Codon optimization of the prM-E coding region generates stable genome-length cDNA clone for a chimeric dengue 2/3 virus that can be propagated in Escherichia coli
2017
Abstract In an effort to generate a chimeric dengue virus containing the prM-E sequence fromDENV-3 and with the rest of the viral genome derived from a DENV-2 strain 16681-3pm by in vitroligation of subgenomic fragments, it was found that the ligated chimeric genome could not be stablypropagated as genome-length cDNA clone in Escherichia coli. Native DENV-3 prM-E coding regionmay contain certain nucleotide sequence that results in plasmid instability when ampli fi ed in E. coli.In this study, we modi fi ed codons in the DENV-3 viral sequence into those that are most frequentlyused in human genome. Employing the Gibson assembly method, the modi fi ed prM-E fragmentwas introduced into the full-length cDNA clone of strain 16681-3pm to replace the correspondingsequence in a DENV-2 genome. The full-length plasmid cDNA clone intended for the generationof a chimeric virus, D3 prMEopt/16661-3pm, was successfully propagated in E. coli. In vitro transcription of this clone produced full-length RNA transcripts that generated infectious viruses whenintroduced into cultured mammalian cells. Based on kinetics experiments, the replication of D3prMEopt/16661-3pm with modi fi ed codons in the prM-E gene was comparable to the one generatedby the in vitro ligation approach. By combining the use of modi fi ed codons and the Gibson assemblymethod, a full-length cDNA clone of a chimeric virus containing the DENV-3 prM-E sequence canbe stably propagated in E. coli, facilitating further manipulation of the viral sequences. Availability ofsuch an infectious cDNA clone would provide a valuable tool for investigating the role of nucleotide/amino acid variations in the DENV-3 prM-E gene in viral replication, immunogenicity, and pathogenicity as well as accelerating dengue vaccine development.
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