Alternative polyadenylation drives isoform-dependent m6A remodeling during Zika virus infection

Viral infection can reprogram host gene expression by selectively remodeling the cellular epitranscriptome. N6-methyladenosine (m6A) is a reversible RNA modification that is dynamically altered during infection by Flaviviridae viruses, including Zika virus (ZIKV), yet the mechanisms underlying transcript-specific m6A changes remain poorly understood. Here, we integrated glyoxal and nitrite-mediated deamination of unmethylated adenosine sequencing (GLORI-seq) and METTL3 RNA immunoprecipitation and sequencing to generate a comprehensive map of m6A dynamics during ZIKV infection. We identified over 2,000 dynamically regulated m6A sites, with m6A-increased genes enriched for JAK/STAT and TGF-{beta} signaling pathways, both central to immune regulation. Many m6A changes arose from ZIKV-induced noncanonical polyadenylation, which generated new RNA isoforms that are preferentially targeted by METTL3. Consistently, depletion of the cleavage stimulation factors CSTF2 and CSTF2T impaired alternative isoform expression and subsequent ZIKV-induced m6A methylation. Overall, this work reveals that ZIKV reshapes the host epitranscriptome through coordinated changes in RNA processing and redirected METTL3 targeting, establishing mechanistic frameworks for infection-induced m6A remodeling.