Construction Of A Recombinant Zika Virus By Using UAA-mediated Amber Codon Suppression Strategy

Genetic code expansion is mainly used in living systems to mark and modify proteins.It can be combined with reverse genetics to site-specifically introduce unnatural amino acids(UAAs)into synthetic proteins and transform the termination codon into a sense codon.In the previous study,the genetic code expansion has been successf?Lly applied to hepatitis D virus(HDV),adenovirus,human immunodeficiency virus type 1(HIV-1)and influenza virus,realizing the surface modification of virus particles and the targeted control of virus replication.The application of Genetic code expansion provides a new idea for the study of targeted reg?Lation and genetic modification of the virus.Meanwhile,it is of great significance to the elucidation of viral pathogenesis and the rational design and modification of vaccine.Zika virus(ZIKV)which belongs to genus flavivirus,family flaviviridae,is a singlestranded and positive-sense RNA virus.ZIKV is a mosquito-borne virus that has attracted worldwide attention since its outbreak.ZIKV,yellow fever virus(YFV),West Nile virus(WNV),dengue virus(DENV),Japanese encephalitis virus(JEV)and tickborne encephalitis virus(TBEV)are important members of the flavivirus family.ZIKV infection can cause severe symptoms,such as fever,rashes,arthritis,conjunctivitis,neonatal microcephaly and Guillain-Barre syndrome(GBS).In February,2016,WHO declared ZIKV infection to be a Public Health Emergency of International Concern.Currently,ZIKV has spread to more than 80 countries around the world,causing a serious threat to global public health.There is no approved clinical vaccine and effective treatment for ZIKV infection.Thus,the rational design and development of a vaccine against ZIKV infection is urgent to us.In this study,we first sought to generate recombinant ZIKVs combining the genetic code expansion and reverse genetics.To seek the appropriate site to be engineered with the termination codon,we first selected the largest viral protein NS5 with important biological functions for structural remodeling analysis.These corresponding sites on ZIKV NS5 protein were replaced by the termination codon(UAG)to generate recombinant ZIKV c DNA clones based on the f?Ll-length infectious clone of ZIKV FSS13025(Gen Bank accession number KU955593).To recover these mutants,the recombinant ZIKV genomic RNA were transfected into two stable cells that harboring the orthogonal t RNAs and t RNA synthases.We further evaluated the viral protein expression and genetic stability of the recombinant ZIKVs in the presence of UAA.This study is mainly divided into the following two parts: 1.Design and construction of recombinant ZIKVs reg?Lated by UAAs.First,we chose several mutant sites located in the MTase domain,the rnadependent RNA polymerase(Rd Rp)domain and the junction region of the two on NS5 protein,the biggest non-structural protein of flavivirus.We also performed sequence alignment of ZIKV and a number of other flaviviruses to predict the appropriate mutation site.Finally,we generated 8 recombinant ZIKV c DNA clones based on the f?Ll-length infectious clone of ZIKV FSS13025 by site-directed mutagenesis.2.Recover and characterization of recombinant ZIKVs reg?Lated by UAAs.We screened 3 recombinant ZIKVs by transfecting the stable cell line harboring orthogonal t RNA and t RNA synthase with 1 ?g of purified UAG-containing ZIKV genomic RNA.At 48 hours post transfection,we monitored viral protein expression using immunofluorescence assay with an antibody targeting ZIKV envelope protein.The recombinant ZIKVs PTC246,PTC272 and PTC561 co?Ld express envelope protein normally in the presence of UAAs and the orthogonal t RNAs and t RNA synthases.The expression of viral NS1 protein in cell supernatant at different time points after transfection was determined by ELISA.The res?Lts showed that the three recombinant ZIKVs co?Ld express NS1 protein and the level of NS1 expression was positively correlated with the time after transfection.Then we found that the replicon of SZ01-272 and SZ01-561 co?Ld replicate normally in transgenic 293 T cells by in the presence of UAAs.In conclusion,our study performed the first application of genetic code expansion to flaviviruses and successf?Lly obtained 3 recombinant ZIKVs reg?Lated by UAAs.The three recombinant ZIKVs have normal replication and infection ability in the presence of UAAs and its protein expression is reg?Lated by UAAs.Our study thus establishes proof-of-concept for a reliable production platform to generate UAA controlled recombinant ZIKV variants for use in a potentially large variety of pathogenic study,vaccine design,therapeutic delivery,and synthetic biology applications.