Effects Of Leucine Zipper Motif On HPIV3 HN Protein Functions

Paramyxoviruses are a group of important viruses that are pathogenic to human beings and animals.They can be epidemic all over the globe.The family of paramyxoviridae is divided into two subfamilies:paramyxovirinae and pneumovirinae. Paramyxovirinae is divided into three genuses:paramyxovirus,morbillivirus and rubulavirus.Paramyxovirus includes human parainfluenza virus(hPIV),Newcastle disease virus(NDV) and Sendai virus(SV);morbillivirus consists of measles virus (MV);and rubulavirus contains mumps virus(MuV).Pneumovirinae only contains pneumovirus which includes respiratory syncytial virus(RSV).Paramyxoviruses mainly induce diseases of respiratory and reproduction systems in human beings,including upper respiratory tract infection,bronchitis,bronchiolitis, pneumonia,conjunctivitis,orehitis and ovaritis,etc.Sometimes,it can cause death. Some virus,such as MV,MuV and RSV,may cause familiar disease in children including morbilli,parotitis and respiratory infection,hPIV3 is the second important pathogen of bronchiolitis and pneumonia in infants.Besides these,it may cause croup and laryngitis.Up to date,no effective anti-hPIV3 measure was found.Besides human beings,paramyxovirus can also infect animals such as poultry,horses and swine.For NDV,an avian virus that is a serious agricultural problem in many regions of the world at present,it is an important pathogen of animal disease.NDV can cause avian and cattle diseases,usually disastrous and wide epidemic.NDV is of other clinical significances.For example,it can induce interferon and can be used to treat tumors.Some viruses are emerging ones,including Nipah virus,Hendra virus,Salem virus,avian pneumovirus and human metapneumovirus belonging to paramyxoviridae. They can cause severe human and animal diseases,infecting many organs or tissues, especially central nervous system.Clinically,fever and headache are common,and the fatality rate is very high.Infection of virus that is similar to Hendra virus bursted out in Singapore and Malaysia.This kind of virus originated from swine,which is familiar to horse Hendra virus.Cell fusion is a critical step in virus multiplication,spread and pathogenesis. Besides paramyxoviridae,many other human pathogenic viruses have envelope glycoprotein that can cause cell fusion,such as herpes simplex virus,human immunodeficiency virus(HIV),etc.Thus,studies on cell fusion of paramyxoviruses possess wide significances in viral theory and practice.The paramyxoviruses are a group of enveloped,single negative-stranded, non-segmented RNA viruses.There occurs envelope outside the viral capsid.The non-segmented genome,with the length of about 15 000 base pairs(bp),encodes 6 kinds of protein,including nucleocapsid protein(NP),phosphoprotein(P),Matrix Protein(M),fusion pritein(F),attachment protein and large molecule protein(L). Among these protein,NP,P and L protein are related with viral genome,which transcribe viral RNA and form active mRNA together.F,M and attachment protein are involved in viral envelope.F and attachment protein,the important protective antigen,compose the viral spike;M protein forms underprop inside the viral envelope. The activity of attachment protein varies in different paramyxovirus.For hPIV,NDV and MuV,their attachment protein possesses both neuraminidase and hemagglutinin acitivity,which were named hemagglutinin-neuraminidase protein(HN).The attachment protein of MV only has the hemagglutinin activity,named hemagglutinin protein(H).RSV attachment protein,named G protein,possesses neither neuraminidase nor hemagglutinin activity.HN protein,penetrating into viral envelope with its N-terminal domain,is homologous tetramer encoded by the gene containing 2 100 nucleotides.HN glycoprotein is divided into head,stalk,transmembrane domain (TM) and cytoplamic tail(CT),responsible for three kinds of function,receptor binding activity,NA activity and cell membrane fusion promotion activity.A flexible site had been observed in head domain that comprised 6β-sheets.It is a bifunctional site,responsible for NA and receptor binding activity,and can switch between them under different conditions.Cell fusion,a key step in paramyxovirus infection,is mediated by HN and F protein together.During viral infection,HN configuration is altered aider the attachment of HN protein to viral receptor on host cell surface,which induces the configuration change of F protein.Heptad repeat motif 1(HR1) and HR2 form steady complex which results in the adjacency of viral envelope and host cell membrane.At the same time,hydrophobic fusion peptides(FP) are released into cell membranes, and the cell fusion process is triggered.During this process,F protein is directly responsible for fusion of the viral and cellular membranes.But F protein can not induce cell fusion alone,which needs the cooperation between F protein and homologous HN protein.Furthermore,the requirement for the HN protein in fusion is virus specific,meaning that F protein can only induce the cell fusion when