Functional characterization of grass carp OXR1 in response to GCRV infection

Oxidation resistance protein 1 (OXR1) protects cells from oxidative injury in several vertebrate systems, but its role during viral infection in teleosts is unknown. We cloned and characterized grass carp OXR1 (Ctenopharyngodon idella; CiOXR1) and examined its response to grass carp reovirus (GCRV). The 959-bp CiOXR1 cDNA contains a 774-bp open reading frame encoding a 257-amino-acid TLDc-domain protein. CiOXR1 transcripts were detected in all tissues examined in healthy grass carp, with the highest basal abundance in brain, followed by heart. Following genotype II GCRV-HZ08 challenge in grass carp, CiOXR1 transcript abundance increased in brain and heart. In vitro, treatment with LPS, poly(I:C), or H₂O₂ increased CiOXR1 transcript abundance in grass carp ovary (GCO) cells. GFP-CiOXR1 localized mainly to the cytoplasm of GCRV-infected GCO cells. Cell-based localization and functional assays used genotype I GCRV-873, which increased DCFH-probe fluorescence. Infected cells transfected with 3HA-CiOXR1 showed lower DCFH fluorescence, lower SOD1 and SOD2 transcript abundance, and reduced accumulation of viral RNA and total-protein markers. TCID₅₀ assays further supported inhibition of infectious-virus production by CiOXR1. Total caspase-3 and caspase-9 protein abundance, their transcript levels, and selected innate immune-response markers were also reduced, whereas no significant difference in STING transcript abundance was detected. Cas13d-mediated knockdown of CiOXR1 yielded reciprocal transcript-level changes. Together, the complementary expression and knockdown results show that CiOXR1 expression is inversely related to GCRV molecular-marker accumulation and accompanies coordinated changes in intracellular oxidation, total caspase abundance, and selected innate immune-response markers. These findings further support an antiviral role for CiOXR1 and provide a foundation for research on the prevention and control of grass carp hemorrhagic disease.