Functional Analyses Of Conserved Amino Acids In The 11-Residue Repeat Of Human Parainfluenza Virus Type 3 Hemagglutinin-Neuraminidase Protein

Background:Human parainfluenza viruses(hPIVs)are a series of single-stranded RNA viruses that belong to Paramyxoviridae,which are also a common class of community-acquired respiratory tract pathogens.According to heredity and antigenicity,hPIVs are divided into 4 serotypes and 2 serosubtypes(hPIV-1 2,3,4a,4b).Human parainfluenza virus type 3(hPIV3)infection rate is high and duration is long,which is the main cause of lower respiratory tract infection leading to bronchiolitis and pneumonias in young children under the age of six months,and it is the second most common pathogen only next to respiratory syncytial virus(RSV).So far,there is no effective prevention and treatment to hPIV3 infection.All kinds of vaccines are in the critical stage of development,which has made important progress in recent years.However,the safe and effective vaccine against human has not come out yet,the main obstacle is that the mechanism of fusion between virus and target cell in the pathogenesis is not clear.The fusion of viral envelope and target cell membrane is the key step in the invasion and infection of paramyxoviruses.The process is mediated by the fusion protein,with the assistance of homologous attachment protein.According to different functions,the attachment protein is divided into three categories:HN,G and H,among which HN is the most common and functional and has been widely concerned by researchers.HN protein exists on viral envelope and host cell membrane in a form of tetramer,the main function of which as follows:?)Recognize sialic acid receptors and attach to the host cell surfaces.?)Exert neuraminidase(NA)activity to promote the spread of progeny virus.?)Specifically trigger the homologous F protein to perform a series of conformational changes and start the fusion process finally.HN protein is composed of four regions:head,stalk,transmembrane and cytoplasmic tail.The globular head is mainly responsible for receptor recognition activity and NA activity,which has been widely studied,while the structure and function of stalk region is not clear,which is thought to play an important role in the protein oligomerization and specific contact with homologous F protein.Recent studies found that the stalks in most of the paramyxovirus attachment proteins form a four-helix bundle(4HB)structure,which has the hydrophobic core composed of the 11-residue repeat sequence pattern(i,i+3,i+4,i+4).This model updated the previously defined conception of heptad repeat region(i,i+3,i+4).A series of studies indicated that the hydrophobic core of 11-residue repeats in HN proteins of different paramyxovirus is partly conserved through sequences alignment and mutants in conserved sites may affect the normal structure and function of HN protein,which provides a new perspective to identification of the key amino acids in stalk region.So far,there has not been a study on the conserved amino acids mutants in the 11-residue repeat of hPIV3 HN protein.In this study,we took hPIV3 HN as the main objective,identified 5 conserved amino acids in the 11-residue repeat through the comparison with highly similar sequences in Newcastle disease virus(NDV)and parainfluenza virus type 5(PIV5)HN proteins,and mutated them into alanine respectively.We determined the critical sites in the 11-residue repeat of hPIV3 HN protein through detection of different activities of each mutant,speculated the main reason of changes and further deepened the understanding of HN stalk region in the fusion mechanism of paramyxovirus.Objectives:1.To determine the critical amino acids in the 11-residue repeat of hPIV3 HN protein.2.To analyze the reasons for the changes of the structure and function of HN protein.3.To further make sure the important roles that stalk domain plays in mechanism of cell fusion.Methods:1.Construction of mutants:sequences alignment in the 11-residue repeats between hPIV3,NDV and PIV5 HN proteins were performed and 5 conserved amino acids were located,which are 1102,Pill,L114,S119 and 1125 in turn.A site-directed mutagenesis method and a homology recombination method were combined to mutate five conservative amino acids into alanine respectively.2.Expression of mutants:cationic transfection reagent was used to transfect the plasmids into baby hamster kidney cells(BHK-21),which are expressed by vaccina virus-T7 transient expression system.3.Analysis of cell fusion promotion activity:mutated pBSK-HN and wildtype;pBSK-F were transfected into BHK-21 together.Giemsa staining was used to observe the formed syncytia and record the fusion of each mutant protein,while reporter gene method was used in quantitative analysis.4.Analysis of receptor recognition activity:mutated pBSK-HN plasmids were transfected into BHK-21,and hemabsorption assay was used to detect the receptor recognition activity of each mutant.Firstly,adsorption of human red blood cells(RBC)was observed and recorded under optical microscope,then the adsorbed erythrocytes were lysed and the receptor recognition activity was detected by colorimetric quantification.5.Analysis of neuraminidase activity:mutated pBSK-HN and wildtype pBSK-F were transfected into BHK-21 together.High speed centrifugation was performed after cell digestion and lysis.Neuraminidase assay kit was used to detect the content of neuraminidase in the supernatant through enzymatic reaction.6.Analysis of semi-fusion activity:mutated pBSK-HN and wildtype pBSK-F were transfected into BHK-21 together and dye transfer was used to detect the semi-fusion activity of each mutant protein.With the suspension of R18 labeled fresh RBC,the semi-fusion state can be observed through the phenomenon that fluorescent probe transferred form RBC surface to target cell membrane.The problem that whether mutant protein can affect the initial stage of membrane fusion could be further explored.Results:1.The results of DNA sequencing showed that the amino acids designated were mutated into alanine respectively and mutant plasmids I102A,P111A L114A,S119A and 1125A were successfully constructed.2.Cell fusion promotion activity of the mutants(I102A,P111A,L114A,S119A and 1125A)decreased to 6%,16%,14%,87%and 4%in turn.There was statistic difference among wildtype and four of the mutants(P<0.01)except S119A for cell fusion promotion activity.3.Receptor recognition activity of the mutants(I102A,P111A,L114A,S119A and I125A)also reduced to 32.2%,77.4%,74.2%,83.9%and 38.7%respectively,among which 1102A and 1125A were significantly lower than that of wildtype(P<0.01),4.There was no statistic differences of neuraminidase activity among wild type and each mutant HN protein(I102A,P111A,L114A,S119A and I125A)(P>0.05),which expressed as percentage were 66.5%,73.1%,69.1%,76.1%and 82.8%individually.5.Semi-fusion activity of all the mutants also decreased to different degrees.S119A is the only mutant still maintain a high level of this activity,while the semi-fusion activity of 1102,P111A,L114A and I125A have sharp decline or basic loss.Conclusions:1.The 11-residue repeat in stalk domain plays a very important role in the cell fusion promotion and receptor recognition activity of hPIV3 HN protein.2.1102,P111,L114 and 1125 were identified as the key amino acids in the 11-residue repeat of human hPIV3 HN protein.3.Crucial residues were inferred to alter the structure and function of HN protein by affecting receptor recognition activity in head domain or the interaction with fusion protein.