NF-B is one of the most important elements that coordinate stress-induced, immune, and inflammatory responses. Myxoma virus, a member of the Poxviridae family responsible for rabbit myxomatosis, codes for several factors that help its survival in the host. In this study, we focused on the product of the M150R gene. We show that the protein has nine ankyrin repeats (ANKs), with the eighth having a close similarity with the nuclear localization signal-containing ANK of I-B␣, which regulates NF-B activity by sequestering it in the cytosol. Because the viral protein is targeted to the nucleus, it was named MNF, for myxoma nuclear factor. This localization was lost when the eighth ANK was removed. In tumor necrosis factor alpha-treated cells, MNF and NF-B colocalized as dotted spots in the nucleus. In vivo experiments with a knockout virus showed that MNF is a critical virulence factor, with its deletion generating an almost apathogenic virus. Detailed histological examinations revealed an increase in the inflammatory process in the absence of MNF, consistent with the interference of MNF with the NF-B-induced proinflammatory pathway. Because MNF has homologs in other poxviruses, such as vaccinia, cowpox, and variola viruses, this protein is probably part of a key mechanism that contributes to the immunogenic and pathogenic properties of these viruses.

Myxoma virus (MYXV), a member of the Poxviridae family, is the agent responsible for myxomatosis, a fatal disease in the European rabbit (Oryctolagus cuniculus). MYXV has a linear double-stranded DNA genome that encodes several factors important for evasion from the host immune system. Among them, four ankyrin (ANK) repeat proteins were identified: M148R, M149R, M150R and M-T5. To date, only M150R and M-T5 were studied and characterized as critical virulence factors. This article presents the first characterization of M148R and M149R. Green Fluorescent Protein (GFP) fusions allowed us to localize them in a viral context. Whereas M149R is only cytoplasmic, interestingly, M148R is in part located in the nucleolus, a unique feature for an ANK repeat poxviral protein. In order to evaluate their implication in viral pathogenicity, targeted M148R, M149R, or both deletions were constructed in the wild type T1 strain of myxoma virus. In vitro infection of rabbit and primate cultured cells as well as primary rabbit cells allowed us to conclude that M148R and M149R are not likely to be implicated in cell tropism or host range functions. However, in vivo experiments revealed that they are virulence factors since after infection of European rabbits with mutant viruses, a delay in the onset of clinical signs, an increase of survival time and a dramatic decrease in mortality rate were observed. Moreover, histological analysis suggests that M148R plays a role in the subversion of host inflammatory response by MYXV.

Myxomatosis in Europe is the result of the release of a South America strain of myxoma virus in 1952. Several attenuated strains with origins in South America or California have since been used as vaccines in the rabbit industry. We sequenced the genome of the SG33 myxoma virus vaccine strain and compared it with those of other myxoma virus strains. We show that SG33 genome carries a large deletion in its right end. Furthermore, our data strongly suggest that the virus isolate from which SG33 is derived results from an in vivo recombination between a wild-type South America (Lausanne) strain and a California MSD-derived strain. These findings raise questions about the use of insufficiently attenuated virus in vaccination.

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.