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 eGFP 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 mcm ⁵ s ² U ₃₄ tRNA modification content in host cells. In ELP1-deficient cells, which exhibit reduced U ₃₄ modifications, ZIKV replication was impaired. Enhancing U ₃₄ 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 U ₃₄ 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 U ₃₄ , to optimize translation of its own proteins and promote viral replication.