Molecular cloning and characterization of Aos1 and Uba2 from the orange-spotted grouper (Epinephelus coioides).

Small ubiquitin-related modifiers (SUMOs) are post-translationally conjugated to other proteins and are essential regulators of a wide range of cellular processes. Covalent attachment of SUMO requires an enzymatic cascade consisting of a single E1-activating enzyme (Aos1 and Uba2 heterodimer), a single E2-conjugating enzyme (Ubc9), and one of several E3 ligases that facilitate transfer of SUMO from Ubc9 to the substrate. In the present study, the Aos1 and Uba2 homologues (EcAos1 and EcUba2) from the orange-spotted grouper (Epinephelus coioides) were cloned and their possible roles in fish immunity were analyzed. The open reading frame (ORF) of EcAos1 contains 1050 base pairs (bp) encoding a 350 amino acid protein with a predicted molecular mass of 38.97 kDa EcAos1 has a nuclear localization signal (NLS) at residues 193-203. The ORF of EcUba2 contains 1950 bp encoding a 650 amino acid protein with a predicted molecular mass of 71.3 kDa EcUba2 has a NLS at residues 608-630. Quantitative real-time polymerase chain reaction analysis indicated that both EcAos1 and EcUba2 were distributed in all examined tissues. The expression levels of EcAos1 and EcUba2 in the spleen and head kidney of E. coioides were differentially up-regulated when challenged with polyinosine-polycytidylic acid. Green fluorescence of both pEGFP-C1-EcAos1 and pEGFP-C1-EcUba2 was distributed in the nucleus of GS cells. When the NLSs of EcAos1 and EcUba2 were deleted, the cellular localizations all changed. Over-expression of EcAos1 and EcUba2 inhibited red-spotted grouper nervous necrosis virus infection and replication. These results are important for better understanding of the SUMO pathway in fish and provide insights into the regulatory mechanism of viral infection in E. coioides under farmed conditions.