Characteristic Analysis Of Two Forms Of L Protein Of Foot-and-Mouth Disease Virus And The Screening Of LncRNA Related To L Protein

Foot-and-mouth disease virus(FMDV)is the pathogen which causes foot-and-mouth disease(FMD)in cloven-hoofed animals such as pigs,cattle,and sheep.Leader(L)protein encoded by FMDV is an important virulence factor.There are two initiation AUGs(Lab AUG and Lb AUG)in L gene,leading to the production of two forms of L protein(Lab and Lb).Until now,it is unclear how Lb AUG affects FMDV replication,and the researches on Lab function are rare.In recent years,a large amount of evidence has shown that lncRNA plays a role in the interaction between virus and host cells.While it has not been determined whether lncRNA is correlated with L protein.In order to analyze the influence of Lb AUG on FMDV replication and obtain FMDV only encoding Lab,explore the characteristics of different forms of L protein,and screen out lncRNAs related to L protein,this thesis was carried out from the following three aspects:1.Different modifications of Lb AUG(UGG,AUC,CUG and AAA)were introduced into the O/HN/CHN/93 full-length infectious plasmid pOFS using reverse genetics technology,and four mutant viruses(K3m,K4m,K5m and K9m)were rescued.Analysis of the biological characteristics of the rescued viruses revealed that four mutant viruses showed similar plaque morphology and replication ability compared with parental virus in BHK-21 cells,while K4m and K9m had lower yields than the WT virus in PK-15 cells.Preliminary studies had proved that the delayed eIF4GI cleavage and reduced interferon(IFN)inhibition were associated with the restricted growth phenotype of K4m and K9m.Further studies demonstrated that the significantly reduced protein translation efficiency in the early stage of viral infection was responsible for the impaired replication of K4m and K9m.In summary,FMDV Lb AUG was not necessary for FMDV survival,but it affected the infectious capacity of FMDV.2.The precise deletion of the La or Lb gene was performed by reverse genetics technology,and mutant FMDV rQLa and rQLb were rescued.The examination of L protein synthesis showed that parental virus expressed Lab and Lb proteins while rQLb did not express Lab or Lb protein.rQLa only synthesized Lb protein,and K4m only synthesized Lab protein.These results indicated that FMDVs encoding different forms of L protein were obtained.In PK-15 cells,rQLa and K4m formed plaques,while rQLb could not.In addition,Lab and Lb proteins,which were synthesized by FMDV or overexpressed by plasmid,both had an ability to cleave eIF4GI and inhibit IFN expression,and were toxic to cells.These results proved that both forms of L protein were important cytotoxicity factors for FMDV.Although rQLa had a stronger ability for eIF4GI cleavage and inhibiting IFN expression than K4m,the replication,cytotoxicity and virulence in suckling mice of rQLa were decreased when comparing with K4m,indicating that La region played a crucial role during FMDV infection.Indirect immunofluorescence assay(IFA)and nuclear/cytosol fractionation assay were performed to detect the subcellular localization of L protein.Two forms of L protein were found to be distributed in the cytoplasm and nucleus,but Lab protein was more prone towards cell membrane while a large of Lb proteins could be distributed in the nucleus,suggesting that the amino acid residues encoded by La gene affected intracellular distribution of L protein.According to the above data,we suggested that the marked toxicity of Lab and Lb protein to host cells and the significant functions of La region mainly determined the occurrence of two species of L protein for FMDV.3.PK-15 cells were infected with parental virus O/HN/CHN/93 and Lb-deleted virus rQLb,and the samples were collected at 3,6 and 8 h post-infection.The lncRNA expression profile in harvested samples was determined by RNA-seq technology.A total of 20044 lncRNAs were screened out,of which 543 were differentially expressed lncRNAs related to L protein.Functional prediction of these differentially expressed lncRNAs revealed that they were involved in enzyme activities,metabolism,membrane structure,apoptosis and inflammation.In summary,a database of lncRNAs which were induced by the FMDV infection in PK-15 cells was established,and differentially expressed lncRNAs related to L protein were screened,which laid foundations for the further study of the pathogenic mechanism of FMDV and the functions of L protein.