coexpressed with the homologous HN protein,but not with the heterologous HN preotein.For example,cell fusion will take place when NDV F and NDV HN or hPIV3 F and hPIV3 HN are coexpressed.Otherwise,there will be no cell fusion observed.The specific membrane fusion has been interpreted as evidence for a necessary interaction between the two kinds of protein,which,in some way,activates the fusion activity of the F protein.The interaction domain between F and HN protein is one of the focuses on envelope glycoproteins of paramyxoviridae in the world.In order to localize an active domain that interacts with homogenous F on HN protein of paramyxoviruses and to understand the molecular mechanism of cell fusion promotion,site-directed mutagenesis and gene recombination were used to get chimeric HN protein.Thus, F-specificity domain of hPIV3 HN protein was localized at a domain that extended from the middle of the membrane anchor to the 82th residue in the ectodomain. Further study showed that hPIV3 chimeric HN protein restored a glycosylation site present in NDV HN,but not in hPIV3 HN.After deletion of this glycosylation site, further mutagenesis and chimeric HN genes analysis showed glycosylation site near the top of hPIV3 HN stalk modulated fusion and shortened the F-specificity domain by 10 amino acids,from the middle of the membrane anchor to the 72th residue in the ectodomain.F-specificity domain in NDV HN protein was localized at the similar domain.In F-specificity domain of paramyxovirus HN protein,there are two conserved leucine zipper motifs at N-terminal and C-terminal,respectively.Many studies showed conserved leucine zipper motif in this domain had great effects on HN protein. The residues from L30 to L44 compose the C-terminal leucine zipper motif of NDV HN.Site-directed mutation in this domain will markedly reduce the cell fusion promotion activity while there is no effect on protein transport.N-terminal leucine zipper motif consists of residues from L96 to L110,which can markedly reduce the cell fusion promotion activity after mutation.Two leucine zipper motifs also occur in the same domain of hPIV3 HN protein.However,up to date,no functional analysis of conserved leucine zipper motif in hPIV3 HN protein F-specificity domain has been reported.In order to identify the effects of leucine zipper motif on hPIV3 HN protein, site-directed mutagenesis was used to mutate the conserved amino acids in the leucine zipper motifs on the basis of previous experiments results.Mutant HN genes were expressed with homogenous F in eukaryocytes.Their cell fusion promoton and receptor binding ability were analyzed with qualitative and quantitative methods, respectively.1.Effects of leueine zipper motif on cell fusion promotion abilityThe motif in TM domain lies from L36 to I50,in which residues L36,L43 and I50 are highly conserved,aligned with other paramyxovirus.Recombinant PCR was used to generate mutant HN genes with corresponding primers for each mutant.Two pairs of primers were designed for one mutation.After PCR,two products with a short homologous sequence that came from the primers will form a complete plasmid automatically in TG1.In order not to mutate other amino acid except the aimed amino acid,no enzyme restricted site was introduced into the primers.PCR product was identified by sequencing after agarose electrophoresis.Thus,3 mutants were obtained, named L36A,L43A and I50A,respectively.To further determine the roles of leucine zipper motif,combined mutant,L36A-L43A,was constructed on the basis of individual mutations.To determine their cell fusion promotion activity,both qualitative and quantitative method,Giemsa staining and reporter gene method,were performed after the mutants transfected BHK21 cells which were infected with vaccine virus vTF7-3 in advance.After corrected by cell surface expression efficiency, mutant HN possessed 66.66%,60.60%and 57.60%of wt HN activity,respectively.In combined mutant L36A-L43A,cell fusion was rarely found.Residues L114 to I128 consist of the leucine zipper motif in the stalk domain close to viral envelope.Among those,L114,L121 and I128 are highly conserved in paramyxovirus.Mutant HN genes were obtained by recombinant PCR with corresponding primers for each mutant.No enzyme restricted site was introduced in the primers in order to mutate exactly the aimed amino acid.The mutant was identified by sequencing after agarose electrophoresis.Thus,3 mutants were obtained, named L114A,L121A and I128A,respectively.To better determine the roles of leucine zipper motif,combined mutant,L121A-I128A,was constructed on the basis of individual mutations.Their cell fusion promotion activity was determined by Giemsa staining and reporter gene method in the vaccinia-T7 RNA polymerase expression system,too.After corrected by cell surface expression efficiency,mutant HN possessed 53.00%,59.10%and 90.90%of wt HN activity,respectively.Cell fusion was rare in combined mutant L121A-I128A.P111,I112,and I125 are also conserved in leucine zipper motif in F-specificity domain of HN