m⁶A RNA methylation modulates Zika virus infection by regulating serine proteases in Aedes albopictus

Epitranscriptomic RNA modifications, particularly N⁶-methyladenosine (m⁶A), have emerged as important regulators of host-virus interctions. However, the role of m⁶A in arbovirus infection within mosquito vectors remains poorly defined. Here, we characterized the m⁶A RNA methylation machinery in Aedes albopictus C6/36 cells and examined its contribution to Zika virus (ZIKV) replication. Arbovirus infection did not significantly alter the transcriptional levels or enzymatic activity of the core m⁶A methyltransferase components METTL3 and METTL14. In contrast, pharmacological inhibition of METTL3 markedly enhanced ZIKV replication, indicating an antiviral role for m⁶A in mosquito cells. Transcriptome-wide analysis of C6/36 cells treated with the METTL3 inhibitor STM2457 revealed extensive changes in gene expression, including the pronounced upregulation of multiple serine proteases, particularly members of the CLIP family. Single-nucleotide-resolution mapping of m⁶A using GLORI-sequencing showed that m⁶A is absent from Zika virus RNA, but readily detectable in the A. albopictus transcriptome. Data analysis defined key features of the mosquito epitranscriptome and demonstrated that m⁶A modifications are enriched within the coding regions of serine protease transcripts, supporting their direct regulation by m⁶A. Functionally, inhibition of serine protease activity using AEBSF resulted in a significant reduction of ZIKV replication. Together, these findings identify m⁶A RNA methylation as a critical regulator of ZIKV infection in mosquito cells and uncover an epitranscriptomic pathway linking m⁶A-dependent control of serine proteases to vector-virus interactions.