Localization Of The Key Amino Acids And Research On The Related Host Factors Of Paramyxovirus Envelope Glycoproteins

Part I:Localization of the key amino acids of NDV envelope glycoproteinsBackgroundThe paramyxoviruses include many pathogenic viruses covering both medical and veterinary areas,such as human parainfluenza viruses(hPIVs),Nipah virus(NiV),Sendai virus(SeV),measles virus(MeV),mumps virus(MuV)and Newcastle disease virus(NDV).Although there are many kinds of paramyxoviruses,the structure of their envelope glycoproteins is relatively conservative.The research results of either virus can be analogized to the others.In this study,we selected NDV and MuV as the research objects:the weak pathogenicity of NDV to human makes it a common model and typical representative to study the structure and function of paramyxovirus envelope glycoproteins;while MuV infecting human was used to study the host factors interacting with paramyxovirus envelope glycoproteins,which can more clearly understand the mechanism of human disease and is conducive to the treatment and prevention of paramyxovirus diseases.Both NDV and MuV belong to the genus Avbulavirus and have high structural similarities.They also cover animal and human pathogenic viruses,which can realize a more comprehensive study of paramyxoviruses.NDV,avian paramyxovirus type 1(APMV-1),is an enveloped,non-segmented,single-stranded,negative-sense RNA virus.The genome encodes the nucleocapsid(N)protein,phosphoprotein(P),matrix(M)protein,fusion(F)protein,haemagglutininneuraminidase(HN)protein and polymerase/large protein(L).The lipid membrane of NDV can fuse with the host cell membrane to form syncytium,so that the virus can invade cells and replicate.Membrane fusion is receptor dependent,pH independent,and requires the cooperation of two glycoproteins,HN and F,embedded in the lipid membrane.There are several different views on the mechanism of HN and F synergy:First,protein-protein interaction exists before receptor binding;Second,protein-protein interactions occur only after receptor binding;Third,some researchers have pointed out that receptor binding proteins are necessary not only for the initial triggering of the F protein,but also for the entire fusion process.Regardless of the mechanism,it is undeniable that HN proteins play an important role in activating F proteins to fusion membrane.NDV HN protein is a multifunctional type II glycoprotein,which exists in the form of autotetramer and has the following three activities:recognition and binding of sialic acid containing receptors;neuraminidase(sialidase)for virus budding;promoting the membrane fusion activity of F protein.HN protein can be structurally divided into cytoplasmic tail,transmembrane,stalk and globular head.The core structure of the globular head domain is β-propeller,which is mainly involved in receptor recognition activity and neuraminidase activity.There are two sialic acid binding sites on the HN protein dimer interface.Studies have shown that the second binding site lacks neuraminidase activity,although the receptor involvement is higher.However,some studies of mutations involving the second binding site have given different results.For example,the F553A mutant resulted in a 70%reduction in receptor-binding capacity and neuraminidase activity compared to the wild-type(wt)HN.Therefore,the function of the second receptor binding site of HN protein needs to be further determined.Although many previous studies have addressed the membrane fusion process,the research on HRB linker functions is relatively rare.Conformational diversity among the conformations of the hPIV3 F protein and NDV F protein was found through a comparison of the crystal structure alignment of hPIV3 solF0 with that of NDV,suggesting that the linker domains might be involved in the rotation and relative orientation of F protein during the membrane fusion process.Subsequently,in research concerning the parainfluenza virus 5(PIV5)F protein structure,the HRB linker exhibited weak electron density in the pre-fusion conformation of the PIV5 F protein,suggesting that the HRB linker is highly flexible.Some researchers found that leucine(L)447 and isoleucine(Ⅰ)449 at the N-terminus of the Simian virus 5 F protein near the HRB linker were both necessary to form stable 6HB.Hence,the HRB linker may be of importance,and its role needs to be further investigated.ObjectiveTo localize the key amino acids in the second receptor binding site of NDV HN protein and HRB linker of F protein,many mutants were constructed by site-mutation and gene recombination and subjected to a series of experiments to detect the influence of mutation on receptor binding activity,neuraminidase activity,fusion(-promoting)activity along with other biological functions,in order to illuminate the roles of these two regions.Methods1.Construction