The enteroviral protease target LSM14A operates outside of P-bodies to augment antiviral innate immunity.

Antiviral innate immune networks in human cells comprise core components that serve as central signaling hubs and several context-dependent modulators whose role may be virus- or tissue-specific. One such modulator is LSM14A, which potentiates innate immune response but is not essential. We recently showed that enteroviruses deploy their protease activity to cleave LSM14A, thereby disabling its antiviral function. In this study, we probe the molecular mechanism by which LSM14A contributes to innate immunity. We show that although LSM14A predominantly localizes to processing bodies (P-bodies; PBs), this localization is not essential for its innate immune function. Likewise, association with peroxisomes does not contribute to its immune activity. Instead, an unbiased systems-level interactomic analysis reveals a distinct cohort of LSM14A-associated proteins that assemble outside canonical PBs and peroxisomes following infection with Sendai virus, a robust inducer of innate immunity. Functional interrogation of these interactors demonstrate that several are essential for LSM14A-dependent amplification of antiviral signaling. Together, these findings uncover a functional axis of LSM14A that operates independently of its canonical subcellular localizations and is mediated through a specialized interaction network, improving our understanding of how this protein reinforces the antiviral innate immune system.