Betanodavirus greasy grouper (Epinephelus tauvina) nervous necrosis viruses (GGNNV) protein alpha, a virus capsid protein, was detected in both nucleolus and cytoplasm of infected cells of Asian sea bass (SB) and transfected cells of SB and Cos-7 with pcDNA3.1/RNA2. To study its subcellular localization, ORF of protein alpha with 338 aa was fused with enhanced green fluorescent protein (EGFP) gene and was detected in transfected cells in the absence of other viral proteins. In both SB and Cos-7 cells, protein alpha was found to localize EGFP to the nucleolus and cytoplasm. Deletion mutants of protein alpha indicated that N-terminal 43 amino acid residues were required to import EGFP-alpha protein into the nucleolus. Further deletions within the 43 amino acid backbone, EGFP/33aa(1-33) and EGFP/30aa(14-43), localized to the nucleolus, suggesting that the 20 amino acids from 14 to 33 of protein alpha were the domain of nucleolus localization. To further determine the nucleolus targeting sequence, deletion mutations within the 20 amino acids of protein alpha were constructed. It was found that the deletion of (23)RRR(25), (29)RRR(31), or (23)RRRANNRRR(31) prevented the accumulation of EGFP fusion proteins into the nucleolus, demonstrating that (23)RRRANNRRR(31) contain the signal required for nucleolar localization. A similar distribution pattern of localization of protein alpha and its deletion mutants in SB and Cos-7 cells suggested that N-terminal residues of protein alpha (23)RRRANNRRR(31) constitute a nucleolus localization signal that functions in both fish and mammalian cells.