of mutants of NDV HN protein:The sequences of second receptor binding site(loop 156-174,loop 191-203,loop 515-527 and loop 547-556)of NDVHN protein were aligned with that of hPIV 1-4,PIV5 and SeV.Ten relatively conserved amino acids were chosen and mutated into alanine(A).In addition,the 4 loops were replaced by the corresponding sequence of hPIV3,respectively,to construct 4 chimeras or deleted to construct 4 deletion mutants.2.Construction of mutants of NDV F protein:The 6 amino acids mutants of NDV F protein HRB linker were constructed in the former research in our lab.They are L436A,L436T,E439A,I450A,S453A and S453N.3.Expression of mutant proteins:Mutant genes were connected into pBSK+vector,and the mutant proteins were expressed in BHK-21 cells with the help of a transient expression system of vaccinia virus-T7 RNA polymerase.4.Expression efficiency detection of mutant proteins:The expression of each mutant was qualitatively and quantitatively detected by indirect immunofluorescence assay(IIFA)and fluorescence-activated cell sorter(FACS),respectively.5.Functional detections of HN mutant proteins:Giemsa staining method,reporter gene method and hemi-fusion assay were utilized to determine the fusion-promoting ability of HN mutant proteins in different fusion stages.The hemadsorption(HaD)test can be used to detect the changes in the receptor recognition activity of mutant HN proteins qualitatively and quantitatively.Changes in neuraminidase activity(NA)of each HN mutant protein were detected using the NA kit.6.Western Blot(WB)was used to detect the hydrolysis of mutant F proteins.7.Statistical analysis:The data of mutants were compared with that of wild type(wt)protein using Student’s t test,and P<0.05 was considered statistically significant.Results1.Function of NDV HN second receptor binding site(1)Construction of mutantsTen mutants were constructed by site-directed mutation PCR:T167A,G171A,C172A,R174A,C196A,D198A,S202A,R516A,Y526A and E547A.The four loops constituting the second receptor binding site were replaced by the corresponding amino acids of hPIV3 HN protein to obtain four chimeras:Ch1,Ch2,Ch3 and Ch4;four loops were deleted to obtain four deletion mutants:Del,De2,De3 and De4.(2)Expression efficiency of mutantsCompared with wt HN protein,there was no significant difference in the cell surface expression efficiency of site-directed mutants.Among chimeras and deletion mutants,only Ch3 and Ch4 were successfully expressed in cells,and there was a statistical difference between the expression efficiency of mutant Ch4 and wt HN protein.(3)Cell fusion promoting activity of mutantsMutant G171A showed a similar ability to promote cell fusion to wt HN protein.T167A,S202A,R516A,Ch3 and Ch4 can promote cell fusion,but their ability,to some extent,decreased.In addition,in the cells transfected with mutants C172A,R174A,C196A,D198A,Y526A and E547A,syncytia could not be observed under the microscope,meaning they almost lost the activity of promoting cell fusion.(4)Receptor binding ability of mutantsCompared with wt HN protein,mutants T167A,G171A,S202A,R516A,Ch3 and Ch4 adsorbed fewerchicken red blood cells.These six mutants were treated with zanamivir,and the receptor binding ability of mutant T167A increased to 107.34%of that of wt HN protein.Mutants C172A,R174A,C196A,D198A,Y526A and E547A did not show obvious receptor binding ability.(5)Neuraminidase activity of mutantsCompared with wt HN protein,neuraminidase activity of each mutant,to some extent,decreased(42.05%to 81.56%),except for mutant T167A,which has similar activity to wt HN protein.After zanamivir was added,the neuraminidase activity of mutants fluctuated to a certain extent,and there was no significant difference in the neuraminidase activity between the mutants and wt HN protein.2.Function of NDVF HRB linker(1)Fusion activity of mutantsThe content mixing degree of cells expressing mutants L436A,L436T and I450A decreased to 5.19%,69.87%and 5.54%of wt F protein,respectively.When the residue S453 was mutated to A or N,the content mixing degree of mutants showed different functional changes:decreased to 87.91%(no significant difference compared with the cells expressing wt F protein)or increased to 139.59%.For mutant E439A,the content mixing degree was similar to that of wt F protein,which is consistent with the data of syncytial formation and semi-fusion experiments in previous studies.(2)Expression efficiency of mutantsThe expression efficiency of mutants L436A,L436T and 1450A decreased to 14.56%,71.06%and 11.45%of that of wt F protein,respectively.And the expression efficiency of mutants E439A,S450A and S453N increased to 123.50%,106.61%and 104.74%of that of wt F protein,respectively.The total protein levels of mutants L436T and I450A were 76.82%and 35.06%of that of wt F protein,while the total protein levels of mutants E439A,S453A and S453N were 127.42%,114.31%and 120.89%of that of wt F protein,respectively.