MNF, an ankyrin repeat protein of myxoma virus, is part of a native cellular SCF complex during viral infection

Abstract

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.

Genetic Screen of a Library of Chimeric Poxviruses Identifies an Ankyrin Repeat Protein Involved in Resistance to the Avian Type I Interferon Response

“…8C). The cullin-1 in the purified eluates from TAP-fpv014-expressing cells, however, migrated slightly slower than in the control lysates (consistent with the 98-kDa ubiquitinated form of cullin-1 observed to interact with the myxoma virus PRANC protein MNF [41]). Even though this cullin-1 copurified with TAP-tagged fpv014 in the eluate, it was not observed in a complex with TAP-tagged fpv014 (predicted mass, 143 kDa or 162 kDa with the addition of Skp1) on the immunoblot, whether due to SDS sensitivity of the complex or to the inability of the antibody to bind complexed cullin-1.…”

“…8B). In this regard, fpv014 behaves like several other members of the ANK/PRANC family from poxviruses that infect mammals, namely, cowpox virus, ectromelia virus, myxoma virus, Orf virus, and vaccinia MVA (41)(42)(43)(44)(45)(46), indicating that fpv014 also functions via the SCF (Skp1, cullin-1, F-box) ubiquitin ligase complex. The PRANC proteins interact indirectly with cullin-1 via Skp1 as an adaptor.…”

“…The PRANC domains of a number of mammalian poxvirus ANK proteins have been shown to mediate interaction with core components of the ubiquitin ligase complex (41,42,45,46,60). It was postulated (40,43) that such interactions would target for ubiquitination ligands captured by the N-terminal ANK domains.…”

“…There is a precedent for this observation, in that two similarly sized bands copurified with green fluorescent-tagged myxoma virus MNF protein. The predominant slower form immunoblotted with an unspecified antiubiquitin antibody (41). There is no indication at this stage whether fpv014 stabilized the modified form of cullin-1 in the cell or whether it was solely (or predominantly) the modified form that copurified with TAP-tagged fpv014.…”

Genetic Screen of a Mutant Poxvirus Library Identifies an Ankyrin Repeat Protein Involved in Blocking Induction of Avian Type I Interferon

“…In the accompanying paper (50), we demonstrated interaction between the fpv014 ANK/PRANC protein involved in ChIFN1 (chicken IFN-␣) resistance and proteins of the SCF (Skp1, Cullin-1, F-box) ubiquitin ligase complex, as has been observed for a number of mammalian poxvirus ANK proteins (51)(52)(53)(54)(55). It is postulated (38) that such interactions allow ANK/PRANC proteins to act as adaptors targeting for ubiquitination (and probable proteasomal degradation) ligands captured by the N-terminal ANK domains.…”