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Ectromelia Virus Encodes A Novel Family Of Ankyrin/F-box Proteins That Manipulate The SCF Ubiquitin Ligase And NF-κB Activation Open Access
- Other title
SCF ubiqutin ligase
- Type of item
- Degree grantor
University of Alberta
- Author or creator
van Buuren, Nicholas J.
- Supervisor and department
- Examining committee member and department
Schultz, Michael (Biochemistry)
Schang, Luis (Biochemistry)
Coscoy, Laurent (Immunology and Pathogenesis, UC Berkeley)
Ostergaard, Hanne (Medical Microbiology and Immunology)
Ingham, Rob (Medical Microbiology and Immunology)
Department of Medical Microbiology and Immunology
- Date accepted
- Graduation date
Doctor of Philosophy
- Degree level
Ectromelia virus (ECTV) is the causative agent of lethal mousepox, and is highly
related to the human pathogen, variola virus, the causative agent of smallpox.
Poxviruses contain large dsDNA genomes that encode numerous open reading
frames that manipulate cellular signalling pathways. We used bioinformatics to
identify a family of four genes encoded by ECTV that contain N-terminal ankyrin
repeats in conjunction with a C-terminal F-box domain. The ECTV encoded
ankyrin/F-box proteins: EVM002, EVM005, EVM154 and EVM165, all interact
with the cellular SCF (Skp1/Cul-1/F-box) ubiquitin ligase complex through an
interaction mediated by their C-terminal F-box domain. These four proteins bind
to the SCF complex in a similar manner to cellular F-box-containing substrate
adaptor proteins. We hypothesize that each of the ECTV encoded ankyrin/F-box
proteins recruits a unique family of target proteins to the SCF complex for
ubiquitylation. The NF-κB signalling cascade is an important mediator of innate
immunity, and is tightly regulated by ubiquitylation. A critical step in the
activation of NF-κB is the ubiquitylation and degradation of the inhibitor of
kappaB (IκBα), by the cellular SCFβ-TRCP ubiquitin ligase. Upon stimulation with
TNFα or IL-1β, orthopoxvirus-infected cells display an accumulation of
phosphorylated IκBα, indicating that NF-κB activation is inhibited during
poxvirus infection at the point of IκBα degradation. Since degradation of IκBα is
catalyzed by the SCFβ-TRCP ubiquitin ligase complex we investigated the role of
the ECTV encoded ankyrin/F-box proteins in the regulation of NF-κB activation.
Expression of Flag-EVM005 inhibited both IκBα degradation and p65 nuclear
translocation in response to TNFα or IL-1β. Regulation of the NF-κB pathway
by EVM005 was dependent on the F-box domain, and interaction with the SCF
complex. Additionally, we created ECTV knockout viruses devoid of each of the
four ankyrin/F-box genes using a novel “Selectable and Excisable Marker”
system. The EVM005 deletion virus was shown to inhibit NF-κB activation
despite lacking the EVM005 open reading frame; however, this virus was
attenuated in two mouse strains. The contribution of EVM005 to virulence is
therefore independent from its ability to inhibit NF-κB activation, and is
potentially linked to unique target proteins ubiquitylated through the SCF
complex during infection.
- 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. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. 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
van Buuren, N., B. Couturier, Y. Xiong, and M. Barry. 2008. Ectromelia virus encodes a novel family of F-box proteins that interact with the SCF complex. J Virol 82:9917-27.Rintoul, J., Wang, J., Gammon, D.B., van Buuren, N., Garson, K., Jardine, K., Barry, M., Evans, D.H., and Bell, J.C. 2011. A selectable and excisable marker system for the rapid creation of recombinant poxviruses. Plos One, 6(9): e24643.Mohamed, M. R., M. M. Rahman, J. S. Lanchbury, D. Shattuck, C. Neff, M. Dufford, N. van Buuren, K. Fagan, M. Barry, S. Smith, I. Damon, and G. McFadden. 2009. Proteomic screening of variola virus reveals a unique NF-kappaB inhibitor that is highly conserved among pathogenic orthopoxviruses. PNAS, 106:9045-50.Teale, A., S. Campbell, N. Van Buuren, W. C. Magee, K. Watmough, B. Couturier, R. Shipclark, and M. Barry. 2009. Orthopoxviruses require a functional ubiquitin-proteasome system for productive replication. J Virol 83:2099-108.
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