Identification and characterization of TBC1D20 in Micropterus salmoides and its role in MSRV infection

Micropterus salmoides rhabdovirus (MSRV) is a major viral pathogen causing severe disease in juvenile largemouth bass. Our previous study identified a potential interaction between the MSRV glycoprotein (MSRV-G) and largemouth bass TBC1D20. TBC1D20, which contains a conserved Tre-2/Bub2/Cdc16 (TBC) domain, is a key regulator of intracellular vesicular trafficking. In this study, we cloned and characterized the TBC1D20 gene from largemouth bass and investigated its role during MSRV infection. Sequence analysis revealed that the TBC1D20 gene comprises 1224 bp and encodes a protein of 407 amino acids. Tissue distribution analysis showed that TBC1D20 was most highly expressed in the intestine, and its expression was upregulated in the intestine, liver, spleen, kidney, and skin following MSRV infection. Molecular docking and co-immunoprecipitation analyses confirmed the interaction between TBC1D20 and MSRV-G. Functional assays in Micropterus salmoides dorsal fin (MSDF) cells demonstrated that TBC1D20 promoted MSRV internalization. However, knockdown of TBC1D20 enhanced viral RNA and protein accumulation, whereas its overexpression suppressed viral RNA and protein accumulation. Collectively, this study suggests the stage-dependent effects of TBC1D20 during MSRV infection and provides novel theoretical clues for deciphering fish antiviral defense mechanisms.