E6AP/UBE3A catalyzes encephalomyocarditis virus 3C protease polyubiquitylation and promotes its concentration...

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 Lys⁴⁸-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.