title: Ectromelia virus encodes a novel family of F-box proteins that interact with the SCF complex.
abstract: Poxviruses are notorious for encoding multiple proteins that regulate cellular signaling pathways, including the ubiquitin-proteasome system. Bioinformatics indicated that ectromelia virus, the causative agent of lethal mousepox, encoded four proteins, EVM002, EVM005, EVM154, and EVM165, containing putative F-box domains. In contrast to cellular F-box proteins, the ectromelia virus proteins contain C-terminal F-box domains in conjunction with N-terminal ankyrin repeats, a combination that has not been previously reported for cellular proteins. These observations suggested that the ectromelia virus F-box proteins interact with SCF (Skp1, cullin-1, and F-box) ubiquitin ligases. We focused our studies on EVM005, since this protein had only one ortholog in cowpox virus. Using mass spectrometry, we identified cullin-1 as a binding partner for EVM005, and this interaction was confirmed by overexpression of hemagglutinin (HA)-cullin-1. During infection, Flag-EVM005 and HA-cullin-1 colocalized to distinct cellular bodies. Significantly, EVM005 coprecipitated with endogenous Skp1, cullin-1, and Roc1 and associated with conjugated ubiquitin, suggesting that EVM005 interacted with the components of a functional ubiquitin ligase. Interaction of EVM005 with cullin-1 and Skp1 was abolished upon deletion of the F-box, indicating that the F-box played a crucial role in interaction with the SCF complex. Additionally, EVM002 and EVM154 interacted with Skp1 and conjugated ubiquitin, suggesting that ectromelia virus encodes multiple F-box-containing proteins that regulate the SCF complex. Our results indicate that ectromelia virus has evolved multiple proteins that interact with the SCF complex.
authors.affiliation: 621 HMRC, Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Nick
authors.initials: N
authors.lastname: van Buuren
authors.forename: Brianne
authors.initials: B
authors.lastname: Couturier
authors.forename: Yue
authors.initials: Y
authors.lastname: Xiong
authors.forename: Michele
authors.initials: M
authors.lastname: Barry
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004481:Ectromelia virus / Q000235:genetics / Q000378:metabolism*; D004482:Ectromelia, Infectious / Q000821:virology; D044783:F-Box Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D044843:SKP Cullin F-Box Protein Ligases / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D014452:Ubiquitins / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D044783:F-Box Proteins; D014452:Ubiquitins; D014764:Viral Proteins; D044843:SKP Cullin F-Box Protein Ligases