protein.3 mutants were obtained with the same methods,named P111A,I112A,and I125A,respectively.Cell fusion promotion ability was assayed with Giemsa staining and reporter gene method.After corrected by cell surface expression efficiency,the mutants had 43.90%,39.40%,and 34.80%of wt HN cell fusion promotion ability,respectively.Every mutant was examined for 3 times under the same condition.The data were analyzed with rank sum test.The H value of rank sum test was 28.78,P＜0.05, suggesting that there was statistical significant difference between mutants.I125A possessed the lowest cell fusion promotion activity,with 34.80%of fusion promotion activity as compared with wt HN.However,I128A had the highest fusion promotion activity,90.90%of wt HNThe FACS was performed to determine expression efficiency of mutant hPIV3 HN protein.The results of FACS indicated that all mutants were expressed on the cell surface successfully although the expression efficiency of all the mutants was lower than that of their relevant wt HN.2.Effects of leucine zipper motif on receptor binding activityThe receptor binding activity of mutant genes was determined by the ability to absorb guinea pig erythrocytes in the vaccinia-T7 RNA polymerase expression system. The BHK21 cells were transfected with the mutants L36A,L43A,I50A,L36A-L43A, L114A,L121A,I128A,L121A-I128A,P111A,I112A and I125A alone.The results showed mutation in leucine zipper motif possessed slight effect on receptor binding ability.The data was corrected by cell surface expression efficiency.In leucine zipper motif in TM domain,mutant L36A possessed 100%receptor binding activity as compared to wt HN;L43A and I50A had 98.99%and 91.92%,respectively. Combined mutant L36A-L43A is 95.96%of wt HN receptor binding activity.In leucine zipper motif in stalk domain,mutant L114A,L121A and I128A possessed 95.96%,92.93%and 100%of wt HN activity,respectively.Combined mutant L121A-I128A had 93.94%activity,compared to wt HN.In addition,mutant P111A, I112A and I125A had 97.98%,95.96%and 85.86%of the activity of wt HN, respectively. Every mutant was examined for 3 times under the same condition.The data were analyzed with rank sum test,and the H value of rank sum test was 17.99,P＞0.05, suggesting that there was no statistical significant difference between groups.All these results showed that mutation of conserved amino acids in F-specificity domain of hPIV3 had some negligible effects on receptor binding ability of hPIV3 HN protein.3.Effects of F protein on receptor binding activityBHK21 cells,infected with vaccine virus vTF7-3 in advance,were transfected with the 11 mutants described previously,coexpreesed with wt hPIV3 F gene. Absorbing guinea pig erythrocytes in the vaccinia-T7 RNA polymerase expression system was applied to determine the receptor binding activity of mutant genes,too. Mutant I125A possessed the lowest receptor binding activity,84.48%as compared to wt HN gene.And mutant L36A had the highest activity,99.14%ofwt HN.Every mutant was examined for 3 times under the same condition.Rank sum test was used to determine the difference between the group transfected with mutant HN alone and the group transfected with mutant HN and wt F gene together.And the H value of rank sum test was 108.00,P＜0.05,which showed there was difference between the two groups,and the group transfected with mutant HN gene alone possessed higher receptor binding activity.The results suggested as followings:Individual mutation L36A,L43A,I50A,L114A,L121A,I128A,P111A,I112A and I125A were obtained successfully.On the basis of individual mutation,combined mutants L36A-L43A and L121A-I128A were constructed.The leucine zipper motif in TM domain and stalk domain close to viral envelope possessed great effects on cell fusion promotion activity of hPIV3 HN protein.The activity will reduce to different level after the conserved residues were mutated.In those individual mutants,mutant I125A had the lowest cell fusion promotion activity, 34.80%as compared to wt HN gene.However,mutant I128A possessed 90.90%of wt HN gene activity,the highest in individual mutation.The activity of other individual mutation lied from 39.40%to 66.66%.Furthermore,cell fusion was rarely found in the two combined mutants.All these suggested further that the domain specific to HN and F protein interaction was localized in the TM domain and stalk domain close to viral envelope,but not in head domain.And this specific domain ended at the residue I125.Complete leucine zipper motif was prerequisite to fusion promotion of HN protein.I125 is the key amino acid for cell fusion promotion activity of hPIV3 HN protein,which played impotant roles in cell fusion promotion ability.All the mutants possessed negligible effects on receptor binding activity of hPIV3 HN protein.Among those,mutant I125A had 85.86%of wt HN gene activity. And the receptor binding activity of other mutation lied from 91.92%to 100%, similar with wt HN.Compared to HN gene expressed alone,the receptor binding activity reduced when F and HN gene coexpressed together.