SmE2D2, a teleost ubiquitin-conjugating enzyme, is involved in the efficient propagation of megalocytivirus

Abstract

Many viruses use the ubiquitin-proteasome system (UPS) of the host for successful infection. The megalocytivirus RBIV-C1 encodes a gene (RNF64L) homologous to the RING domain ubiquitin ligase E3. In this study, we identified and functionally characterized an RNF64L interacting protein, the E2 ubiquitin-conjugating enzyme (SmE2D2) in turbot (Scophthalmus maximus). Phylogenetic tree analysis clustered SmE2D2 into the class I E2 enzyme family, which contains five E3 interaction residues and one active site (Cys85). Real-time PCR analyses revealed ubiquitous expression of SmE2D2, with expression levels that varied in the different stages of RBIV-C1 infection. An in vitro ubiquitination assay showed that SmE2D2, but not a mutant SmE2D2 (SmE2C85S), had ubiquitin-conjugating enzyme activity to catalyze the formation of enzyme-polyUb conjugates in the presence of RNF64L. Overexpression of SmE2D2, but not SmE2C85S, significantly promoted the replication of RBIV-C1 in turbot. The results demonstrate that SmE2D2 is an E2 ubiquitin-conjugating enzyme that can be hijacked by RBIV-C1 to promote efficient replication.