Encephalomyocarditis Virus 3C Protease Research Articles

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

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 does not influence the in vivo 3Cpro concentration.

Encephalomyocarditis virus 3C protease attenuates type I interferon production through disrupting the TANK-TBK1-IKKε-IRF3 complex.

TRAF family member-associated NF-κB activator (TANK) is a scaffold protein that assembles into the interferon (IFN) regulator factor 3 (IRF3)-phosphorylating TANK-binding kinase 1 (TBK1)–(IκB) kinase ε (IKKε) complex, where it is involved in regulating phosphorylation of the IRF3 and IFN production. However, the functions of TANK in encephalomyocarditis virus (EMCV) infection-induced type I IFN production are not fully understood. Here, we demonstrated that, instead of stimulating type I IFN production, the EMCV-HB10 strain infection potently inhibited Sendai virus- and polyI:C-induced IRF3 phosphorylation and type I IFN production in HEK293T cells. Mechanistically, EMCV 3C protease (EMCV 3C) cleaved TANK and disrupted the TANK–TBK1–IKKε–IRF3 complex, which resulted in the reduction in IRF3 phosphorylation and type I IFN production. Taken together, our findings demonstrate that EMCV adopts a novel strategy to evade host innate immune responses through cleavage of TANK.

Encephalomyocarditis Virus 3C Protease Relieves TRAF Family Member-associated NF-κB Activator (TANK) Inhibitory Effect on TRAF6-mediated NF-κB Signaling through Cleavage of TANK

TRAF family member-associated NF-κB activator (TANK) is a negative regulator of canonical NF-κB signaling in the Toll-like receptor- and B-cell receptor-mediated signaling pathways. However, functions of TANK in viral infection-mediated NF-κB activation remain unclear. Here, we reported that TANK was cleaved by encephalomyocarditis virus 3C at the 197 and 291 glutamine residues, which depends on its cysteine protease activity. In addition, encephalomyocarditis virus 3C impaired the ability of TANK to inhibit TRAF6-mediated NF-κB signaling. Interestingly, we found that several viral proteases encoded by the foot and mouth disease virus, porcine reproductive and respiratory syndrome virus, and equine arteritis virus also cleaved TANK. Our results suggest that TANK is a novel target of some viral proteases, indicating that some positive RNA viruses have evolved to utilize their major proteases to regulate NF-κB activation.

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

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.

SUMOylation Promotes PML Degradation during Encephalomyocarditis Virus Infection

The promyelocytic leukemia (PML) protein is expressed in the diffuse nuclear fraction of the nucleoplasm and in matrix-associated structures, known as nuclear bodies (NBs). PML NB formation requires the covalent modification of PML to SUMO. The noncovalent interactions of SUMO with PML based on the identification of a SUMO-interacting motif within PML seem to be required for further recruitment within PML NBs of SUMOylated proteins. RNA viruses whose replication takes place in the cytoplasm and is inhibited by PML have developed various strategies to counteract the antiviral defense mediated by PML NBs. We show here that primary fibroblasts derived from PML knockout mice are more sensitive to infection with encephalomyocarditis virus (EMCV), suggesting that the absence of PML results in an increase in EMCV replication. Also, we found that EMCV induces a decrease in PML protein levels both in interferon-treated cells and in PMLIII-expressing cells. Reduction of PML was carried out by the EMCV 3C protease. Indeed, at early times postinfection, EMCV induced PML transfer from the nucleoplasm to the nuclear matrix and PML conjugation to SUMO-1, SUMO-2, and SUMO-3, leading to an increase in PML body size where the viral protease 3C and the proteasome component were found colocalizing with PML within the NBs. This process was followed by PML degradation occurring in a proteasome- and SUMO-dependent manner and did not involve the SUMO-interacting motif of PML. Together, these findings reveal a new mechanism evolved by EMCV to antagonize the PML pathway in the interferon-induced antiviral defense.

N‐Terminal ubiquitination of the encephalomyocarditis virus 3C protease

The EMCV 3C protease is a substrate for ubiquitination and rapid degradation by the 26S proteasome. While the 3C protease is usually ubiquitinated at internally located lysine residues, we report here that N-terminally ubiquitinated 3C protease conjugates accumulate under conditions of reduced ubiquitin C-terminal hydrolase (UCH) activity. Examinations of the in vitro ubiquitination of a catalytically active, no-lysine version of the EMCV 3C protease (3C[K-R] protease) revealed that the protein is incorporated into ubiquitinated conjugates only in the presence of ubiquitin aldehyde, an UCH inhibitor. Reaction mixtures containing methylated ubiquitin and ubiquitin aldehyde were found to support the synthesis of a monoubiquitinated 3C[K-R] protease product. Our results show that N-terminally conjugated ubiquitin is preferentially removed from the 3C protease by UCH activity. A protein destruction signal, previously shown to be required for efficient EMCV 3C protease ubiquitination, was found not to be required for N-terminal ubiquitination. Mouse fibroblast cell lines stably transfected with genes encoding either the wild type or 3C[K-R] protease were found to accumulate either 3C protein under promoter-inducing conditions only in the presence of a proteasome inhibitor, suggesting that the N-terminal ubiquitination of the 3C protease occurs in vivo and results in its destruction by the proteasome.

