Targeting endolysosomal acidification inhibits poxvirus entry and replication.

BACKGROUND: Orthopoxviruses, including monkeypox virus (MPXV) and cowpox virus (CPXV), pose significant public health threats, yet treatment options remain limited. Host-targeted antivirals could circumvent viral resistance by disrupting essential host pathways. Vacuolar H⁺-ATPase (V-ATPase), a proton pump critical for endolysosomal acidification, is implicated in poxviral entry and replication, making it a promising therapeutic target. METHODS: We screened 268 antiviral compounds for activity against CPXV and identified Diphyllin, a V-ATPase inhibitor. In vitro assays (plaque reduction, time-of-addition, qPCR, TEM) evaluated its mechanism. V-ATPase dependency was confirmed using inhibitors (Bafilomycin A1, KM91104) and TMEM175 channel activators (DCPIB, arachidonic acid). Combination treatment assays in vitro were performed to evaluate the potential synergistic inhibitory effects of Diphyllin and Tecovirimat on viral infection. In vivo efficacy was assessed in CPXV-infected mice through analysis of viral titers, histopathology, cytokine levels, as well as survival experiments to evaluate the therapeutic benefit of Diphyllin alone and in combination with Tecovirimat. RESULTS: Diphyllin potently inhibited CPXV and vaccinia virus (VACV) replication (EC₠₀ = 0.69 µM) without cytotoxicity. It blocked post-entry stages by disrupting V-ATPase-mediated endolysosomal acidification, suppressing viral uncoating and morphogenesis. Other V-ATPase inhibitors and TMEM175 activators similarly restricted infection, validating endolysosomal pH as a key vulnerability. In mice, Diphyllin reduced viral loads in spleen, liver, and adipose tissue, attenuated inflammation, and downregulated pro-inflammatory cytokines. Importantly, Diphyllin treatment improved the survival of CPXV-infected mice, demonstrating a measurable therapeutic benefit. Moreover, combination therapy with Tecovirimat further lowered the effective drug doses and yielded additional survival benefits, supporting the therapeutic potential of this ingredient. CONCLUSIONS: Diphyllin is a broad-spectrum anti-poxviral agent targeting host endolysosomal acidification. Its efficacy in vitro and in vivo, coupled with synergistic effects of TMEM175 activation, highlights host pH modulation as a therapeutic strategy. These findings support further development of V-ATPase inhibitors for treating poxvirus infections. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12964-026-02705-6.