Controlling the Replication of a Genomically Recoded HIV-1 with a Functional Quadruplet Codon in Mammalian Cells

Abstract

Large efforts have been devoted to genetic code engineering in the past decade, aiming for unnatural amino acid mutagenesis. Recently, an increasing number of studies were reported to employ quadruplet codons to encode unnatural amino acids. We and others have demonstrated that the quadruplet decoding efficiency could be significantly enhanced by an extensive engineering of tRNAs bearing an extra nucleotide in their anticodon loops. In this work, we report the identification of tRNA mutants derived from directed evolution to efficiently decode a UAGA quadruplet codon in mammalian cells. Intriguingly, the trend of quadruplet codon decoding efficiency among the tested tRNA variants in mammalian cells was largely the same as that in E. coli. We subsequently demonstrate the utility of quadruplet codon decoding by the construction of the first HIV-1 mutant that lacks any in-frame amber nonsense codons and can be precisely activated by the decoding of a genomically embedded UAGA codon with an unnatural amino acid. Such conditionally activatable HIV-1 mutant can likely facilitate both fundamental investigations of HIV-1 as well as vaccine developments. The use of quadruplet codon, instead of an amber nonsense codon, to control HIV-1 replication has the advantage in that the correction of a frameshift caused by a quadruplet codon is much less likely than the reversion of an amber codon back into a sense codon in HIV-1.

Harnessed viruses in the age of metagenomics and synthetic biology: an update on infectious clone assembly and biotechnologies of plant viruses

“…Insertion of non‐sense and unnatural codons into essential virus genes can condition the replication of engineered viruses to the presence of specific chemical and genetic suppressors. Unnatural amino acids and quadruplet codons were used to generate live replication‐incompetent viruses as safer vaccines (Chen et al ., ; Si et al ., ). In plants, viral expression of the cytochrome P450 SU1 conferred conditional plant sensitivity to the R7402 proherbicide (Whitham et al ., ).…”

Insights into genome recoding from the mechanism of a classic +1-frameshifting tRNA

“…While most recoding employs stop codons as sites for incorporating non-proteinogenic amino acids, only two stop codons can be simultaneously recoded due to the cellular need to reserve the third stop codon for termination of protein synthesis. The use of quadruplet codons as additional sites for incorporating non-proteinogenic amino acids has thus emerged as an attractive alternative 1 , 2 . Recoding at a quadruplet codon requires a +1-frameshifting tRNA that is aminoacylated with the non-proteinogenic amino acid of interest.…”

Genetic‐Code‐Expansion Strategies for Vaccine Development

“…To decrease the likelihood of PTC HIV-1 variants to revert to a virulent state, the use of a nonsense quadruplet codon to encode for a ncAA was explored. [58] For this, an OTS adapted to decode UAGA codons with N ɛ -(tert-butoxycarbonyl)-l-lysine (N ɛ -BocLys, Figure 4B) in Escherichia coli could be repurposed for the incorporation of this ncAA in mammalian (HEK 293T) cells. [59] To avoid mistranslation of five in-frame UAG-A sequences in the HIV-1 genome, which could interfere with viral assembly, Guo and co-workers created an HIV-1 variant that lacked these sequences and displayed replication and infectivity levels comparable to the parent strain.…”