Identification of E6AP/UBE3A as a ubiquitin‐protein ligase that catalyzes encephalomyocarditis virus 3C protease ubiquitylation (952.1)

Abstract

The 3C proteases of encephalomyocarditis virus (EMCV) and hepatitis A virus (HAV) are rapidly degraded by the ubiquitin‐26S protease system, which limits the 3C protease concentration that develops in infected cells. We have discovered that at least one UbcH7‐dependent ubiquitylation pathway is important for EMCV replication success determination. To identify the UbcH7‐dependent E3 ubiquitin‐protein ligase(s) involved in tagging the 3C proteases for destruction, we developed a protein purification scheme that begins with mouse cell lysate and ends with a ubiquitin‐UbcH7 affinity column. Analysis by mass spectroscopy of the proteins eluted from this column revealed the presence of four E3s, one of which is the HECT E3 E6AP/UBE3. The ability of this E3 to recognize the EMCV 3C protease as a substrate was confirmed using reconstituted mixtures containing purified E6AP. The major product of these reactions was monoubiquitylated 3C protease rather than polyubiquitylated conjugates. Whether this accurately reflects E6AP action in vivo remains to be determined, but it is possible that E6AP must function with other E3s to catalyze 3C protease polyubiquitylation in infected cells. Either UbcH7 or UbcH5 support E6AP‐catalyzed EMCV 3C protease ubiquitylation, with a slight kinetic preference for UbcH7. While the HAV 3C protease is also ubiquitylated by a UbcH7‐dependent E3, our results show that this E3 is not E6AP.Grant Funding Source: Supported by NIH Award 1R15AI099134‐01.