MNF, an ankyrin repeat protein of myxoma virus, is part of a native cellular SCF complex during viral infection
© Blanié et al; licensee BioMed Central Ltd. 2010
Received: 9 December 2009
Accepted: 8 March 2010
Published: 8 March 2010
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-κB in the nucleus of TNFα-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.
Myxoma virus (MYXV), a member of the Poxviridae family, is the agent responsible for myxomatosis, a fatal disease in the European rabbit (Oryctolagus cuniculus). Due to its host specificity, MYXV is a useful model to study in vivo the mechanisms by which the numerous virus-encoded virulence factors control host response to infection. Among the virulence factors, MYXV genome encodes four proteins which share the same structure: M148R, M149R, MNF and M-T5 [1–4]. They present Ankyrin repeats (ANK) and a C-terminal PRANC domain . The Ankyrin repeat, a 33-residue sequence domain, is one of the most common protein-protein interaction motifs found in nature . Extensively identified in eukaryotes and bacteria, ANK motif is relatively rare among viruses, with the exception of poxviruses, especially chordopoxviruses . Thus, poxviruses generally encode 4 or 5 ANK proteins, and more than 80% of these ANK proteins present an F-box-like domain at their C-terminus . This domain is shorter than the typical cellular F-box and lacks helix 3 . Furthermore, the poxviral F-box-like domain is located at the C-terminus of ANK protein whereas cellular F-boxes are typically located in the N-terminus of the protein. In addition, cellular F-boxes are associated with a second protein-protein interaction domain such as WD40 or Leucine Rich Repeat but no association with ANK motif has been reported yet. Due to these significant differences with eukaryotic F-boxes, the poxviral F-box domain has been defined as a new motif: the PRANC domain (Pox protein Repeats of Ankyrin C-terminal) .
The F-box motif mediates protein-protein interactions and was first described as a recognition motif in the E3 ubiquitin ligase complex, known as SCF (Skp, Cullin, F-box) . The SCF complex is a multi-protein complex that mediates the ubiquitination of substrates mainly destined for degradation by the 26S proteasome . Targeted proteins are recognized by a variety of F-box proteins and are delivered to the E3 ligase complex via an adaptor protein, Skp1 . Interaction between the adaptor protein and the F-box protein occurs via the F-box motif. The SCF complex plays a critical role in the selective degradation of regulatory proteins that mediate various cellular functions, such as signal transduction and cell cycle regulation . Ten poxvirus ANK proteins have been shown to contain a functional F-box: myxoma virus protein M-T5 , orf virus proteins OV008, OV123, OV126, OV128 and OV129 , vaccinia virus protein MVA186R  and ectromelia virus proteins ECTV002, ECTV005 and ECTV154 . Hence, among MYXV proteins, only M-T5 was identified as a cellular binding partner of Cullin-1 to date. Enhanced ubiquitination of p27/Kip1 and subsequent degradation via the proteasome pathway was observed through the interaction of M-T5 and Cullin-1. Consistent with this interaction, M-T5 was shown to promote cell cycle progression beyond the G0/G1 checkpoint during virus infection. In this study, we focused on MNF. MNF gene expresses a nuclear protein that presents nine ANK repeats, one of which exhibits significant sequence similarity with IκBα nuclear localization signal. Moreover, in transfection, MNF colocalises with the transcription factor NF-κB in the nucleus of TNFα-stimulated cells. Functionally, infection of rabbits with an MNF-deleted MYXV induced very mild clinical signs, with few discrete secondary myxomas, no respiratory infection, and no lethality. Histological analysis of the primary myxoma and parotid lymph node showed that deletion of MNF gene allowed a more rapid and quickly resolved inflammation . To summarize, MNF was shown to inhibit the pro-inflammatory pathway, seemingly through an interaction with NF-κB pathway.
For the first time, endogenous components of SCF (Cullin-1 and Skp1) were co-precipitated with an ANK myxoma virus protein, virally expressed under control of its own promoter, and without over-expression of any protein. Thus, our data demonstrate that MNF interacts with cellular SCF ubiquitin ligase complexes, in real infectious context. Many viral proteins have been shown to interact with subunits of the SCF complex. In the case of ANK poxviral proteins, they seem to act as substrate adaptors, thus directing the ubiquitin ligase complex toward new targets or protecting cellular targets from ubiquitination. However, the substrates are mostly unknown. In some instances, viral proteins can also be targeted by SCF for their own destruction. The only identified substrates ubiquitinated by SCF containing an ANK/PRANC poxviral protein are p27  and Akt , which are bound to the host SCF complex via Skp1 and MYXV M-T5 in infected cells. On the other hand, it has been shown that the 68k-ank protein contains another critical domain that may function independently from SCF ubiquitin ligase complex formation , suggesting that ANK/PRANC poxviral proteins may have multiple roles. In addition, poxviruses encode ANK proteins that do not present PRANC motif, such as Vaccinia virus K1L which mediates host-range function in RK-13 cells via ANK repeat. Moreover, K1L may interact with a cellular GTPase-activating protein  and inhibits host NFκB activation by preventing IκBα degradation . The identification of proteins bound by the N-terminal ANK domains and the outcome of these proteins could also greatly enhance our understanding of the multiple functions of the ANK/PRANC poxviral proteins during infection.
SBl was supported by funds from the French Ministry of Research and Technology. We thank Brigitte Peralta and Josyane Loupias for excellent technical assistance. We are grateful to B. Séverac for critical reading of the manuscript.
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