FIG. 1.
WSSV222 is a RING-containing E3 ligase. (A) Sequence alignment of the N-terminal portion WSSV222 with related proteins. The WSSV222 RING domain is of the C3H2C3 type (CX2CX9-39CXHX2HX2CX4-48CX2C), similar to that of Arabidopsis (ARA-T) and ORF MSV251 (Melanoplus sanguinipes entomopoxvirus; MSV). Black highlights consensus residues for RING structure among the three species. (B) Schematic representation of WSSV222 protein and mutant constructs. (C) In vitro conjugation assay using anti-ubiquitin antibody P4D1, as described in Materials and Methods. A panel of different E2 enzymes was screened for activity in the presence of 222RING. The negative control reaction was performed in the absence of E2 (No E2). (D) Both full-length WSSV222 and RING domain of WSSV222 have polyubiquitination activity under a specific E2 enzyme, UbcH6. The negative control reaction was performed in the absence of E1 or E2. (E) Effects of mutations in WSSV222 on the in vitro conjugation reaction. Purified mutant and wild-type 222RING proteins were subjected to an in vitro ubiquitination assay using E1, UbcH6, and ubiquitin. The input amount of E3 ligase was the same, detected by anti-His6 antibody. Ubi, ubiquitin.