Flaviviruses , including Zika virus (ZIKV), are a significant global health concern, yet no licensed antivirals exist to treat disease. The small M (Membrane) protein plays well-defined roles during viral egress, yet remains within virion membranes following release and maturation. However, it is unclear whether M plays a functional role in this setting. Here, we show that M forms oligomeric membrane-permeabilising channels in vitro , with increased activity at acidic pH and sensitivity to the prototypic channel-blocker, rimantadine. In turn, rimantadine blocked an early stage of ZIKV cell culture infection. Molecular dynamics (MD) generated rationalised structure-based channel models, comprising hexameric arrangements of dual trans -membrane protomers. Interestingly, His28 protonation increased channel opening, consistent with activation within acidifying endosomes. Models contained a predicted lumenal binding site for rimantadine, as well as a second target region on the membrane-exposed periphery. In silico screening enriched for repurposed drugs/compounds predicted to bind to one or other site. Multiple hits displayed potency in vitro and in cell culture, supporting the relevance of channel models. Finally, rimantadine effectively blocked ZIKV viraemia in a preclinical model, supporting that M constitutes a physiologically relevant antiviral target, for either repurposing rimantadine, or the development of new ZIKV therapies.