Degradation of the encephalomyocarditis virus and hepatitis A virus 3C proteases by the ubiquitin/26S proteasome system in vivo

We have isolated stably transfected mouse embryonic fibroblast cell lines that inducibly express either the mature encephalomyocarditis virus (EMCV) or hepatitis A virus (HAV) 3C protease and have used these cells to demonstrate that both proteins are subject to degradation in vivo by the ubiquitin/26S proteasome system. The detection of 3C protease expression in these cells requires inducing conditions and the presence of one of several proteasome inhibitors. Both 3C proteases are incorporated into conjugates with ubiquitin in vivo. HAV 3C protease expression has deleterious effects on cell viability, as determined by observation and counting of cells cultured in the absence or presence of inducing conditions. The EMCV 3C protease was found to be preferentially localized to the nucleus of induced cells, while the HAV 3C protease remains in the cytoplasm. The absence of polyubiquitinated EMCV 3C protease conjugates in nuclear fraction preparations suggests that localization to the nucleus can protect this protein from ubiquitination.

Kinetic Analysis of the Conjugation of Ubiquitin to Picornavirus 3C Proteases Catalyzed by the Mammalian Ubiquitin-protein Ligase E3α

The 3C proteases of the encephalomyocarditis virus and the hepatitis A virus are both type III substrates for the mammalian ubiquitin-protein ligase E3alpha. The conjugation of ubiquitin to these proteins requires internal ten-amino acid-long protein destruction signal sequences. To evaluate how these destruction signals modulate interactions that must occur between E3alpha and the 3C proteases, we have kinetically analyzed the formation of ubiquitin-3C protease conjugates in a reconstituted system of purified E1, HsUbc2b/E2(14Kb), and human E3alpha. Our measurements show that the encephalomyocarditis virus 3C protease is ubiquitinated in this system with K(m) = 42 +/- 11 microm and V(max) = 0.051 +/- 0.01 pmol/min whereas the parameters for the ubiquitination of the hepatitis A virus 3C protease are K(m) = 20 +/- 5 microm and V(max) = 0.018 +/- 0.003 pmol/min. Mutations in the destruction signal sequences resulted in changes in the rate at which E3alpha conjugates ubiquitin to the altered 3C protease proteins. The K(m) and V(max) values for these reactions change proportionally in the same direction. These results suggest differences in rates of conjugation of ubiquitin to 3C proteases are primarily a k(cat) effect. Replacing specific encephalomyocarditis virus 3C protease lysine residues with arginine residues was found to increase, rather than decrease, the rate of ubiquitin conjugation, and the K(m) and V(max) values for these reactions are both higher than for the wild type protein. The ability of E3alpha to catalyze the conjugation of ubiquitin to both 3C proteases was found to be inhibited by lysylalanine and phenylalanylalanine, demonstrating that the same sites on E3alpha that bind destabilizing N-terminal amino acids in type I and II substrates also interact with the 3C proteases.

Identification and Characterization of a Protein Destruction Signal in the Encephalomyocarditis Virus 3C Protease

The amino acid sequence LLVRGRTLVV, which is probably located in a strand-turn-strand structure, has been identified as a protein destruction signal in the rapidly degraded encephalomyocarditis virus 3C protease. Mutations within this sequence reduced the susceptibility of the 3C protease toward ubiquitination and degradation in rabbit reticulocyte lysate. This signal is transferable, since poliovirus 3C protease, which is a poor ubiquitin-mediated proteolytic system substrate, was found to be ubiquitinated and degraded when the signal sequence was either generated at an internal location in the protein or fused to the N terminus. An evaluation of the behavior of 3C protease proteins containing mutations in the signal region indicates that considerable variability in the primary structure is tolerated, although the conservation of certain features appears to be required for signal function. Two E3 ubiquitin-protein ligases that recognize the encephalomyocarditis virus 3C protease as a substrate were also partially purified. One of these was identified as the previously described E3alpha, and this was shown to require the destruction signal sequence to catalyze efficiently the ubiquitination of the 3C protease. The other is a Ubc5-dependent E3 that appears to recognize a different, unidentified feature of the 3C protease.

The encephalomyocarditis virus 3C protease is a substrate for the ubiquitin-mediated proteolytic system.

