Identification of poly(I:C) interacted proteins and their regulation in transcription level against bacterial infection in zebrafish

Introduction Polyinosinic-polycytidylic acid (poly(I:C)), a synthetic double-stranded RNA (dsRNA) analog, activates innate immunity against infectious and non-infectious diseases through interactions with both DNA and proteins. However, the full spectrum of poly(I:C)-binding proteins and their roles remains unclear. Methods Affinity-proteomics was used to identify poly(I:C)-interacting proteins in zebrafish, with four candidates validated by microscale thermophoresis (MST). Gene expression of all 27 targets was analyzed by qRT-PCR in poly(I:C)-treated fish and in fish surviving or dying after Vibrio alginolyticus or Edwardsiella tarda challenge. Results Twenty-seven poly(I:C)-interacting proteins involved in diverse cellular processes were identified. Four candidatesprolyl 4-hydroxylase (P4HTM), acidic leucine-rich nuclear phosphoprotein 32 family member E (ANP32E), F-box only protein 2 (FBXO2), and ribosomal protein large P2 (RPLP2)were validated for binding via microscale thermophoresis, with P4HTM's functional modulation by poly(I:C) further demonstrated. At the gene expression level, 10 targets were upregulated, 9 of which showed elevated levels in surviving fish but declined in dying fish after Vibrio alginolyticus or Edwardsiella tarda challenge, implicating them as anti-infective biomarkers Discussion These findings reveal poly(I:C)'s broader protein targets than previously recognized, offering new insights into its multifaceted biological functions in antibacterial defense in fish, with broader implications for understanding evolutionarily conserved dsRNAhost interactions in vertebrate innate immunity.