPIV-3HN glycoprotein-mediated membrane fusion promotion and HN-F interaction.Materials and methods:1. Construction and transfection of hPIV-3HN mutants:Mutants were constructed by combination of fusion PCR and recombination. The presence of the desired mutation was confirmed by DNA sequencing. LipofectamineTM2000(invitrogen) transfections were performed in the vaccinia virus-T7RNA polymerase expression system, using1μg of each plasmid for transfection.2. Radiolabeling, immunoprecipitation and co-immunoprecipitation assay:In immunoprecipitation assay, the total HN and F proteins were labeled with100μCi of Expre35S35S labeling mix. One aliquot of each sample was digested with PNGase F, and the other was not. In co-immunoprecipitation assay, cell surface proteins were labeled Expre35S35S labeling mix and biotinylated with sulfo-NHS-SS-biotin. Proteins were eluted by boiling in protein loading buffer and then analyzed by10%SDS-PAGE. The percentage of total cell surface HN that co-immunoprecipitated with F was quantified with a Perkin Elmer Cyclone Plus.3. Cell surface expression and functions characteristics assay of mutants:The cell surface expression efficiency was quantitated by flow cytometry with a mixture of two MAbs specific for the hPIV-3HN protein. Qualitative and quantitative assay of fusion promotion function was performed by Giemsa staining and the reporter gene method, respectively. The HAD activity was determined by the ability of HN protein to adsorb erythrocytes. The NA activity was determined colorimetrically using sialyllactose. The hemifusion assay was performed by the spread of the fluorescence-labeled lipid probe octadecyl rhodamine B (R18) from RBCs membranes into membranes of cells expressing wt or mutated HN and F proteins.4. Dynamic measurement of membrane fusion:Fresh human erythrocytes (RBCs) were labeled with R18. BHK-21cells co-transfected with HN and F genes were then washed and incubated with R18labeled RBCs (0.1%hematocrit) with occasional gentle rocking to ensure an even spread. The fluorescence was measured continuously in a spectrofluorometer from0to300s. Fit curves were drawn to describe dynamic changes in the membrane fusion process.5. Statistical analysis:Statistical analysis was performed by SPSS16.0software. The results are expressed as mean±the standard deviation for three independent experiments. T-test was used to compare the data of mutated HN protein with the data of wt HN protein and P<0.05demonstrated the difference was statistically significant.Results:1. Construction and functions characteristics assay of N-glycosylation-defective mutants.Four single mutants (Gl, G2, G3and G4). three double mutants with alterations in two of the sites used (G12. G14. G24) and a triple mutant G124were constructed. The results showed that G1. G2. G4. G12, G14. G24and G124in the undigested samples migrated slightly faster than the wt HN rotein on polyacrylamide gels, but G3mutant protein and wt HN protein migrated to the same position. After digestion with PNGase F, each mutated protein co-migrated with wt HN protein at a higher rate. Single and multiple mutations deficient in receptor recognition activity diminished the fusion promotion activity but had no significant effect on NA activity and the ability to interact with homologous F protein at the cell surface. Also, the F protein that co-expressed with multiple mutated HN protein was inefllciently cleaved. Amino acid substitutions in the N-linked glycosylation sites of HN also diminished the hemi fusion activity and ability of initial fusion promotion activity.2. Construction and function characteristics assay of conserved amino acid substitutions in receptor binding/NA active sites. Mutations in receptor binding/NA active site I (R192A, D216A, E409A, R424A, R502A, Y530A, E549A) or site Ⅱ (N551A and H552A) were constructed. Cell fusion promotion efficiency of each mutant protein decreased to some extent except N551A (112.2%of wt HN). The mutated proteins were expressed at more than90%of the wt HN level. There was no statistic difference of cell surface expression between wt and each mutant HN protein. The avidity of HN-F interaction was not affected by mutations in receptor binding/NA active sites, but the E409A, R424A, R502A and H552A mutants reduced the percent of F protein cleavage to54.8%,46.3%,56.3%and43.4%of wt HN, respectively. Receptor binding activity of each mutant protein was reduced to different extent except N551A (100.1%of wt HN). There was different neuraminidase activity among each mutant HN protein. R502A decreased most to18.6%, but the neuraminidase activity of both E549A and N551A were similar to that of wt hPIV3HN. Amino acid substitutions in receptor binding/NA active sites of HN except N551A also diminished the hemifusion activity and ability of initial fusion promotion activity.3. Construction and function characteristics assay of conserved amino acid substitutions in the hexapeptide.N252A, R253A, K254A, S255A, C256A and S257A were constructed. There was no statistic difference of cell surface expression and HN-F interaction between wt and each mutant HN protein. N252A, R253A, K254A and C256A reduced the percent of F protein cleavage to59.1%,17.3%,65.0%and13.2%of wt HN, respectively. Fusion promotion activity, HAD and NA activity of each mutant protein was reduced to different extent except S257A which were similar to that of wt hPIV3HN. Amino acid substitutions in hexapeptide except S257A also diminished the hemifusion activity and ability of initial fusion promotion activity.4. Construction and function characteristics assay of conserved amino acid substitutions in the three tripeptides.E409A, G410A, R411A, G476A, V477A, Y478A, Y530A, T531A and T532A were constructed. Cell fusion promotion efficiency of each mutant HN protein decreased to some extent. There was no statistic difference of cell surface expression and HN-F interaction between wt and each mutant HN protein. E409A, R411A, G475A, V477A, Y478A and H552A reduced the percent of F protein cleavage to54.8%,35.5%,35.5%,31.4%,29.8%and11.8%of wt HN, respectively. HAD and NA activity of each mutant protein was reduced to different extent. Also, amino acid substitutions in three tripeptides diminished the hemifusion activity and ability of initial fusion promotion activity.Conclusion:1. N308, N351and N523were indeed utilized for N-linked carbohydrate attachment.2. Mutations in single or multiple glycosylation sites deficient in receptor recognition activity diminished the fusion promotion activity. There is a direct correlation between the extent of membrane fusion and the strength of the HN-receptor interaction.3. Conserved amino acids in the receptor binding/NA active site I of hPIV-3HN protein play an important role in cell fusion promotion activity. R502is a key amino acid.4. H552is a key amino acid in the receptor binding/NA active site II of hPIV-3HN protein.5. The hexapeptide of hPIV-3HN is part of the receptor binding/NA active site in molecule and mutaions in the domain except S257play an important part in syncytium formation. R253is a key amino acid in the hexapeptide.6. The three conserved tripeptides play an important role in cell fusion promotion activity. T532is a key amino acid in the three conserved tripeptides.7. HN and F proteins arrive at the cell surface as a complex before, and after receptor binding. Efficient F-activation does not require the receptor binding activity as the wt level, and54.2%of wt HN level of HAD activity that T531-mutated HN protein exhibited could fully trigger the activation of F protein to initiate membrane fusion.8. Low levels of syncytium formation may correlate with the decreased initial promotion fusion capacity. The earliest membrane fusion not only depends on the cleavage of F protein, but also requires receptor-binding HN proteins that regulate the extent of initial fusion process by the interaction with the F protein.