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

Abstract

The encephalomyocarditis virus (EMCV) 3C protease (3Cpro) 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 3Cpro-polyubiquitylating pathway. Following the identification of UbcH7/UBE2L3 as a participant in 3Cpro ubiquitylation, we purified a UbcH7-dependent 3Cpro-ubiquitylating activity from mouse cells, which we identified as E6AP. In vitro reconstitution assays demonstrated that E6AP catalyzes the synthesis of 3Cpro-attached Lys48-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 3Cpro concentration regulation. We also discovered that ARIH1 functions with UbcH7 to catalyze EMCV 3Cpro monoubiquitylation, but this activity not influence the in vivo 3Cpro concentration.

Encephalomyocarditis Virus Abrogates the Interferon Beta Signaling Pathway via Its Structural Protein VP2

“…However, throughout evolution viruses have acquired numerous strategies to evade the host immune system (32), notably the type I IFN antiviral response. Previous studies have shown the ability of EMCV to evade host immunity via interfering with IFN signaling (10)(11)(12)(13)(14)(15). To our knowledge, we are the first to show the ability of the EMCV structural protein, VP2, to antagonize the components of the RLR/MDA5 signaling pathway.…”

“…We and others have used HEK293 cells as a model cell line for EMCV infection studies (10,11,35,36). We are aware of the limitation that using this cell line poses as it is inherently virally transformed and expresses the E1A oncogene from adenovirus (Adv) and also the artificial nature of using a cell line model.…”

“…EMCV structural proteins play essential roles in virus entry, uncoating and assembly, whereas non-structural proteins form the main components in the viral replication complex. Notably, EMCV L pro , 2C and 3C play important roles in blocking interferon (IFN) expression and downstream antiviral signaling (10)(11)(12)(13)(14)(15).…”

DDX56 antagonizes IFN-β production to enhance EMCV replication by inhibiting IRF3 nuclear translocation

“…and VP2, are known to block innate immune signaling pathway activation[5][6][7][22][23][24]. Hostproteins are also involved in EMCV infection.…”