E6AP/UBE3A catalyzes encephalomyocarditis virus 3C protease polyubiquitylation and promotes its concentration reduction in virus-infected cells

Abstract 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 3C pro 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.