Using the yeast two-hybrid system to screen and identify interacting factors with the envelope protein of fish necrosis virus

Nervous necrosis virus (NNV) is a significant pathogen affecting a wide range of marine fish species. The capsid protein (CP) of NNV, as its major structural protein, not only participates in viral particle assembly but also plays a critical role in viral entry into host cells and the subsequent pathogenesis. However, the host protein factors that interact with CP remain largely uncharacterized. To investigate the mechanism of nervous necrosis virus (NNV) infection in fish and, identify CP-interacting factors, total RNA was extracted from SSN-1 cells to construct a yeast two-hybrid cDNA library. The NNV CP gene was PCR-amplified and cloned into the pBT3-STE bait vector. Using CP as bait, we screened the SSN-1 cDNA library via yeast two-hybrid assay. The library capacity reached 7.2×10⁶ CFU with 100% recombination rate and average insert size >1000 bp. Initial screening yielded 24 positive clones. After sequencing, alignment, and pairwise validation, seven candidate interacting proteins were identified. Functional analysis indicated their roles in immune regulation, transcription, stress response, and signal transduction. This study successfully identified CP-interacting factors in NNV-susceptible SSN-1 cells, providing data to elucidate NNV infection mechanisms.