Structural And Functional Analysis Of The Roles Of Influenza C Virus Membrane Proteins In Assembly And Budding

Influenza C virus is a common human pathogen that causes mild respiratory symptoms of child and young people.The virus has a worldwide distribution and the majority of humans develop antibodies against the virus early in life.Human are the main reservoir of influenza C virus,but the virus occasionally infects pigs.Influenza C virus is a single-stranded,negative-strand segmented RNA virus,belongs to the Orthomyxoviridae family.And it consists of 7 RNA fragments,which has same genome structure to influenza D virus and similar genome structure to influenza A and B viruses.Influenza A and B viruses consist of8 RNA fragments,but influenza C virus contain only one spike protein HEF,so it means HEF might combines the function of HA and NA in influenza A and B viruses.Through the electron microscope we found that the surface of influenza C virus particles is covered with a regular network of HEF trimers that consist of mainly hexagons.However,the function of reticular structure has not been investigated.Since proteins HA,NA,M1 and M2 play an important role in the budding process of influenza A and B viruses,we speculated that proteins HEF,CM1 and CM2 may play an important role in the budding process of influenza C virus too.Nevertheless,the budding mechanism of influenza C virus has not been reported yet.In order to investigate the role of these three membrane proteins HEF,M1 and CM2 of influenza C virus in the process of virus assembly and budding.First,the genes of HEF,CM1 and CM2 proteins were cloned from the reverse genetic system plasmids of influenza C virus(C/JJ/50)and inserted into the protein expression plasmid p CAGGS.Then the reconstructed plasmids were transfected into CHO cells to perform a co-localization analysis.It was shown that all these three membrane proteins can be transported to the plasma membrane when expressed independently,which is the budding site of the virus.The HEF and CM1 proteins are mainly located on the cell plasma membrane,and their distribution is irregular,while a stronger fluorescence signal was detected for CM2 inside cells.Super resolution microscopy revealed 250 nm-sized HEF accumulations,which are insensitive to cholesterol extraction and cytochalasin treatment.It is proved that the HEF accumulation is caused by the lateral interaction between HEF trimers.HA of influenza A virus binds to lipid rafts on the plasma membrane in the budding process of influenza A virus,but the HEF of influenza C virus does not bind to lipid rafts.So,the budding mechanism of influenza C virus may be more complicated.To further explored the influence of membrane protein on virus morphology,we found that expression of CM1 and CM2 caused release of membrane-enveloped particles,long filaments(CM2)and ellipsoid vesicles(CM1).HEF expression mediates release of spherical vesicles exhibiting the characteristic hexagonal HEF clusters by Cryo-electron microscope.So,it shown that clustering is an intrinsic property of HEF.To further explored whether CM1 interact with the cytoplasmic tail of HEF to stabilize the aggregation of HEF.Therefore,the cytoplasmic tail of the HEF was deleted,and the recombinant virus was rescued by reverse genetic system to verify the key sites which may cause the HEF clustering to form a reticular structure.To compare the growth curves of wild type and mutant viruses on MDCKI cells,it was shown that deletion of the short cytoplasmic tail of HEF reduced virus titers and the number of HEF clusters in virions suggesting that an interaction with CM1 stabilizes the HEF clusters.To determine the role of HEF clustering on influenza C virus budding,we substituted charged amino acids(R322,E350 and D353 to Ala)on the surface which might mediate lateral interactions in the closed HEF conformation.The recombinant viruses were rescued by reverse genetics system.Exchange of R322+E350 prevented virus rescue,R322+D353 reduced virus titers and the number of HEF clusters in virions.According to the report recently,the head of HEF1 can be rotated by 20° to form a more open conformation.This rotation will cause other HEF1-HEF1 contacts between the trimers.These sites also be potential factors causing the hexagonal HEF clusters.Two regions 101-106(YLYQGC)and 208-214(TQFGTYE)that mediate contacts between trimers.Mutation of two regions(Y101S+L102S+Y103S,3S;F210S+Y213S,2S;Y101S+L102S+Y103S+F210S+Y213S,5S)prevented rescue of infectious virus particles.Afterwards,293 T cells were transfected with these mutant plasmids and found that the expression level of R322,R322+E350,2S,3S and 5S was reduced significantly.Next,we analyzed the intracellular transportation of these mutants by flow cytometry and fluorescence co-localization analysis.We found that most proteins of 2S,3S,5S and R322+E350 still stay in the endoplasmic reticulum(ER)and cannot be transported to the cytoplasm membrane.So,HEF with these mutations supposed to prevent lateral interactions revealed intracellular trafficking defects.Overall,we propose that lateral interactions between the ectodomains of HEF trimers are one driving force for virus budding,but CM2 and especially CM1 also play important roles.This study is the first time to explore the role of the membrane proteins HEF,CM1 and CM2 of influenza C virus in the budding process,which is significant for further studying and understanding of the budding mechanism of influenza C virus.