Zika virus reprograms the host tRNA epitranscriptome to adapt translation to A-ending codon bias

The codon usage bias of the Zika virus (ZIKV) genome is skewed towards AA-ending codons, which are preferentially decoded by U34-modified cognate tRNAs. This contrasts with the human host's preference for AG-ending codons, suggesting that ZIKV may exploit specific tRNA modifications to optimize protein synthesis within human cells. To test this hypothesis, we used codon-biased GFP sensors and found that ZIKV infection transiently increased the expression of AA-biased GFP at the expense of AG-biased GFP. Mass spectrometry analysis further showed that ZIKV virus infection increases mcm5U34 and mcm5s2U34 tRNA modification content in host cells. In ELP1-deficient cells, which exhibit reduced U34 modifications, ZIKV replication was impaired. Enhancing U34 modification through using a small molecule known to restore ELP1 expression, rescued viral replication in these cells. Moreover, CRISPR/Cas9 and shRNA-mediated knockdown of key enzymes involved in U34 modification, ELP1, ALKBH8, and CTU1, significantly reduced ZIKV replication. Collectively, these results provide strong evidence that ZIKV reprograms the host tRNA epitranscriptome and exploits host cell tRNA modifications, particularly at the wobble position U34, to optimize translation of its own proteins and promote viral replication.