Study On The Construction Of Replication-Controllable Recombinant Foot-and-Mouth Disease Virus Via Genetic Code Expansion Technology

The genetic code expansion is an umbrella term for the strategies that enable the ribosomal incorporation of unnatural amino acids(UAAs)into peptides or proteins of interest,by using orthogonal aminoacyl t RNA synthetase/t RNA pairs(aa RS/t RNA)to identify nonsense codon(stop codons or quadruplet codons)on m RNA.This orthogonal aa RS/t RNA pair exclusively recognizes and readthroughs nonsense codons in the process of protein translation and allows for site-selective incorporation of the specific UAAs into the amino acid sequence,constituting an orthogonal translation system(OTS).At present,more than 200 different UAAs have been genetically encoded and site-specific incorporated into proteins in various cells and organisms,which are widely used in many biological research fields such as protein labeling and modification.Moreover,the UAAs are incorporated into specific amino acid sites of viral proteins to achieve the surface modification of virus particles and the targeted control of virus replication,which convert the wild-type viruses into live but avirulent vaccines and represent a revolution in vaccinology.In this study,Foot-and-mouth disease virus(FMDV)was used as a model system to explore the feasibility of generating recombinant FMDV harboring prematuration termination codon(PTC).The genetic code of FMDV genome was expanded via transgenic cell line containing orthogonal translation machinery by using the genetic code expansion and the reverse genetics technology.The virus packaging efficiency and genetic stability of the recombinant PTC-FMDV were further evaluated.The results are as followings:1.Generation and genetic stability analysis of recombinant PTC-FMDVIn this study,we first expanded the genetic code of the FMDV genome via a transgenic cell line containing an orthogonal translation machinery.It was demonstrated that the transgenic cells stably integrated the orthogonal pylt RNA/pyl RS pair into the genome and enabled efficient,homogeneous incorporation of unnatural amino acids into target proteins in mammalian cells.Next,we constructed 129single-PTC FMDV mutants and four dual-PTC FMDV mutants after considering the tolerance,location,and potential functions of those mutated sites according to tertiary structure analysis of viral proteins.Amber stop codons were individually substituted the selected amino acid codons in four viral proteins(3D,L,VP1 and VP4)of FMDV.The PTC-FMDV mutants were successfully rescued,but the amber codon unexpectedly showed a highly degree of mutation rate during PTC-FMDV packaging and replication.There was no significant correlation between the mutation of amber stop codon and the conservation of amino acids in viral proteins.Further studies on the dual-PTC-FMDV mutants have revealed that increasing the number of amber codons can reduce the efficiency of virus packaging,but there was also the possibility of reversion during virus passaging.2.Generation and genetic stability analysis of recombinant TAGA-FMDVIn this study,in order to solve the problem of PTC-FMDV reverse mutation and explore the feasibility of using quadruple codon decoding strategy to control FMDV replication,we first constructed a stable transgenic cell line with the function of decoding TAGA quadruplet codon.Then,the VP1-N46,VP4-F76,L-Y42,3D-H14 and 3D-M16 amino acid sites of FMDV were selected on the basis of previous experimentation.The recombinant TAGA-FMDV was assembled and rescued in presence of UAAs to produce live virus particles.The results of real-time q PCR and IFA indicated that the viral RNA and protein expression were detected in the cells transfected with recombinant rescue plasmid,indicating that the TAGA-FMDV could be rescued successfully.However,the genetic stability analysis of the TAGA quadruplet codons introduced into the recombinant FMDV genome revealed that these quadruplet codons were genetically unstable in L-Y42 and VP1-N46 mutants,and it has the possibility of mutation or reversion to a sense codon during virus packaging and replication.Furthermore,the sequencing results indicated that the TAGA quadruplet codons introduced into the FMDV genome were also mutated or reverted after third passage of VP4-F76,3D-H14 and 3D-M16 mutants.In summary,in this study we successfully constructed two orthogonal translation cell line that could effectively identify and decode amber stop codons and quadruple codons respectively.The recombinant PTC-FMDV packaging plasmid was constructed via genetic code expansion technology.More than 130PTC-FMDV mutants were systematically screened and successfully rescued with orthogonal translation cell lines,which proved that the concept of genetic code expansion in the generation of PTC-FMDV is feasible.However,the application of this technique to PTC-FMD vaccine development is still challenging,and further optimization is needed to solve the problem of genetic instability.In short,this study established a new and stable technical platform for the generation of recombinant PTC virus,and our study may also provide a valuable reference for future research in other Picornaviridae viruses,highlighting the possibility and challenge for the future use of this emerging technique.