MNF, an ankyrin repeat protein of myxoma virus, is part of a native cellular SCF complex during viral infection.
Myxoma virus (MYXV), a member of the Poxviridae family, is the agent responsible for myxomatosis, a fatal disease in the European rabbit (Oryctolagus cuniculus). Like all poxviruses, MYXV is known for encoding multiple proteins that regulate cellular signaling pathways. Among them, four proteins share the same ANK/PRANC structure: M148R, M149R, MNF (Myxoma Nuclear factor) and M-T5, all of them described as virulence factors. This family of poxvirus proteins, recently identified, has drawn considerable attention for its potential role in modulating the host ubiquitin-proteasome system during viral infection. To date, many members of this novel protein family have been shown to interact with SCF components, in vitro. Here, we focus on MNF gene, which has been shown to express a nuclear protein presenting nine ANK repeats, one of which has been identified as a nuclear localization signal. In transfection, MNF has been shown to colocalise with the transcription factor NF-kappaB in the nucleus of TNFalpha-stimulated cells. Functionally, MNF is a critical virulence factor since its deletion generates an almost apathogenic virus. In this study, to pursue the investigation of proteins interacting with MNF and of its mechanism of action, we engineered a recombinant MYXV expressing a GFP-linked MNF under the control of MNF native promoter. Infection of rabbits with MYXV-GFPMNF recombinant virus provided the evidence that the GFP fusion does not disturb the main function of MNF. Hence, cells were infected with MYXV-GFPMNF and immunoprecipitation of the GFPMNF fusion protein was performed to identify MNF's partners. For the first time, endogenous components of SCF (Cullin-1 and Skp1) were co-precipitated with an ANK myxoma virus protein, expressed in an infectious context, and without over-expression of any protein.
Blanié
Sophie
S
INRA, UMR 1225 Interactions Hôtes - Agents Pathogènes F-31076 Toulouse, France.
Gelfi
Jacqueline
J
Bertagnoli
Stéphane
S
Camus-Bouclainville
Christelle
C
D000818:Animals; D017089:Ankyrin Repeat; D002460:Cell Line; D002522:Chlorocebus aethiops; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D047468:Immunoprecipitation; D009233:Myxoma virus / Q000502:physiology*; D011213:Poxviridae Infections / Q000473:pathology / Q000662:veterinary; D011485:Protein Binding; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D044843:SKP Cullin F-Box Protein Ligases / Q000378:metabolism*; D014412:Tumor Virus Infections / Q000473:pathology / Q000662:veterinary; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D037521:Virulence Factors; D049452:Green Fluorescent Proteins; D044843:SKP Cullin F-Box Protein Ligases