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Updated: Apr 23, 2026
In-vitro Reconstitution of Bacterial Ubiquitination and VCP/p97-mediated Elimination
Published on: January 2, 2026
The poxvirus encoded ubiquitin ligase, p28, is regulated by proteasomal degradation and autoubiquitination
Kelly Mottet1, Bettina Bareiss1, Craig D Milne1
1Li Ka Shing Institute of Virology, Department of Medical Microbiology and Immunology, 621 HMRC, University of Alberta, Edmonton, Alberta, Canada T6G 2S2.
Abstract:
Virus manipulation of the ubiquitin-proteasome system has become increasingly apparent. Ubiquitin is a 76 amino acid protein that is post-translationally conjugated to target proteins, while poly-ubiquitination subsequently leads to degradation via the 26S proteasome. Target specificity is determined by a large family of ubiquitin ligases. Poxviruses encode p28, a highly conserved ubiquitin ligase expressed in a wide range of poxviruses (J. Virol. 79:597). Here we investigate the relationship between p28 and ubiquitination. Confocal microscopy indicated that orthologs of p28 co-localized with ubiquitin at the virus factory. Flow cytometry assays further demonstrated that p28 was regulated by proteasomal degradation. Moreover, when the ubiquitin ligase activity of p28 was disrupted by mutating the RING domain conjugated ubiquitin still localized to the viral factories, indicating that an unknown ubiquitin ligase(s) was responsible for regulating p28. Our observations indicate that p28 is a ubiquitin ligase that is regulated by ubiquitination and proteasomal degradation.
Insights
Poxviruses utilize p28, a ubiquitin ligase, which is regulated by ubiquitination and proteasomal degradation. This study reveals p28
Area of Science:
- Virology
- Molecular Biology
- Cellular Biology
Background:
- Viruses extensively manipulate the ubiquitin-proteasome system for replication.
- Ubiquitin ligases control protein ubiquitination and degradation, crucial for cellular processes.
- Poxviruses encode p28, a conserved ubiquitin ligase found in various poxviruses.
Purpose of the Study:
- To investigate the relationship between poxvirus p28 and the ubiquitination process.
- To determine how p28 itself is regulated within the host cell.
- To identify the ubiquitin ligase responsible for p28 regulation.
Main Methods:
- Confocal microscopy to visualize p28 and ubiquitin localization.
- Flow cytometry assays to assess p28 regulation by proteasomal degradation.
- Site-directed mutagenesis of the p28 RING domain to disrupt ligase activity.
Main Results:
- Poxvirus p28 orthologs co-localized with ubiquitin at viral factories.
- p28 was found to be regulated by proteasomal degradation.
- Disruption of p28's ubiquitin ligase activity did not prevent ubiquitin localization to viral factories, suggesting other ligases are involved.
Conclusions:
- Poxvirus p28 functions as a ubiquitin ligase.
- p28 is subject to ubiquitination and subsequent proteasomal degradation.
- An unidentified ubiquitin ligase(s) regulates p28 activity within the viral factory.
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