A novel fish CRADD inhibits Singapore grouper iridovirus (SGIV) infection by mediating apoptosis and modulating SGIV CARD (VP48)-caspase-2 interaction

The bipartite linker protein CRADD is a molecule that contains death domain (DD)- and caspase recruitment domain (CARD), and remarkably assists in inducing apoptosis and activating caspase-2. This study describes a novel fish CRADD (EcCRADD) isolated from the orange-spotted grouper (Epinephelus coioides), meanwhile demonstrating its inhibitory effect in viral replication. Our findings suggest that EcCRADD can form long-chain filamentous structures that may be involved in apoptosis triggered by nuclear fragmentation. EcCRADD engages the pro-apoptotic protein EcRIP1 (receptor interacting protein 1) through the DD domain and activates caspase-2 through the CARD domain. This bridging allows EcCRADD to promote Singapore grouper iridovirus (SGIV)-induced apoptosis and synergistically inhibits SGIV replication in vitro. SGIV contains a CARD protein encoded by ORF048L (VP48). In contrast to EcCRADD, the overexpression of VP48 significantly promotes SGIV replication and inhibits SGIV-induced apoptosis. Furthermore, we found that VP48 may utilize caspase-2 as a scaffolding molecule to activate NF-κB and promote cell survival. However, EcCRADD could activate p53 in the presence of caspase-2 and inhibit this VP48-induced NF-κB activity in a dose-dependent manner. Lastly, EcCRADD inhibits the nuclear expression of VP48 via caspase-2. EcCRADD competes with VP48 to bind with caspase-2. Our study provides insights into host–virus interactions in fish, specifically that EcCRADD inhibits virus replication via the caspase-2-activated p53 pathway and interferes with the VP48-activated NF-κB signaling pathway.