Ectromelia Virus Encodes A Novel Family Of Ankyrin/F-box Proteins That Manipulate The SCF Ubiquitin Ligase And NF-κB Activation

  • Author / Creator
    van Buuren, Nicholas J.
  • 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.

  • Subjects / Keywords
  • Graduation date
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
  • Institution
    University of Alberta
  • Degree level
  • Department
    • Department of Medical Microbiology and Immunology
  • Specialization
    • Virology
  • Supervisor / co-supervisor and their department(s)
    • Barry, Michele
  • Examining committee members and their departments
    • Ostergaard, Hanne (Medical Microbiology and Immunology)
    • Schang, Luis (Biochemistry)
    • Ingham, Rob (Medical Microbiology and Immunology)
    • Coscoy, Laurent (Immunology and Pathogenesis, UC Berkeley)
    • Schultz, Michael (Biochemistry)