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-!B in the nucleus of TNFa-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

M148R and M149R are two virulence factors for myxoma virus pathogenesis in the European rabbit

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. 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

Time scale evolution of avipoxviruses

AbstractAvipoxviruses are divided into three clades: canarypox-like viruses, fowlpox-like viruses, and psittacinepox-like viruses. Several molecular clock and demographic models available in the BEAST package were compared on three avipoxvirus genes (P4b, cnpv186 and DNA polymerase genes), which enabled to determine that avipoxviruses evolved at a rate of 2–8×10−5substitution/site/year, in the range of poxviruses previously reported evolution rates. In addition, the date of mean time of divergence of avipoxviruses from a common ancestor was extrapolated to be about 10,000–30,000years ago, at the same period as modern poxvirus species. Our findings will facilitate epidemiological investigations on avipoxviruses’ spread, origin and circulation

Les vecteurs viraux : outils modernes de vaccination

Les vaccins destinés aux animaux appartiennent à deux grandes catégories : les vaccins à agents vivants, et ceux à agents inertes. Depuis quelques années, dans chacune de ces catégories, les innovations technologiques ont considérablement amélioré et diversifié les stratégies vaccinales disponibles en fonction des contraintes liées à des préoccupations tant d’innocuité, que d’efficacité ou encore de nature économique. C’est dans ce cadre que l’INRA a depuis de nombreuses années orienté les efforts de recherche vers l’élaboration de nouveaux vaccins s’appuyant sur la mise au point de vecteurs viraux adaptés à diverses espèces animales et susceptibles de répondre aux exigences des filières animales. Dans cette revue, nous décrivons ainsi les principes d’obtention et le développement de vecteurs vaccinaux fondés sur l’emploi de poxvirus animaux à spectre d’hôte étroit (virus myxomateux), d’adenovirus humains ou animaux défectifs (c'est-à-dire ayant perdu toute capacité à se multiplier chez l’hôte) ainsi que de rhabdovirus de poissons modifiés par génétique inverse. Des exemples d’application de vaccination non seulement contre des maladies animales d’intérêt économique, mais aussi dans le cadre de modèles de pathologie comparée permettent d’illustrer le potentiel indiscutable de ces vecteurs viraux et d’envisager leur emploi pour le contrôle de maladies animales émergentes ou réémergentes en Europe

In vitro permissivity of bovine cells for wild-type and vaccinal myxoma virus strains

Myxoma virus (MYXV), a leporide-specific poxvirus, represents an attractive candidate for the generation of safe, non-replicative vaccine vector for non-host species. However, there is very little information concerning infection of non-laboratory animals species cells with MYXV. In this study, we investigated interactions between bovine cells and respectively a wild type strain (T1) and a vaccinal strain (SG33) of MYXV. We showed that bovine KOP-R, BT and MDBK cell lines do not support MYXV production. Electron microscopy observations of BT-infected cells revealed the low efficiency of viral entry and the production of defective virions. In addition, infection of bovine peripheral blood mononuclear cells (PBMC) occurred at a very low level, even following non-specific activation, and was always abortive. We did not observe significant differences between the wild type strain and the vaccinal strain of MYXV, indicating that SG33 could be used for new bovine vaccination strategies

Protection against myxomatosis and rabbit viral hemorrhagic disease with recombinant myxoma viruses expressing rabbit hemorrhagic disease virus capsid protein

Two myxoma virus-rabbit hemorrhagic disease virus (RHDV) recombinant viruses were constructed with the SG33 strain of myxoma virus to protect rabbits against myxomatosis and rabbit viral hemorrhagic disease. These recombinant viruses expressed the RHDV capsid protein (VP60). The recombinant protein, which is 60 kDa in size, was antigenic, as revealed by its reaction in immunoprecipitation with antibodies raised against RHDV. Both recombinant viruses induced high levels of RHDV- and myxoma virus-specific antibodies in rabbits after immunization. Inoculations by the intradermal route protected animals against virulent RHDV and myxoma virus challenges

Characterisation of a non-pathogenic and non-protective infectious rabbit lagovirus related to RHDV

The existence of non-pathogenic RHDV strains was established when a non-lethal virus named rabbit calicivirus (RCV) was characterised in 1996 in Italy. Since then, different RNA sequences related to RHDV have been detected in apparently healthy domestic and wild rabbits, and recently a new lagovirus was identified in Australia. We have characterised from seropositive healthy domestic rabbits a non-lethal lagovirus that differs from RHDV in terms of pathogenicity, tissue tropism and capsid protein sequence. Phylogenetic analyses have revealed that it is close to the Ashington strain and to the RCV, but distinct. We proved experimentally that it is infectious but non-pathogenic and demonstrated that, contrary to the other described non-pathogenic lagoviruses, it induces antibodies that do not protect against RHDV. Our results indicate the existence of a gradient of cross-protection between circulating strains, from non-protective, partially protective to protective strains, and highlight the extent of diversity within the genus Lagovirus