The encephalomyocarditis virus (EMCV) 3C protease (3C(Pro)) is one of a small number of viral proteins whose concentration is known to be regulated by the cellular ubiquitin-proteasome system. Here we report that the ubiquitin-conjugating enzyme UbcH7/UBE2L3 and the ubiquitin-protein ligase E6AP/UBE3A are components of a previously unknown EMCV 3C(Pro)-polyubiquitylating pathway. Following the identification of UbcH7/UBE2L3 as a participant in 3C(Pro) ubiquitylation, we purified a UbcH7-dependent 3C(pro)-ubiquitylating activity from mouse cells, which we identified as E6AP. In vitro reconstitution assays demonstrated that E6AP catalyzes the synthesis of 3C(Pro)-attached Lys(48)-linked ubiquitin chains, known to be recognized by the 26S proteasome. We found that the 3CPro accumulates to higher levels in EMCV-infected E6AP knockdown cells than in control cells, indicating a role for E6AP in in vivo 3C(Pro) concentration regulation. We also discovered that ARIH1 functions with UbcH7 to catalyze EMCV 3C(Pro) monoubiquitylation, but this activity does not influence the in vivo 3C(Pro) concentration. (C) 2017 Elsevier Inc. All rights reserved.