Wolbachia outcompetes mosquito-borne viruses by reprogramming host ATPSβ1-driven ATP metabolism toward symbiotic proliferation.

Wolbachia endosymbionts confer broad-spectrum resistance to mosquito-borne viruses. While the antiviral metabolites in Wolbachia-Aedes have been identified during viral replication, the core metabolic gene underlying this defense across the entire viral infection cycle remains undefined. Here, we demonstrated for the first time that Wolbachia strongly suppressed the emerging alphavirus Venezuelan equine encephalitis virus, extending its known antiviral spectrum. Transcriptomic and functional analyses revealed ATP synthase beta subunit 1 (ATPSβ1) as a key metabolic hub hijacked by both MBV and the symbiont. Without Wolbachia symbiosis, dengue virus and VEEV induced ATPSβ1 to promote viral binding via the viral envelope protein and increased intracellular ATP production for viral replication. Conversely, Wolbachia overrode ATPSβ1-mediated viral binding and redirected ATPSβ1-driven ATP synthesis to fuel its proliferation, strengthening antiviral activity. Pharmacologically, adenosine increased Wolbachia wAlbB density and potently suppressed viral infection in Ae. aegypti and Ae. albopictus cells. Oligomycin reduced intracellular ATP and inhibited viral infection in Wolbachia-free Ae. aegypti cells. Collectively, Wolbachia repurposes dual-functional ATPSβ1, which governs MBV binding and replication, into a potent antiviral effector through a metabolic tug-of-war over host ATP during symbiont-virus competition. Moreover, we highlight interventions targeting the ATPSβ1-driven metabolism as promising strategies to curb MBV transmission.