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Permanent link (DOI): https://doi.org/10.7939/R3HQ3S41P

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Characterization of the Poxviral Encoded Ubiquitin Ligase p28 Open Access

Descriptions

Other title
Subject/Keyword
p28
Poxvirus
Ubiquitin ligase
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Lehman, Bettina, J
Supervisor and department
Ingham, Rob (MMI)
Examining committee member and department
Foley, Edan (MMI)
Cao, Jingxin (Medical Microbiology, University of Manitoba)
LaPointe, Paul (Cell Biology)
Ingham, Rob (MMI)
Evans, David (MMI)
Department
Department of Medical Microbiology and Immunology
Specialization
Virology
Date accepted
2015-05-14T09:24:29Z
Graduation date
2015-11
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
Many cellular processes are regulated by the ubiquitin proteasome system (UPS), which utilizes the polypeptide ubiquitin to mark proteins for destruction via the 26S proteasome. Since the UPS plays an important role in cellular homeostasis, many viruses have evolved strategies to regulate the process of ubiquitination to their own advantage. Poxviruses also manipulate the UPS by encoding E3 ubiquitin ligases to target cellular or viral proteins for ubiquitination. This allows poxviruses to exploit the UPS and manipulate a wide range of cellular processes. p28, a poxvirus encoded ubiquitin ligase, was first described in ectromelia virus (ECTV). p28 contains an N-terminal KilA-N DNA binding domain and a C-terminal RING-domain that is necessary for the ubiquitin ligase activity. Buller and colleagues demonstrated that deletion of p28 from ECTV results in a dramatic reduction in virus virulence, indicating its importance during viral infection. p28 is a highly conserved ubiquitin ligase that is expressed in a wide range of poxviruses, including Avipoxviruses. One distinguishing feature of the Avipoxviruses is their large genome, as exemplified by the prototypical members of fowlpox virus (FWPV) (288 kbp) and canarypox virus (CNPV) (365 kbp), encoding a number of yet uncharacterized proteins. FWPV and CNPV each contain two p28-like ubiquitin ligases. In contrast, non-Avipoxviruses encode only one p28-like ubiquitin ligase. FWPV and CNPV also encode additional KilA-N only genes lacking a RING domain; an observation not seen in other members of the poxvirus family. The data presented in this thesis demonstrate that both p28 homologues in FWPV, FWPV150 and FWPV157 are functional ubiquitin ligases located at the virus factories. Intriguingly, expression of FWPV150 started early in infection, while FWPV157 was expressed late. We further demonstrated that each of the eight KilA-N domain-containing proteins were expressed during FWPV infection. Since many cellular and viral ubiquitin ligases are regulated through ubiquitination, we investigated whether this was true of p28. Our data confirmed a role for the RING domain for the selfubiquitination of p28, but we further found that p28 is also regulated by another E3 ubiquitin ligase(s). It remains to be investigated whether ubiquitination changes p28 activity or if the ubiquitination is solely required for the proteasomal degradation of p28 at a certain stage during infection. Finally, we conducted a mass spectrometry screen for p28 substrates, and identified heat shock protein 70 (HSP70) as an interaction partner of p28 during VACV infection. While we were unable to show that HSP70 is a substrate of p28, we demonstrated HSP70 enrichment at the virus factory in the presence of p28 when compared with VACVCop infected cells alone. This effect of HSP70 enrichment was conserved within the investigated p28 homologues. We further found that p28 residues 184-204 were important for recruiting HSP70 from the cellular nuclear and cytoplasmic pool to the virus factory. The importance of the p28 mediated HSP70 recruitment to the virus factory is to be determined. In summary, this thesis characterized the expression, cellular localization and ubiquitin ligase activity of p28 homologues in FWPV. In addition, p28 RING and KilAN mutants were characterized in terms of their ubiquitin ligase activity and interaction with ubiquitin and the UPS; in the end a mass spectrometry screen and follow up validation confirmed HSP70 as an interaction partner of p28.
Language
English
DOI
doi:10.7939/R3HQ3S41P
Rights
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
Citation for previous publication
Virology. 462-463:60-70Virology. 468-470: 363-78.

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