Poxviruses are an extensive family of large DNA viruses, including the human pathogen variola virus, which causes smallpox. Vertebrate poxviruses encode numerous proteins of unknown function that contain an ankyrin repeat (ANK) domain. ANK domains have been shown to mediate protein-protein interactions and are present in a large number of cellular proteins involved in pathways such as transcription, cell cycle regulation and development. Recently, an F-box-like motif, which conserved key residues of the cellular F-box motif, was identified at the C-terminus of these proteins. Most cellular F-box proteins are specificity factors of SCF1 ubiquitin ligases, recruiting substrate proteins to the SCF1 complex where they are poly-ubiquitinated. F-box proteins use their N-terminal F-box to bind the SCF1 complex, and a second interaction motif, such as a leucine-rich-repeat domain, but never an ANK domain, to recruit the substrate. Poxviral ANK/F-box-like proteins exhibit a reversed domain order compared to cellular F-box proteins, and a novel domain combination of ANK domain and putative F-box. The aim of this study was to examine the functionality of the poxviral F-box-like motif and to assess whether poxviral ANK/F-box-like proteins could function as specificity factors of cellular ubiquitin ligases.
Using immunoprecipitation of transiently expressed recombinant proteins, several poxvirus F-box-like motifs that represented the sequence variation and differences in motif length observed among poxvirus ANK/F-box-like proteins, were tested and found to be functional F-box domains. The poxviral ANK/F-box proteins interacted with all three subunits of the SCF1 complex, Skp1 (directly), Cul1 and Rbx1 (indirectly) regardless of the manner of SCF1 expression (transient over-expression or endogenous). A representative poxviral F-box was found to be both necessary and sufficient for binding SCF1, while the ANK domain of the same protein was expendable. SCF1 ubiquitin ligases bound by a poxviral ANK/F-box protein remained competent in forming poly-ubiquitin chains, indicating that these poxviral proteins are not inhibitors of SCF1.
This evidence strongly suggests that poxvirus ANK/F-box proteins function as specificity factors of SCF1 ubiquitin ligases. Individual chordopoxviruses each encode several discrete ANK/F-box proteins. The five ANK/F-box proteins of one poxvirus, orf virus, were competent in binding SCF1, and when transiently expressed, were found to exhibit different sub-cellular localization patterns that were consistent in two cell lines and in the presence and absence of orf virus infection. Furthermore, the ANK domains of the five orf virus ANK/F-box proteins were distinct in primary amino acid sequence and in predicted tertiary structure. Together theses findings indicate that the five orf virus ANK/F-box proteins interact with different binding partners.
In summary, this study identifies a novel class of F-box proteins encoded by chordopoxviruses that exhibits a reversed domain order and a novel domain combination of ANK domain with a truncated, but functional, F-box motif. Bioinformatic analysis, structure modeling, and microscopy findings suggest that chordopoxvirus ANK/F-box proteins function as specificity factors of SCF1 ubiquitin ligases. Several viruses use specificity factors to target cellular anti-viral factors to the ubiquitination system, a virulence mechanism that now seems likely to also exist in poxviruses.
| Identifer | oai:union.ndltd.org:ADTP/222190 |
| Date | January 2009 |
| Creators | Sonnberg, Stephanie, n/a |
| Publisher | University of Otago. Department of Microbiology & Immunology |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://policy01.otago.ac.nz/policies/FMPro?-db=policies.fm&-format=viewpolicy.html&-lay=viewpolicy&-sortfield=Title&Type=Academic&-recid=33025&-find), Copyright Stephanie Sonnberg |
Page generated in 0.0024 seconds