Evaluation of the Susceptibility of the 3C Proteases of Hepatitis A Virus and Poliovirus to Degradation by the Ubiquitin-Mediated Proteolytic System

Gladding, Rebecca L.; Haas, Amy L.; Gronros, D L; Lawson, T. Glen

doi:10.1006/bbrc.1997.7251

Cited by 19 publications

(29 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Wild type HAV 3C protease was expressed in E. coli from pETHAV3C and purified as described previously (21). The mutated HAV 3C(ϩA37) and 3C(D37R) proteins were expressed from pETHAV3C A37ϩ and pETE3C D37R , but the presence of the mutations necessitated an altered purification scheme.…”

Section: Construction Of Plasmids Containing 3c Protease Coding Sequementioning

confidence: 99%

“…The ability of these proteins to serve as substrates for ubiquitination was evaluated by incubating 7 l of transcription-translation reaction mixtures in a final volume of 20 l containing 20 mM HEPES-KOH, pH 7.5, 1 mM dithiothreitol, 0.1 mM methylated ubiquitin, 0.1 mg/ml cycloheximide, and 60% by volume reticulocyte lysate containing an energy-generating system at 30°for 40 min (21)(22)(23). Aliquots of the reaction mixtures were analyzed by 12% SDS-PAGE and fluorography.…”

Section: Evaluation Of the Susceptibility Of Poliovirus 3c Protease Pmentioning

confidence: 99%

“…The poliovirus 3C protease has been shown to be a very poor substrate for the ubiquitin/26 S proteasome system (21). The poliovirus 3C protease contains, in a position analogous to that of the EMCV and HAV 3C protease destruction signal sequences, the sequence 28 LGVHDNVAIL 37 (23).…”

Section: Kinetic Characterization Of E3␣-catalyzed Conjugation Of Ubimentioning

confidence: 99%

“…The 3C proteases produced by the encephalomyocarditis virus (EMCV) and the hepatitis A virus (HAV), both members of the picornavirus family, have been shown to serve as substrates for E3␣-dependent ubiquitination (21)(22)(23). 2 The ten-amino acid sequence 34 LLVR-GRTLVV 43 , located in what is probably a strand-turn-strand structure, has been discovered to function as a protein destruction signal in the EMCV 3C protease (22).…”

mentioning

confidence: 99%

“…2 The ten-amino acid sequence 34 LLVR-GRTLVV 43 , located in what is probably a strand-turn-strand structure, has been discovered to function as a protein destruction signal in the EMCV 3C protease (22). The HAV 3C protease contains the sequence 32 LGVKDDWLLV 41 in a location homologous to that of the EMCV protein destruction signal sequence, and this sequence has been shown to be required for the ubiquitination and degradation of the HAV 3C protein (21,23). 3 The identification of two substrate proteins recognized by E3␣, both of which contain precisely mapped, internal sequences known to be required for E3␣-dependent ubiquitin conjugation, provides excellent model systems for detailed studies of the interactions that take place between E3␣ and substrate proteins lacking a destabilizing N-terminal amino acid.…”

