INRA, UMR 1225 Interactions Hôtes - Agents Pathogènes F-31076 Toulouse, France.
Virol J. 2010 Mar 8;7:56. doi: 10.1186/1743-422X-7-56.
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.
粘液瘤病毒（MYXV），痘病毒科的一员，是引起粘液瘤病的病原体，这种疾病是欧洲兔（Oryctolagus cuniculus）的致命疾病。与所有痘病毒一样，MYXV 以编码多种调节细胞信号通路的蛋白而闻名。其中，有四种蛋白具有相同的 ANK/PRANC 结构：M148R、M149R、MNF（粘液瘤核因子）和 M-T5，它们都被描述为毒力因子。这种痘病毒蛋白家族最近被发现，由于其在病毒感染过程中可能调节宿主泛素-蛋白酶体系统的作用而引起了相当大的关注。迄今为止，许多这类新型蛋白家族的成员已被证明可以与 SCF 成分在体外相互作用。在这里，我们重点关注 MNF 基因，该基因已被证明表达一种具有九个 ANK 重复的核蛋白，其中一个被鉴定为核定位信号。在转染实验中，MNF 已被证明与 TNFalpha 刺激细胞中的转录因子 NF-kappaB 在核内共定位。功能上，MNF 是一种关键的毒力因子，因为其缺失会产生几乎无致病性的病毒。在这项研究中，为了进一步研究与 MNF 相互作用的蛋白及其作用机制，我们构建了一种表达 GFP 连接的 MNF 的重组 MYXV，该 MNF 由 MNF 天然启动子控制。用 MYXV-GFPMNF 重组病毒感染兔子，证明 GFP 融合不会干扰 MNF 的主要功能。因此，用 MYXV-GFPMNF 感染细胞，然后进行 GFP 融合蛋白的免疫沉淀，以鉴定 MNF 的相互作用伙伴。这是首次在感染背景下，用 GFP 融合蛋白共沉淀到内源性 SCF（Cullin-1 和 Skp1）成分，而没有任何蛋白的过表达。
