Abstract

Neurotropic alphavirus infection is characterized by neuronal injury and sustained neuroinflammation, yet the viral determinants linking membrane perturbation to host inflammatory damage remain poorly defined. Here, we show that the Western equine encephalitis virus (WEEV) 6K protein functions as an ER-localized viroporin, displaying stable single-channel activity with broad cation permeability. Consistent with its ion channel activity, 6K expression perturbs intracellular Ca²⁺ store homeostasis and is associated with cellular injury phenotypes. Mechanistically, 6K predominantly engages MLKL-mediated necroptosis, identifying programmed necrotic signaling as a principal pathway underlying 6K-driven injury under these conditions. Importantly, genetic deletion or pharmacological inhibition of MLKL significantly attenuates 6K-associated cellular damage and inflammatory release, establishing MLKL-dependent execution as a key modifiable node in viroporin-induced host injury. We further identify HYH09-D4 as a protective small molecule that dampens MLKL activation and limits tissue damage associated with 6K expression. Together, these findings define WEEV 6K as a host-facing viroporin that links ionic dysregulation to MLKL-dependent necroptosis and suggest that targeting MLKL may represent a broadly applicable strategy to mitigate host injury driven by 6K-encoding alphaviruses.