Mechanistically Revealing The Role Of TRNA-Ser-GCU Iso-decoder In Promoting Translation Of Duck Hepatitis A Virus In Chicken Embryo Cells

Duck Virus Hepatitis(DVH)is mainly caused by Duck Hepatitis A Virus(DHAV).Duckling infection by this virus can cause acute infectious disease.Vaccination of DHAV live attenuated vaccine can induce a strong immune response,thus preventing ducklings from DHAV infection.Serial passaging of DHAV in chicken embryos is an empirical approach to the development of such vaccines.However,how duck-origin DHAV adapted in chicken embryos and the underly mechanisms remain largely unknown.In this study,we aimed to investigate how DHAV circumvents host translation barrier in chicken embryo cells that allow duck-origin DHAV to adapt to chicken host.By deciphering tRNA and DHAV codon dependence,we revealed that a gene-locus specific tRNA iso-decoder dominantly promoted DHAV translation in chicken thus its duck-to-chicken fitness.Key findings were listed as follows:1.The translation level of wild-type DHAV was significantly enhanced after passaging the virus to chicken embryo cellsWild-type DHAV and chicken-adapted CH60 strains were used to infect chicken embryos and chicken embryo cells respectively.Compared with wild-type DHAV,the chicken-adapted CH60 strain could cause the death of chicken embryos and serious cytopathic effects in chicken embryo cells in 48 hpi.Western blot results showed that the protein expression of chicken-adapted DHAV in chicken embryo cells was significantly higher than that of wild-type DHAV infection groups.2.The chicken-adapted DHAV can induce the remodeling of the host tRNA in chicken embryo cellsAfter infecting chicken embryo cells with wild-type DHAV and chicken-adapted CH60 strains,we found extensive up-regulation of the whole host tRNA in DHAV CH60 strain infected CEF for 48 hpi.Combined analysis of the codon usage bias of multiple duck-origin DHAV strains and chicken-adapted strains which including CH strain and CH60 strain with the dynamic changes of host tRNAome showed that the chicken-adapted CH60 strain had significant enrichment of some codons,such as Ser-AGC codon and Cys-UGC codon,and the corresponding host tRNA were also up-regulated significantly after infection.These results suggest that these two tRNAs may facilitate viral protein expression and host tRNAome remodeling is associated with DHAV adaptation from duck to chicken.3.tRNA-Ser-GCU is a key host tRNA that promotes the translation of DHAV in vitro and in vivoThe impact of tRNA-Ser-GCU on chicken-adapted DHAV infection was validated in CEF,chicken embryo hepatocytes(CPH),and chicken embryo infection models.In addition,Serine and Cysteine deprivation assays showed that only Serine deprivation could inhibit the protein expression of the CH60 strain in a dose-dependent manner.These results indicated that nuclear-encoding tRNA-Ser-GCU is a key tRNA iso-decoder that promotes the translation of DHAV in the chicken host.4.Overexpression of tRNA-Ser-GCU iso-decoder promotes the translation of chickenadapted DHAV in chicken embryo cells,while the tRNA anticodon mutation and backbone replacement inhibit the translationTransient and lentivirus-mediated overexpression of four tRNA-Ser-GCU iso-decoders were performed in CEF and DF-1 respectively.The results showed that overexpression of the second tRNA-Ser-GCU gene encoded by chromosome 7 significantly promoted the translation of chicken-adapted DHAV in chicken embryo cells.In addition,transfection of two copies of tRNA-Ser-GCU-7-2 further promoted protein expression of chicken-adapted DHAV in chicken embryo cells compared with transfection of one copy of wild-type tRNASer-GCU-7-2.However,the translation of chicken-adapted DHAV was inhibited by tRNAmSer-ACU and tRNAm-Ala-GCU in chicken embryo cells.5.The death time of chicken embryos transgenic with tRNA-Ser-GCU mutants was prolonged,as well as pathological progressAfter inoculations of the lentivirus containing the artificial tRNAm-Ser-GCA,tRNAmAla-GCU,and the natural variant tRNA-Ser-GCU,we found that the embryos transfected with the natural variant tRNA-Ser-GCU all died within 48 hpi,and the embryo bodies showed severe hemorrhagic lesions.While the tRNA anticodon mutation and backbone replacement could prolong the chicken embryos’ survival time after infected in 96 hpi.The body showed chronic edema,especially in the abdomen and legs.In conclusion,after serial passaging of DHAV on chicken embryos,the tRNA-Ser-GCU iso-decoder can regulate the translation of chicken-adapted DHAV in chicken embryo cells in a codon-dependent manner.