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

Epitranscriptomic RNA modifications, particularly N6-methyladenosine (mA), have emerged as important regulators of host-virus interctions. However, the role of mA in arbovirus infection within mosquito vectors remains poorly defined. Here, we characterized the mA 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 mA methyltransferase components METTL3 and METTL14. In contrast, pharmacological inhibition of METTL3 markedly enhanced ZIKV replication, indicating an antiviral role for mA 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 mA 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 mA modifications are enriched within the coding regions of serine protease transcripts, supporting their direct regulation by mA. Functionally, inhibition of serine protease activity using AEBSF resulted in a significant reduction of ZIKV replication. Together, these findings identify mA RNA methylation as a critical regulator of ZIKV infection in mosquito cells and uncover an epitranscriptomic pathway linking mA-dependent control of serine proteases to vector-virus interactions. Author SummaryMosquito-borne viruses such as Zika virus pose a major threat to global public health. Successful transmission of these viruses depends not only on infection in humans, but also on their ability to replicate efficiently inside mosquito vectors. Chemical modifications of RNA, collectively known as epitranscriptomic marks, have recently emerged as important regulators of gene expression and virus-host interactions. Among these, N6-methyladenosine (m6A) is the most abundant internal RNA modification in eukaryotic cells. While m6A has been extensively studied in mammalian systems, its role in mosquito antiviral responses remains poorly understood. In this study, we investigated how m6A RNA methylation influences Zika virus infection in mosquito cells derived from Aedes albopictus. We found that reducing m6A levels enhances viral replication, indicating that this RNA modification restricts infection in mosquito cells. Notably, Zika virus RNA itself does not contain detectable m6A modifications. Instead, m6A regulates the expression of specific mosquito genes, including a group of serine proteases that influence viral replication. Pharmacological inhibition of these proteases significantly impaired virus growth, identifying them as key downstream effectors. Our findings reveal an antiviral role for m6A in mosquito cells and uncover a previously unrecognized epitranscriptomic pathway that shapes mosquito-virus interactions. Understanding how RNA modifications regulate arbovirus infection in vectors may open new avenues for strategies aimed at limiting virus transmission.