mentioning

confidence: 99%

See 4 more Smart Citations

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

Lawson¹,

Sweep²,

Schlax³

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

The 3C proteases of the encephalomyocarditis virus and the hepatitis A virus are both type III substrates for the mammalian ubiquitin-protein ligase E3␣. 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 E3␣ 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 E3␣. Our measurements show that the encephalomyocarditis virus 3C protease is ubiquitinated in this system with K m ‫؍‬ 42 ؎ 11 M 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 M and V max ‫؍‬ 0.018 ؎ 0.003 pmol/min. Mutations in the destruction signal sequences resulted in changes in the rate at which E3␣ 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 E3␣ 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 E3␣ that bind destabilizing N-terminal amino acids in type I and II substrates also interact with the 3C proteases.The selection of proteins for destruction by the ubiquitin 26 S/proteasome pathway depends upon specific interactions that occur between the targeted substrates and enzymes involved in the formation of the ubiquitin-target protein conjugates. A hierarchical family of pathways, each composed of at least three enzymes, accomplishes the attachment of ubiquitin to proteins destined to be degraded (1-3). Common to all of these pathways is the ubiquitin-activating enzyme, E1, 1 which recruits free ubiquitin through the ATP-dependent formation of a thiolester bond between a cysteine in the E1 and the C-terminal glycine of the ubiquitin molecule. This ubiquitin is then transferred to one of several members of the E2 family of proteins that are referred to as ubiquitin carrier proteins or ubiquitin-conjugating enzymes. Finally, the ubiquitin is transferred from the E2 to the target substrate protein through the action of an ubiquitin-protein ligase, or E3. Although each E2 protein appears to function with several specific ubiquitinprotein ligases, each E3 can specifically interact with only a limited number of substrate proteins. Regardless of the E3 involved in the ubiquitination process, following the conjugation of the first ubiquitin molecule to a primary amine on the subst...

show abstract

Section: Construction Of Plasmids Containing 3c Protease Coding Sequementioning

confidence: 99%

Section: Evaluation Of the Susceptibility Of Poliovirus 3c Protease Pmentioning

confidence: 99%

Section: Kinetic Characterization Of E3␣-catalyzed Conjugation Of Ubimentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

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

Lawson¹,

Sweep²,

Schlax³

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

The ubiquitin‐proteasome system in positive‐strand RNA virus infection

Choi

Wong

Marchant

et al. 2012

Reviews in Medical Virology

View full text Add to dashboard Cite

SUMMARY Positive‐stranded RNA viruses, like many other viruses, have evolved to exploit the host cellular machinery to their own advantage. In eukaryotic cells, the ubiquitin‐proteasome system (UPS) that serves as the major intracellular pathway for protein degradation and modification plays a crucial role in the regulation of many fundamental cellular functions. A growing amount of evidence has suggested that the UPS can be utilized by positive‐sense RNA viruses. The UPS eliminates excess viral proteins that prevent viral replication and modulates the function of viral proteins through post‐translational modification mediated by ubiquitin or ubiquitin‐like proteins. This review will discuss the current understanding of how positive RNA viruses have evolved various mechanisms to usurp the host UPS to modulate the function and stability of viral proteins. In addition to the pro‐viral function, UPS‐mediated viral protein degradation may also constitute a host defense process against some positive‐stranded RNA viral infections. This issue will also be discussed in the current review. Copyright © 2012 John Wiley & Sons, Ltd.

show abstract

Inhibition of hepatitis E virus replication by proteasome inhibitor is nonspecific

Zhou

Peppelenbosch

2014

Arch Virol

View full text Add to dashboard Cite

The ubiquitin proteasome system plays important role in virus infection. A previous study showed that the proteasome inhibitor MG132 could potentially affect hepatitis E virus (HEV) replication. In this study, we found that MG132 could inhibit HEV and hepatitis C virus (HCV) replication-related luciferase activity in subgenomic models. Furthermore, treatment with MG132 in a HEV infectious model resulted in a dramatic reduction in the intracellular level of HEV RNA. Surprisingly, MG132 concurrently inhibited the expression of a luciferase gene used as a control as well as a wide range of host genes. Consistently, the total cellular RNA and protein content was concurrently reduced by MG132 treatment, suggesting a nonspecific antiviral effect.

show abstract

Evaluation of the Susceptibility of the 3C Proteases of Hepatitis A Virus and Poliovirus to Degradation by the Ubiquitin-Mediated Proteolytic System

Cited by 19 publications

References 33 publications

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

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

The ubiquitin‐proteasome system in positive‐strand RNA virus infection

Inhibition of hepatitis E virus replication by proteasome inhibitor is nonspecific

Contact Info

Product

Resources

About