A UbcH7‐mediated pathway is critical for the conjugation of ubiquitin to picornaviral 3C proteases

Abstract

The polyprotein processing 3C proteases of the encephalomyocarditis virus (EMCV) and hepatitis A virus (HAV) are rapidly degraded by the ubiquitin/26S protease system. We have identified three distinct ubiquitin‐conjugating pathways that catalyze 3C protease ubiquitination: one that includes the E2/E3 pair UbcH1/E3α, one requiring UbcH5a, and a UbcH7‐dependent pathway. As part of an effort to understand how these pathways function together to mark the 3C proteases for proteasome‐mediated degradation, we have carried out experiments using reconstituted in vitro ubiquitination reaction systems to evaluate the contribution each pathway makes to the conjugation of polyubiquitin chains to the EMCV and HAV 3C proteases. Our results show the UbcH7‐dependent pathway plays a critical role in the initial conjugation of ubiquitin molecules to the 3C proteases. Initial 3C protease recognition by the UbcH7‐dependent E3 ubiquitin‐protein ligase, or UbcH7/E3 pair, requires that a previously identified ten‐amino acid "destruction signal" in the 3C proteases be intact. The subsequent ubiquitin chain extension process, however, requires the presence of all three complete pathways. Reaction mixtures lacking one or two of the complete individual ubiquitin‐conjugating pathways failed to robustly support 3C protease polyubiquitination. This work was supported NSF grant award MCB‐0210188.