(3)Hydrolysis efficiency of mutantsThe hydrolysis efficiency of mutant L436T is the highest,117.18%of that of F protein.The protein hydrolysis efficiency of mutants E439A,I450A,S453A and S453N decreased,which were 89,68%,66.21%,97.91%and 92.84%of that of wt F protein,respectively.No protein bands were detected in mutant L436A.Conclusions1.The key amino acids in the second receptor binding site of HN protein(T167,G171,C172,R174,C196,D198,S202,R516,Y526 and E547)can affect the protein’s cell fusion promoting activity by affecting the receptor binding ability and/or neuraminidase activity of HN protein.2.The second receptor binding site of HN protein is involved in the early stage of membrane fusion,and the effect caused by mutation may also be related to the inability of the head of HN protein,where the second receptor binding site is,to complete the signal transduction of conformational change.3.The key amino acids in the HRB linker of NDV F protein(L436,E439,1450 and S453)can affect the protein expression and/or hydrolysis,participating in maintaining the stability of 6HB structure and promote the transformation of F protein from pre-fusion conformation to post-fusion conformation.4.The mutation of F protein HRB linker can affect the polarity,hydrophilicity and rigidity of amino acids,change the flexibility of the domain,and then affect the fusion activity of the protein.Part II: Research on the related host factors of MuV envelope glycoproteinsBackgroundMumps virus(MuV)is the causative agent of mumps,which is a common childhood illness characterized by painful swelling of the parotid glands,and is often accompanied by severe complications such as orchitis,aseptic meningitis,pancreatitis and deafness.Despite the long-term usage of vaccine for MuV3 there are still many outbreaks of mumps that cannot be ignored around the world.MuV belongs to the genus Avbulavirus,family Paramyxoviridae.The genomic RNA of mononegaviruses is encapsidated by the N protein,forming the ribonucleocapsid.The viral RNA-dependent RNA polymerase(RdRp),which is composed of L protein and its cofactor P protein,binds to ribonucleocapsid and forms the viral ribonucleoprotein(RNP)complex.The RNP complex,but not the naked genome,functions as an active template for both transcription and genome replication,which is linked to the virion membrane by M protein.The outer surface of the virus is covered with viral glycoproteins consisting of the HN protein,which binds sialic acid to allow virus to attach to cells,and F protein,which induces viral and cellular membranes to fuse during virus entry.In the late steps of viral replication,M protein recruits viral particle components including the RNP complex and glycoproteins in the vicinity of the plasma membrane,and coordinates assembly and budding of MuV virions.During the whole life cycle of MuV,many host factors participate in the different processes.Some host proteins have been determined previously,for instance,R2 TP complex that can regulate RNA synthesis,heat shock protein(Hsp)72 that can modulate degradation of the P protein and Hsp90 that ensures efficient virus replication.Enveloped virus particles are formed at the specific region of the plasma membrane where viral and host proteins have assembled.However,there have been few reports on the host factors involved in the late steps,and the molecular mechanisms underlyingthese steps of the MuV life cycles remain for the most part unknown.Interaction studies between assembly-associated viral proteins and host factors can help to illuminate the molecular mechanism of the assembly and trafficking of the virion.Most host factors in live cells have specific subceilular localization patterns to ensure their function.Proximity labeling is a useful tool for studying protein localization and interactions.In proximity labeling,a promiscuous enzyme such as APEX,BioID,or TurboID is genetically targeted to protein complex of interest.Then,biotin-derived small molecule substrates are added to initiate biotinylation of endogenous proteins within a few nanometers of the promiscuous enzyme,via an activated biotin adenylate intermediate in the case of TurboID.After cell lysis,biotinylated proteins are harvested using streptavidin beads and identified by mass spectrometry.ObjectiveHere,based on the proximity labeling,we searched for the host factors located in the vicinity of the cytoplasmic tails of the F and HN proteins.Some host factors were identified and subjected to a series of experiments to try to understand their role in the late steps of MuV propagation and further clarify the molecular mechanism of paramyxovirus replication.Methods1.Construction of plasmids: The plasmids of vims gene were amplified from293 T cells infected with MuV