WSSV403 is a viral E3 ubiquitin ligase. (A) Both full-length WSSV403 and 403RING can be expressed in E. coli with pQE vector and expression is confirmed by Western blot with anti-histidine antibody. 1: Total cell lysate from un-induced E. coli; 2: Total cell lysate from E. coli expressing 403RING; 3: Total cell lysate from E. coli expressing WSSV403. (B) A panel of different E2 enzymes was screened for activity in the presence of 403RING. The negative control reaction was performed in the absence of E2. -E2: negative control without E2 conjugating enzyme; sE2: shrimp E2 Pvubc; 5a, 5b, 5c, 6, 9 and 12: Ubc 5a, Ubc5b, Ubc5c, Ubc6, Ubc 9 and Ubc 12 are variant E2 enzymes from human. (C) In vitro conjugation assay using anti-WSSV403 and anti-ubiquitin antibody P4D1. WSSV403 can be polyubiquitinated in the presence of Pvubc, a shrimp E2 ubiquitin conjugation enzyme.