The encephalomyocarditis virus 3C protease has been shown to be rapidly degraded in infected cells and in vitro in rabbit reticulocyte lysate. The in vitro degradation, at least, is accomplished by a virus-independent, ATP-dependent proteolytic system. Here we identify this proteolytic system as the ubiquitin-mediated system. Incubation of the 3C protease in rabbit reticulocyte or cultured mouse cell lysate preparations, alone or in the presence of added ubiquitin or methylated ubiquitin, resulted in the generation of new higher molecular weight species. These new products were shown to be 3C protease-ubiquitin conjugates by their ability to bind antibodies against both the 3C protease and ubiquitin. Supplemental ubiquitin also stimulated the degradation of the 3C protease in these preparations. Large 3C protease-polyubiquitin conjugates were observed to accumulate in reticulocyte lysate in the presence of adenosine 5'-O-(3-thiotriphosphate), an inhibitor of the 26 S multicatalytic protease. This, combined with the fact that the proteolytic activity could be removed from the lysate by sedimentation, implicates the multicatalytic protease in the degradation of the 3C protease-ubiquitin conjugates. It was also found that the slow rate of degradation of a model polyprotein, which resembles the stable viral 3CD diprotein produced in vivo, is likely due to the fact that the polyprotein is a poor substrate for the ubiquitin-conjugating system.

The Encephalomyocarditis Virus 3C Protease Is Rapidly Degraded by an ATP-Dependent Proteolytic System in Reticulocyte Lysate

The encephalomyocarditis virus 3C protease has been observed to undergo rapid degradation, both in vivo in mouse cells and in vitro in reticulocyte lysate. Experiments were carried out to characterize the turnover of the 30 protease in reticulocyte lysate. 3C protease prepared in reticulocyte lysate by in vitro translation and processing of a precursor polyprotein could be separated from the proteolytic activity responsible for its degradation. This implies the 3C protease is not directly involved in its own proteolysis. Active 30 protease flanked by only a few amino acids was degraded at a rate identical to that of a similar protein containing an inactivated catalytic site. This indicates that 3C protease activity is not indirectly required for the proteolytic process. Other vital proteins, including the 3D polymerase and capsid proteins, were relatively stable in the lysate. In addition, polyprotein precursors containing 3C protease with an inactive catalytic site and various flanking proteins displayed distinctly different stabilities. These results suggest that the reticulocyte proteolytic system functions in a selective manner toward the vital proteins. The effects of several proteolytic inhibitors on the lysate proteolytic system were evaluated. The results of these experiments indicate that the rapid degradation of the EMC virus 3C protease requires the hydrolysis of ATP.

Inducible expression of encephalomyocarditis virus 3C protease activity in stably transformed mouse cell lines

An inducible expression vector system has been developed to facilitate the study of the effects of individual virus gene products on cell function. The vector utilizes the mouse metallothionein promoter carried on the bovine papillomavirus genome. Conditions which optimize the induced expression of open reading frames inserted downstream from the mouse metallothionein promoter have recently been described. In this communication we describe the use of this system to clone and express the encephalomyocarditis virus 3C protease in cultured mouse cells. Stably transformed cell lines could be induced to produce levels of 3C protease activity comparable to those observed during normal virus infection. In spite of this, no effects on cellular protein synthesis rate or membrane permeability were observed. It was also discovered that 3C protease as well as 3C protease-containing polyproteins are turned over. This was true not only in the induced cell clones, but also during the normal course of encephalomyocarditis virus infection, as well as in translation systems in vitro. This phenomenon was highly specific for this family of polypeptides, perhaps explaining their apparent lack of cytotoxic effects.

Encephalomyocarditis virus 3C protease: efficient cell-free expression from clones which link viral 5' noncoding sequences to the P3 region.

All picornaviral peptides are derived by progressive posttranslational cleavage of a giant precursor polyprotein. Translation of encephalomyocarditis virus (EMC) RNA in rabbit reticulocyte extracts produces active viral peptides, including protease 3C, which is responsible for many cleavage reactions within the processing cascade. DNA plasmids containing 5' noncoding sequences of EMC linked to other portions of the viral genome were constructed and transcribed into RNA. Like virion RNA, the clone-derived transcripts directed efficient protein translation in vitro. The 5'-linked constructions may represent examples of a general method for cell-free expression of any cloned gene segment. One construction produced a self-cleaving P3 region precursor, which contained active 3C protease. A genetically engineered insertion within the 3C sequences eliminated endogenous self-cleavage activity without altering the ability of the P3 peptide to serve as substrate in bimolecular reactions with added 3C. Another plasmid encoding the L-VP0 portion of the capsid region was used to demonstrate that scission between the leader peptide (L) and capsid protein VP0 can be catalyzed by 3C. The enzyme responsible for this step was previously unidentified. A rapid purification scheme for isolation of 3C from EMC-infected HeLa cells is also presented.