Odate.The cDNA of M,N,F and HN protein were obtained by reverse transcription(RT)-PCR,cloned into pCAGGS vector,and added FLAG or HA label.TurboID was cleaved at 73/74 amino acids and connected to F and HN plasmids,respectively.The plasmid of host factor was amplified from A549 cells and connected to pCAGGS-HA vector.2.Determination of virus titer: Virus titration was carried out by plaque assay.3.Detection of protein expression and interaction: The expression of viral protein and host factor and interaction between them were detected by WB,UFA and immunoprecipitation(IP).4.Preliminary determination of host factors: The host factors near the target protein were biotinylated with the help of TurboID by proximity labeling.After the protein was purified,the relevant host factors were identified by mass spectrometry(MS)and interactome analysis.5.Gene silencing of host factors: Small interfering RNA(siKNA)was used to silence the expression of host factors.6.Multi-step growth study of viras: After gene silencing,the recombinant virus expressing fluorescence was inoculated.At 0,24,48,72 and 96 hour post inoculation(hpi),the fluorescence was detected,the supernatant was collected for virus titration,the cells were collected to extract RNA for real-time fluorescence quantitative PCR,and the protein was collected to detect the protein expression.7.Cell fusion assay: On the one hand,cell fusion can be reflected by detecting the expression of pAcGFPl-Cl plasmid in A549 cells.On the other hand,293 T cells expressing dual-split-proteins(DSPs)were used.The DSPs are green fluorescence protein(GFP)and Renilla luciferase,which can quantitatively reflect cell fusion.8.The expression of F protein detected by WB: The N glycans were digested by endoglycosidase Endo H and PNGase F;HeLa/MGATl knockout(KO)cells,which lack hybrid/complex type glycans,were used to detect the glycosylation of F protein;EZ-Link Sulfo-NHS-Biotin was used to biotinylate the cell surface proteins and detect the cell surface expression of F protein.9.Statistical analysis: The data were compared using Students’ test,and P<0.05 was considered statistically significant.Results1 Verification of proximity labeling system Through IIFA and WB,it was determined that F-FLAG-Tb(C)and Tb(N)-HA-HN can be biotinylated,which means the split TurboID can be recombined and activated.Therefore,the proximity labeling system was successfully constructed,which can be used to search for the host factors located in the vicinity of the cytoplasmic tails of the F and HN proteins.2.Host factors near the cytoplasmic tails of F and HN proteinsAccording to the results of LC-MS/MS and interactome analysis,24 host factors near the cytoplasmic tails of MuV F and HN proteins were determined.3.Host factors related to virus proliferationAfter gene silencing of 24 host factors,5 host factors related to MuV propagation,BET1,EMD,LRRC59,USE1 and VAPA,were determined by fluorescence detection and virus titration.4.Construction of host factor plasmidsThe sequences of host factors were amplified from A549 cells and connected to pCAGGS-HA vector.Plasmids pCAGGS-HA-BETI,pCAGGS-HA-EMD5 pCAGGSHA-LRRC59,pCAGGS-HA-USEl and pCAGGS-VAPA-HA were successfully constructed.5.Multi-step growth study of virusAt 96 hpi,the fluorescence in BET1 and USE1 gene silencing cells decreased significantly;after 48 hpi,viral RNA(mRNA and genomic RNA),protein expression and MuV titer decreased significantly in USE1 gene silencing cells;in BET1 gene silencing cells,protein expression and virus titer decreased.6.Host factors interacting with glycoproteinsThrough IIFA,IP and WB,it was determined that BET1 and USE1 can interact with F protein in endoplasmic reticulum(ER).BET1 and USE1 gene silencing can weaken the cell fusion effect of F and HN proteins,which may be due to the abnormal expression of F protein caused by gene silencing.7.The influence of USE1 on the proper glycosylation of MuV F proteinThe glycosylation of F protein was detected by endoglycosidase and HeLa/MGATl KO cells.The results showed that F protein could not complete the proper attachment of complex type glycans after USE1 knockdown,but the transport of F protein to the cell surface was not interfered.Conclusions1.Using proximity labeling system with split-TurboID can determine the host factors close to the cytoplasmic tails of F and HN protein.2.SNARE protein,BET1 and USE1,interacted with F protein in ER.3.USE1 influenced viral transcription and genome complication,and BET1 or USE1 knockdown depressed MuV propagation.4.BET1 or USE1 knockdown affected the ER-to-GoIgi anterograde and retrograde transport of F protein,causing the impaired fusion activity of F protein co-expressing with HN protein.5.USE1 was involved in MuV F protein maturation through influencing the complex type glycosylation and intra-Golgi transport of protein.